Objective:To analyze the drug resistance and virulence characteristics of KPC-2-producing carbapenem-resistant Klebsiella pneumoniae(CRKP). Methods:A total of 26 non-repeating CRKP strains clinically isolated from a Class Ⅲ hospital in Kunming from August 2021 to March 2022 were collected. Mass spectrometry and the VITEK 2 Compact system were used to identify the bacteria and perform drug susceptibility tests. PCR was used to amplify the drug resistance and virulence genes carried by the strains. These CRKP strains were divided into a hypervirulent CRKP(CR-hvKP) group and a CR-non-hvKP group according to the characteristic virulence genes of hypervirulent Klebsiella pneumoniae. The virulence phenotypes of CRKP were investigated by wire drawing test, serum resistance test and siderophore qualitative and quantitative tests. The whole genomes of CRKP-67 (a CR-hvKP strain) and CRKP-94 (a CR-non-hvKP strain) were sequenced by the Illumina high-throughput sequencing platform, to further analyze the drug resistance genes, virulence genes, and virulence plasmidds carried by the strains. Results:The drug sensitivity results indicated that all 26 strains were resistant to carbapenem, cephalosporins, fluoroquinolones and β-lactam/β-lactam inhibitor complexes. The resistance rates to amicacin, cotrimoxazole and gentamicin were 61.54%(6/26), 57.69%(15/26) and 73.08%(9/26), respectively. Regarding the drug resistance gene amplification results, the carrying rates of blaKPC-2, blaNDM-1, blaOXA-48, blaVIM, blaIMP, blaSME, blaSHV, blaCTX-M and blaTEM were 100.00%(26/26), 0, 0, 0, 0, 100.00%(26/26), 100.00%(26/26), 15.38% (4/26) and 73.08%(19/26), respectively. In the 26 strains, the carrying rates of toxic genes entB, entC, ureA, uge, wabG, ycf, irp1, irp2, mrkD, fimH and ybtS were 100.00%(26/26), while the carrying rates of virulence genes kfuB, iroN, aero, magA and alls were 0. The positive rate of string test was 66.7%(6/9) in the CR-hvKP group and 0 in the CR-non-hvKP group. The serum killing test showed a high sensitivity rate of 77.78%(7/9), a low sensitivity rate of 11.11%(1/9), and a serum resistance rate of 11.11%(1/9) in the CR-hvKP group. In the CR-non-hvKP group, the high sensitivity rate was 29.41%(5/17); the low sensitivity rate was 17.65%(3/17), and the serum resistance rate was 52.94%(9/17). There was no statistical significance between the two groups( P>0.05). The qualitative results of siderophore showed that all strains produced yellow chelating circles with slightly different color depth and size. The quantitative results of siderophore experiment showed that the average siderophore production level of CR-hvKP group was 40.74%, and that of CR-non-hvKP group was 28.21%. The level was higher in the CR-hvKP group than in the CR-non-hvKP group, and the difference was statistically significant( P<0.05). Whole-genome sequencing results showed that CRKP-67 was ST11 type and contained 3 plasmids. Among them, plasmid pCRKP-67-A carried a series of virulence genes, including iucABCD, iutA, rmpA, rmpA2, iroB and peg344, which were highly virulent characteristic genes. Plasmid pCRKP-67-B carried blaKPC-2, blaCTX-M, blaSHV, blaTEM and other drug-resistant genes. Plasmid pCRKP-67-C carried sul2, tetR, tetA and other drug-resistant genes. The CRKP-94 was of ST340 type and contained a drug-resistant plasmid carrying blaKPC-2, blaCTX-M, blaSHV, blaTEM and other drug-resistant genes. Conclusions:CRKP strains are highly resistant, and are only sensitive to a few antibiotics, and carry a variety of drug resistance genes. The main resistance mechanism to carbapenem antibiotics is the presence of the blaKPC-2 gene, which is located on the plasmids, which results in the spread of carbapenem resistance. The types and quantity of virulence genes carried by the CR-hvKP strain are more and greater respectively than those carried by the CR-non-hvKP strain. The co-existence of drug-resistant and virulence plasmids in CR-hvKP strains may lead to the co-transmission of high drug resistance and hypervirulence, which should be highly valued by relevant departments.

