Objective To investigate the resistance-related genes of Shigella sonnei with decreased susceptibility to fluoroquinolones.MethodsA total of 131 strains of Shigella sonnei were analyzed for their antimicrobial susceptibility.Mutations within the quinolone resistance determining regions (QRDR) of gyrA and parC were detected by polymerase chain reaction (PCR) and PCR products were then sequenced. Meanwhile, the plasmid-mediated quinolone resistance (PMQR) genes,qnr and aac(6')-Ib-cr were screened by PCR.ResultsResistance rates of 131 Shigella sonnei isolates to nalidixic acid,tetracycline,ampicillin and trimethoprim-sulfamethoxazole were 100.0％,93.9％,93.2％ and 92.8％,respectively.All strains were susceptible to norfloxacin,ciprofloxacin,levofloxacin,while 94％ nalidixic acid-resistant Shigella sonnei strains showed reduced susceptibilities to fluoroquinolones.All of nalidixic acid-resistant Shigella sonnei strains presented a single mutation at codon 83 (Ser→Leu) of gyrA genes,but no mutations were detected in parC gene.And PMQR genes qnr and aac (6’)-Ib-cr were not detected.Conclusions The nalidixic acid-resistant Shigella sonnei strains with reduced susceptibility to fluoroquinolones are common in the clinical practice,which may mainly due to a single mutation at codon 83 (Ser→Leu) of gyrA genes.

Objective To evaluate antimicrobial activity of fosfomycin combined with tigecycline against Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae and study the mechanism of drug resistance to fosfomycin. Methods Broth microdilution method was used to independently determine the minimum inhibitory concentrations (MIC)of fosfomycin and tigecycline against 42 Klebsiella pneumoniae isolates (including 20 KPC-producing and 22 KPC non-producing isolates).Checkerboard design method was applied to evaluate combined effect of different concentrations on antimicrobial susceptibility and calculate the fractional inhibitory concentration index (FICI).FICI=MICfosfomycin joint/MICfosfomycin monotherapy +MICtigecycline joint/MICtigecycline monotherapy .Related interpretation criteria were as following:FICI≤0.5 means synergy;0.5 2 means antagonism.The fosfomycin resistant genes in KPC-producing Klebsiella pneumoniae isolates were screened.The data were analyzed by t test.Results Antimicrobial susceptibility testing results indicated the fosfomycin and tigecycline susceptibility rates in KPC-producing isolates were 35 .0%(7/20)and 70.0% (14/20 ),respectively.The susceptibility rates of drug combination increased to 50.0% (10/20 )and 95 .0% (19/20 ),respectively,with both MIC decreased.MIC of tigecycline decreased significantly after combination therapy and showed a statistical significance compared with the MIC of monotherapy (t = - 2.596,P = 0.013 ),whereas there was no significant difference between single and combined therapy of fosfomycin (t=-1 .274,P =0.211).FICI indicated that a total of 60.0%isolates showed synergy and additive effects between two antimicrobial agents,followed by indifference (40.0%),but there was no antagonism effect.Among 22 KPC non-producing isolates,there were 54.5 %showing indifference effects,followed by additive (31 .8%)and synergy (13.6%)effects.No antagonism effect was found.The study also identified two isolates with fosA resistant gene which located on the same plasmid as well as the bla KPC gene.The plasmid sizes in the two isolates were 138.9 kb and 104.5 kb, respectively.Conclusions KPC-producing Klebsiella pneumoniae are more susceptible to tigecycline. Combined use of two antimicrobial agents mainly exerts synergy and additive effect rather than antagonism,which may suggest the combination therapy strategy could inhibit the activity of KPC-producing Klebsiella pneumoniae .

Objective To investigate the effect of resistance nodulation division (RND) efflux pumps on reduced susceptibility to tigecycline in carbapenem-resistant Acinetobacter baumanii.Methods Totally 631 isolates of Acinetobacter baumanii were collected from 16 hospitals in 7 provinces in 2010.Genes oxa-51 and oxa-23 were detected by PCR method,and the ST profiles were determined by multilocus sequence typing (MLST).The disk susceptibility assay was used to determine the inhibition zone diameters of β-lactams,aminoglycosides,macrolides,tetracyclines,carbapenems,tigecycline and polymyxin.The minimum inhibitory concentration (MIC) of tigecycline was determined by E-test in strains with inhibition zone diameters ≤ 12 mm on tigecycline.The expression of operon genes adeB,adeG and adeJ was determined with efflux pump inhibitor NMP (N-methyl-2-pyrrolidone) for detection of efflux pump inhibitor phenotype.The isolates of Acinetobacter baumanii which were resistant both to tigecycline and carbapenems and with the inhibited phenotype of efflux pump inhibitor were collected as the experiment group,the isolates which were susceptible to tigecycline but resistant to carbapenems were collected as the control group,and ATCC 19606 was used as the reference strain.The expressions of adeABC,adeFGH and adeIJK were quantified by q-PCR at the transcriptional level.Genes adeR,adeS and adeL were amplified and sequenced using PCR method to find polymorphic locus and insertion sequences.Results There were 32 isolates of Acinetobacter baumanii with reduced susceptibility to tigecycline and carbapenem-resistant.Eight isolates were with the inhibited phenotype by efflux pump inhibitor.And 4 strains which were susceptible to tigecycline but resistant to carbapenems were selected as the control.The expressions of adeABC in A518,Z1219 and A527 of experiment group were 13-fold,5-fold and 7-fold higher than reference strain ATCC19606,respectively.The expressions of adeFGH and adeIJK were up-regulated slightly in some isolates.Transcript of adeABC was not found in control group strains A207 and A1731,and the expressions of adeABC,adeFGH and adeIJK were not up-regulated in other isolates.The single nucleotide polymorphism (SNP) was detected in adeR (E220K) and adeS (A130D),respectively.ISAab1 insertion sequence was identified in adeS of adeABC-over expressed isolates.No mutation was found in adeL.Conclusion High expression of adeABC pump may play an important role in tigecycline resistance in carbapenem-resistant Acinetobacter baumannii,mainly due to the insertion of ISAba1 sequence in its regulator gene adeS,but other mechanism of tigecycline resistance may not be excluded.

Objective To compare zinc-finger BED domain-containing 3 ( ZBED3 ) expression in various tissues of C57BL/6J mice and the effects of liraglutide, glucose, and insulin on the levels of ZBED3 protein expression in C57BL/6J mice and db/db mice. Methods The mRNA level of ZBED3 in various tissues of C57BL/6J mice was measured by realtime PCR. The protein level of ZBED3 was measured by using western blot. Results ZBED3 mRNA levels were detected in muscle, spleen, kidney, brain, heart, lung, and liver of C57BL/6J mice, yielding the highest expression in muscle. Additionally, The liver ZBED3 levels were higher in db/db mice compared with C57BL/6J mice (P<0. 01). Furthermore, the protein expression of ZBED3 was significantly increased in liver tissues of db/db mice treated with high concentrations of liraglutide, glucose or insulin(P<0. 05), however, the expression of ZBED3 only responded to high concentration of glucose in liver tissues of C57BL/6J mice. Conclusion ZBED3 may act as a novel factor in regulating glucose metabolism. The expression of ZBED3 can be regulated by liraglutide, glucose, and insulin. Thus, ZBED3 may play an important role in conditioning of hyperglycemia.

Objective To characterize the prevalence of plasmid-mediated quinolone resistance determinants qnrA in extended-spectrum β-lactamase(ESBL)-producing Escherichia coli and Klebsiella pneumonia.Methods PCR was used to amplify qnrA gene in ESBL-rpoducing isolates(including 263isolates of Escherichia coli and 99 isolates of Klebsiella pneumonia).Conjugation experiments and southern blot hybridization were employed to definitude the location of the genes in ZJ96 isolate of Klebsiella pneumonia which had positive qnrA and CTX-M genes.Shot gun sequencing was performed for analyzing the complete nucleotide sequence of pKP96,a plasmid containing qnrA and CTX-M-24 genes in ZJ96 isolate.Results qnrA was detected in 5 out of 263(1.9%)Escherichia coli isolates and 8 out of 99(8.1%)Klebsiella pneumonia isolates.pKP96,a conjugative plasmid including qnrA gene and CTX-M-24 gene presented in ZJ96 isolate.The sequence of the plasmid pKP96 displayed the qnrA,CTX-M-24,aac(6')-Ib-cr,tetA and int Ⅰ 1 genes.Conclusion The plasmid-mediated genes,such as qnrA and CTX-M,may facilitate the prevalence of multi-drug resistant strains.