Objective:To analyze the drug resistance and virulence characteristics of KPC-2-producing carbapenem-resistant Klebsiella pneumoniae(CRKP). Methods:A total of 26 non-repeating CRKP strains clinically isolated from a Class Ⅲ hospital in Kunming from August 2021 to March 2022 were collected. Mass spectrometry and the VITEK 2 Compact system were used to identify the bacteria and perform drug susceptibility tests. PCR was used to amplify the drug resistance and virulence genes carried by the strains. These CRKP strains were divided into a hypervirulent CRKP(CR-hvKP) group and a CR-non-hvKP group according to the characteristic virulence genes of hypervirulent Klebsiella pneumoniae. The virulence phenotypes of CRKP were investigated by wire drawing test, serum resistance test and siderophore qualitative and quantitative tests. The whole genomes of CRKP-67 (a CR-hvKP strain) and CRKP-94 (a CR-non-hvKP strain) were sequenced by the Illumina high-throughput sequencing platform, to further analyze the drug resistance genes, virulence genes, and virulence plasmidds carried by the strains. Results:The drug sensitivity results indicated that all 26 strains were resistant to carbapenem, cephalosporins, fluoroquinolones and β-lactam/β-lactam inhibitor complexes. The resistance rates to amicacin, cotrimoxazole and gentamicin were 61.54%(6/26), 57.69%(15/26) and 73.08%(9/26), respectively. Regarding the drug resistance gene amplification results, the carrying rates of blaKPC-2, blaNDM-1, blaOXA-48, blaVIM, blaIMP, blaSME, blaSHV, blaCTX-M and blaTEM were 100.00%(26/26), 0, 0, 0, 0, 100.00%(26/26), 100.00%(26/26), 15.38% (4/26) and 73.08%(19/26), respectively. In the 26 strains, the carrying rates of toxic genes entB, entC, ureA, uge, wabG, ycf, irp1, irp2, mrkD, fimH and ybtS were 100.00%(26/26), while the carrying rates of virulence genes kfuB, iroN, aero, magA and alls were 0. The positive rate of string test was 66.7%(6/9) in the CR-hvKP group and 0 in the CR-non-hvKP group. The serum killing test showed a high sensitivity rate of 77.78%(7/9), a low sensitivity rate of 11.11%(1/9), and a serum resistance rate of 11.11%(1/9) in the CR-hvKP group. In the CR-non-hvKP group, the high sensitivity rate was 29.41%(5/17); the low sensitivity rate was 17.65%(3/17), and the serum resistance rate was 52.94%(9/17). There was no statistical significance between the two groups( P>0.05). The qualitative results of siderophore showed that all strains produced yellow chelating circles with slightly different color depth and size. The quantitative results of siderophore experiment showed that the average siderophore production level of CR-hvKP group was 40.74%, and that of CR-non-hvKP group was 28.21%. The level was higher in the CR-hvKP group than in the CR-non-hvKP group, and the difference was statistically significant( P<0.05). Whole-genome sequencing results showed that CRKP-67 was ST11 type and contained 3 plasmids. Among them, plasmid pCRKP-67-A carried a series of virulence genes, including iucABCD, iutA, rmpA, rmpA2, iroB and peg344, which were highly virulent characteristic genes. Plasmid pCRKP-67-B carried blaKPC-2, blaCTX-M, blaSHV, blaTEM and other drug-resistant genes. Plasmid pCRKP-67-C carried sul2, tetR, tetA and other drug-resistant genes. The CRKP-94 was of ST340 type and contained a drug-resistant plasmid carrying blaKPC-2, blaCTX-M, blaSHV, blaTEM and other drug-resistant genes. Conclusions:CRKP strains are highly resistant, and are only sensitive to a few antibiotics, and carry a variety of drug resistance genes. The main resistance mechanism to carbapenem antibiotics is the presence of the blaKPC-2 gene, which is located on the plasmids, which results in the spread of carbapenem resistance. The types and quantity of virulence genes carried by the CR-hvKP strain are more and greater respectively than those carried by the CR-non-hvKP strain. The co-existence of drug-resistant and virulence plasmids in CR-hvKP strains may lead to the co-transmission of high drug resistance and hypervirulence, which should be highly valued by relevant departments.

Objective To explore the coronary flow reserve(CFR)in patients with coronary slow flow(CSF)and the diagnostic value of non-invasive myocardial work indices derived from echocardiography for CSF.Methods A retrospective study was conducted on 65 patients who underwent coronary angiography at the Zhongshan Hospital(Xiamen Branch),Fudan University due to angina pectoris,coronary artery risk factors,or electrocardiographic abnormalities from August 2020 to November 2023.Patients were divided into two groups based on the corrected TIMI frame count(cTFC):the CSF group(n=35)and the normal coronary blood flow velocity group(control group,n=30).Both groups underwent an adenosine triphosphate(ATP)drug load test to measure their coronary flow reserve(CFR).Conventional indices and myocardial work indices via echocardiography and two-dimensional speckle-tracking imaging(2D-STI)were acquired:left ventricular end-diastolic diameter(LVEDD),left ventricular end-systolic diameter(LVESD),left ventricular ejection fraction(LVEF),E/e'ratio,global longitudinal strain(GLS),global constructive work(GCW),global wasted work(GWW),global work index(GWI),and global work efficiency(GWE).Receiver operating characteristic(ROC)curves were used to evaluate the diagnostic value of myocardial work indices for CSF.Results There was no significant difference in CFR values between the two groups,but the proportion of CSF group with CFR less than 2 was higher than that of the control group(P=0.023).Compared with the control group,the CSF group showed significantly lower levels of GLS,GWI,and GCW(P＜0.05).ROC curve analysis revealed that the GLS diagnostic threshold for CSF was-19.5%,with a sensitivity of 64.7%,specificity of 78.6%,and AUC of 0.793.Among the myocardial work indices,the AUC of GWI was the highest(0.825),with a sensitivity of 88.2% and specificity of 75.0% .Conclusions Some CSF patients retain coronary microcirculatory blood flow reserve function,but the proportion of patients with reduced CFR function is increasing.The left ventricular myocardial work indices can identify early myocardial work abnormalities and monitor myocardial ischemic damage in CSF patients.