Objective To analyze the epidemiological characteristics of Klebsiella pneumoniae car-bapenemase(KPC)-producing Escherichia coli(E. coli)strains isolated in Hangzhou,China. Methods A total of 25 KPC-producing Escherichia coli strains were collected from four hospitals in Hangzhou from July 2012 to January 2014. Antibiotic susceptibility of the isolates to 22 common antimicrobial agents was deter-mined by using Kirby-Bauer(K-B)disk diffusion method. PCR analysis and gene sequencing were used for bla KPC gene screening. The modified Hodge test was performed to detect the production of carbapenemase. Pulsed-field gel electrophoresis(PFGE)and multi-locus sequence typing(MLST)were used for homology analysis. Results All of the 25 clinical isolates were confirmed to be KPC-producing E. coli strains,harbo-ring the blaKPC-2 gene. These KPC-producing isolates showed high drug resistance rates and were resistant to almost all β-lactam antibiotics. PFGE typing classified the 25 isolates into three main homologous clone groups,including clone group A(4 isolates),clone group B(5 isolates)and clone group C(2 isolates), and some single clones(14 isolates). MLST typing classified the isolates into eight ST types,including ST131(14 isolates),ST167(3 isolates),ST2003(3 isolates),ST410(1 isolate),ST457(1 isolate), ST1463(1 isolate),STnew1(1 isolate)and STnew2(1 isolate). The typing results of PFGE and MLST were consistent with each other. Conclusion The prevalent KPC-producing E. coli strains in Hangzhou, China were ST131 type,which were resistant to multiple antibiotics and had been detected in several hospi-tals. The epidemic of KPC-producing E. coli strain often occurred at some special wards,such as Intensive Care Unit(ICU)and emergency ICU.

Objective:To construct a novel biomimetic calcium phosphate (BioCaP) scaffold loaded with bone morphogenetic protein-2 (BMP-2),and to investigate its role in the osteogenesis of human adipose-derived stem cells (hASCs) in vitro and in vivo.Methods:The BioCaP scaffold coprecipitated with BMP-2 (BMP-2-BioCaP) was constructed in this study.Field emission scanning electron microscopy (SEM) was used to analyze the morphology of the surfaces.The release kinetics was measured to evaluate the slow-release characteristics in vitro.BMP-2-BioCaP was immersed in proliferation medium (PM) or osteogenic medium (OM),respectively.The supernatants were collected and used to culture hASCs in vitro.Cell numbers were determined using the cell-counting kit-8 (CCK-8) to assess the cell proliferation.After 7 and 14 days,alkaline phosphatase (ALP) staining and quantification were performed to test the activity of ALP.After 14 and 21 days,the calcification deposition was determined by alizarin red S (ARS) staining and quantification.The expressions of the osteoblast-related genes were tested on day 4 and day 14.In the in vivo study,6 nude mice were used and implanted subcutaneously into the back of the nude mice for 4 groups:(1) BioCaP scaffold only,(2) BioCaP scaffold + hASCs,(3) BMP-2-BioCaP scaffold,(4) BMP-2-BioCaP scaffold + hASCs (test group).After 4 weeks of implantation,hematoxylin-eosin (HE) staining was performed to evaluate the in vivo osteogenesis of hASCs.Results:SEM observations showed that BioCaP and BMP-2-BioCaP scaffold were entirely composed of straight,plate-like and sharp-edged crystal units,and the length of the crystal units varied between 5 and 10 μm.Release kinetics analysis demonstrated that BMP-2 incorporated with BioCaP could be released at certain concentration and last for more than 21 days,and the accumulative protein release could reach 20％.CCK-8 assays showed that cell proliferation was not significantly affected by BMP-2BioCaP.ALP activity was higher by the induction of OM + BMP-2-BioCaP than of the other groups (P ＜0.01).More mineralization deposition and more expressions of osteoblast-related genes such as Runt-related transcription factor 2 (RUNX2),ALP,osteopontin (OPN) and osteocalcin (OC) were determined in the OM + BMP-2-BioCaP group at different time points (P ＜0.01).HE staining showed that,in the test group and BMP-2-BioCaP scaffold group,the extracellular matrix (ECM) with eosinophilic staining were observed around hASCs,and newly-formed bone-like tissues could be found in ECM around the scaffold materials.Moreover,compared with the BMP-2-BioCaP scaffold group,more bone-like tissues could be observed in ECM with typical structure of bone tissue in the test groups.No obvious positive results were found in the other groups.Conclusion:BMP-2-BioCaP scaffold could achieve slow-release of BMP-2 and promote the osteogenic differentiation of hASCs in vitro and in vivo.The novel tissue-engineered bone composed of hASCs and BMP-2-BioCaPis promising for the repair of bone defect.

BACKGROUNDTo explore the sequential variation of pulmonary flow spectrum and its value on evaluation of risk for pulmonary resection in perioperative patients with lung cancer.

METHODSForty-nine patients with lung cancer who underwent pneumonectomy (12 cases) and lobectomy (37 cases) were observed for the values of Doppler pulmonary flow spectrum before operation, on the 3-5 days and 8-10 days postoperatively. Moreover the patients were divided into different groups according to the different operative procedures and with or without postoperative cardiac arrhythmia.

RESULTSDoppler pulmonary flow spectrum changed in all cases who underwent pneumonectomy and lobectomy from 3 to 5 days postoperatively. These changs included prolonged preejection period (PEP), shortened acceleration time (ACT), increased PEP/ACT ratio, increased pulmonary artery mean pressure (PAMP), and increased pulmonary vascular resistance (PVR). There were significant differences comparing with those before operation ( P < 0.01). The patients who underwent lobectomy recovered to the same level of pre operation on the 8th to 10th postoperative days. However, the changes of pulmonary flow spectrum continuously existed in the patients who underwent pneumonectomy on the 8th to 10th postoperative days. There were significant differences of pulmonary flow spectrum between patients with postoperative arrhythmia and without postoperative arrhythmia before operation.

CONCLUSIONSPulmonary hemodynamic obviously changes after pulmonary resection in the patients with lung cancer and the changes last longer in pneumonectomy patients. Patients with postoperative cardiac arrhythmia have marked pulmonary hemodynamic changes before operation. Doppler pulmonary flow spectrum can not only be used to analyse the pulmonary hemodynamic changes for those cases undergoing pulmonary resection after operation, but also to evaluate the risk of pulmonary resection before operation.

Plasmids remain important microbial components mediating the horizontal gene transfer (HGT) and dissemination of antimicrobial resistance. To systematically explore the relationship between mobile genetic elements (MGEs) and antimicrobial resistance genes (ARGs), a novel strat-egy using single-molecule real-time (SMRT) sequencing was developed. This approach was applied to pooled conjugative plasmids from clinically isolated multidrug-resistant (MDR) Klebsiella pneu-moniae from a tertiary referral hospital over a 9-month period. The conjugative plasmid pool was obtained from transconjugants that acquired antimicrobial resistance after plasmid conjugation with 53 clinical isolates. The plasmid pool was then subjected to SMRT sequencing, and 82 assem-bled plasmid fragments were obtained. In total, 124 ARGs (responsible for resistance toβ-lactam, fluoroquinolone, and aminoglycoside, among others) and 317 MGEs [including transposons (Tns), insertion sequences (ISs), and integrons] were derived from these fragments. Most of these ARGs were linked to MGEs, allowing for the establishment of a relationship network between MGEs and/or ARGs that can be used to describe the dissemination of resistance by mobile elements. Key elements involved in resistance transposition were identified, including IS26, Tn3, IS903B, ISEcp1, and ISKpn19. As the most predominant IS in the network, a typical IS26-mediated multi-copy composite transposition event was illustrated by tracing its flanking 8-bp target site duplica-tions (TSDs). The landscape of the pooled plasmid sequences highlights the diversity and complex-ity of the relationship between MGEs and ARGs, underpinning the clinical value of dominant HGT profiles.

The close relationship between gastric cancer (GC) and type 2 diabetes mellitus (T2DM) has garnered significant attention. On one hand, T2DM may play a role in the development and progression of GC, correlating with poor patient outcomes. On the other hand, after radical surgery for GC, T2DM can be effectively managed, potentially improving tumor prognosis. In recent years, bariatric and metabolic surgery (BMS) has revolutionized T2DM treatment for obese and overweight patients. Comparative analyses reveal similarities between surgical approaches for gastric cancer and BMS, leading to the emergence of the onco-metabolic surgery (OMS) concept, which suggests that radical tumor resection and T2DM remission in GC patients can be potentially achieved through a single procedure. However, there are notable differences between OMS and BMS, including target populations, surgical details, and perioperative management. Therefore, optimizing the application of the OMS concept in GC patients holds significant clinical importance. This article provides a review to facilitate the better implementation of this concept in practice.