Objective:To investigate the mechanism by which non-enterotoxin-producing Bacteroides fragilis (NTBF) inhibits TNF-α-induced inflammatory responses in human normal colonic epithelial cells hcoEPIC, and explore new probiotic therapies for the prevention and treatment of colitis. Methods:The co-culture system of NTBF and hcoEPIC cells was established, and the adhesion and invasion ability of NTBF were detected, respectively. TNF-α was added to induce cellular inflammation after 4 h of co-culture of NTBF and hcoEPIC cells, and cell survival and apoptosis were detected by the CCK-8 assay and the AnnexinⅤ-FITC/PI assay respectively after 24 h. Key proteins of the NF-κB signalling pathway in hcoEPIC cells in different treatment groups were detected by Western blot and RT-qPCR, and the expression of downstream cytokines of this pathway incluing IL-1β, TNF-α and IL-10 were detected by ELISA. The effect of NTBF intervention on dextran sodium sulfate(DSS)-induced colitis mice was assessed by in vivo animal experiments. Results:NTBF adhered to hcoEPIC cells, and was non-toxic to the cells. Compared with control group, NTBF treatment alone did not affect cell survival and apoptosis of hcoEPIC cells ( P>0.05), but significantly reduced cell damage and apoptosis induced by TNF-α ( P<0.05); Compared with the TNF-α treatment alone group, the expression levels of p-NF-κB p65 and p-IκBα protein as well as NF-κB and IκBα mRNA were significantly reduced ( P<0.05); the production of IL-1β and TNF-α in the cell supernatant was reduced and the release of IL-10 was increased ( P<0.05). Animal experiments demonstrated that NTBF was indeed effective in alleviating DSS-induced colitis in ulcerative colitis model mice, which was mainly manifested by inhibiting weight loss, lowering DAI scores, improving colonic shortening, and attenuating colonic pathological damage in colitis-induced mice. Conclusions:NTBF may inhibit TNF-α-induced inflammatory responses in colonic epithelial cells by down-regulating the NF-κB pathway.

Objective:To investigate the effect of instantaneous flow rate on the consistency of diagnostic accuracy of severe degenerative mitral regurgitation (DMR) using proximal isovelocity surface area (PISA).Methods:From June 2019 to June 2021, 75 patients with DMR who underwent echocardiography in Department of Echocardiography of Zhongshan Hospital, Fudan University were prospectively enrolled. The instantaneous flow rate of DMR during the systolic phase was calculated using M-mode PISA(PISA M-mode), and a time-integrated curve was plotted. Regurgitant volume (RVol) and effective regurgitant orifice area (EROA) were calculated by traditional PISA (PISA max), pair PISA (PISA pair), and PISA M-mode, respectively. RVol acquired from cardiac magnetic resonance (CMR) volumetric method in 22 patients of the enrolled patients. The correlation and consistency of RVol acquired between the three PISA methods and CMR were compared. Agreement of diagnostic accuracy of severe mitral regurgitation (sMR) acquired between the three PISA methods and multi-parameter algorithm by American Society of Echocardiography (ASE) was analyzed using Cohen′s Kappa analysis. Results:The curve of instantaneous flow rate of DMR showed unimodal pattern with the peak at mid-late systolic phase. The correlation of RVol acquired between PISA methods and CMR was moderate for PISA max and PISA pair ( r=0.77, 0.80, both P<0.001), whereas PISA M-mode presented strong correlation with CMR ( r=0.87, P<0.001). RVol acquired from PISA max was larger than that of CMR[(69.1±37.1) ml vs (49.0±29.0)ml, P=0.002]. Both PISA max and PISA pair were shown moderate agreement of diagnostic accuracy of sMR with ASE multi-parameters algorithm (RVol: κ=0.496, 0.525, both P<0.001; EROA: κ=0.570, 0.578, both P<0.001), while PISA M-mode presented strong agreement (RVol: κ=0.867 and EROA: κ=0.802, both P<0.001). Conclusions:Based on the unimodal pattern of instantaneous flow rate in patients with DMR, PISA max may significantly overestimate RVol, exposing a significant proportion of patients with DMR to unnecessary MR surgery. PISA M-mode presents better correlation and consistency with CMR on the quantification of RVol compared with PISA max and PISA pair, and may improve the diagnostic accuracy of quantification of sMR using PISA.