Objective To examine the quinolone resistance and mechanisms of resistance,including prevalence of transferable mechanisms of quinolone resistance(TMQR)genes in carbapenem-resistant Klebsiella pneumoniae(CRKP).Methods Antimicrobial susceptibility was tested for CRKP isolated from 7 hospitals in China during 2019 by broth microdilution method.Genome sequencing analysis was performed on Illumina HiseqX sequencing platform.The virulence genes,multilocus sequence typing(MLST),chromosome quinolone resistance-determining region(QRDR)variation,and prevalence of TMQR genes in CRKP were analyzed.Results Overall,98.4％and 99.2％of the 481 CRKP strains were non-susceptible to levofloxacin and ciprofloxacin,respectively.ST11(77.8％)and ST15(15.8％)were the dominant clones.Among the 303 CRKP strains carrying virulence gene(CV-CRKP),ST11 type and ST15 type strains accounted for 92.1％and 5.0％,respectively,while ST11 and ST15 type strains accounted for 53.4％and 34.3％in the 178 CRKP strains without the virulence gene(non-CV-CRKP).All of the ST11 and ST 15 type strains were resistant to quinolones associated with gyrA and parC mutation.All of the quinolone-sensitive strains were not ST11 type or not ST15 type.TMQR analysis showed that qnrS1(61.1％,294 strains),qnrB4(10.0％,48 strains)and aac(6)-Ib-cr(18.7％,90 strains)were most prevalent in CRKPs,while qnrB1(4 strains),qnrB2(2 strains),qnrD1(2 strains)and qnrB6(1 strain)were infrequently identified in CRKPs.Specifically,qnrB4 was mainly identified in high-level levofloxacin-resistant strains(MIC≥8 mg/L),while aac(6)-Ib-cr was found mainly in ciprofloxacin-resistant strains(MIC＞8 mg/L).Conclusions Clinical CRKPs were highly resistant to quinolone.The rate of QRDR variation were high in quinolone resistant CRKP strains,with high TMQR gene carriage.

OBJECTIVE To establish the fingerprints of Ligusticum sinense from different habitats ，screen differential components and determine their contents. METHODS Using Z-ligustilide as reference ，HPLC fingerprints of 12 batches of L. sinense were established by using Similarity Evaluation System of Chromatographic Fingerprints of TCM （2012 edition）；common peaks were identified and their similarities were evaluated. Cluster analysis （CA），principal component analysis （PCA）and orthogonal partial least squares-discriminant analysis （OPLS-DA）were performed to screen differential components with variable importance in the projection （VIP）＞1 as standard ；meanwhile，the contents of above differential components were determined by the same HPLC method. RESULTS There were 17 common peaks in the fingerprints of 12 batches of L. sinense ，and their similarities ranged 0.989-1.000. A total of 9 common peaks were identified ，i.e. chlorogenic acid （peak 1），ferulic acid （peak 2）， senkyunolide Ⅰ（peak 7），coniferyl ferulate （peak 9），E-ligustilide（peak 13），senkyunolide A （peak 14），Z-ligustilide（peak 17）. CA results showed that 12 batches of L. sinense were divided into 3 categories，S1-S5（Wuning）were clustered into one category，S6-S8（Ruichang）were clustered into one category ，S9-S12（De’an）were clustered into one category ；the VIP values of peaks 2，13，14 and 17（corresponding to ferulic acid ，E-ligustilide，senkyunolide A ，and Z-ligustilide respectively ）were all greater than 1，respectively. In S 1-S5，S6-S8 and S 9-S12 samples，the contents of ferulic acid were 0.488-0.533，0.603-0.658 and 0.415-0.433 mg/g，respectively；senkyunolide A were 1.184-1.295，1.450-1.588 and 1.307-1.377 mg/g，respectively；E-ligustilide were 0.118-0.125，0.130-0.135 and 0.223-0.229 mg/g，respectively；Z-ligustilide were 7.200-7.681，8.076-8.643 and 4.508-4.996 mg/g， respectively；the differences between two groups were statisti-cally significant （P＜0.05）. CONCLUSIONS Established ARS-11）；fingerprint is simple and accurate ，and can be used for overall quality evaluation of L. sinense from different habitats by combining with multivariate statistical analysis. Ferulic acid ， senkyunolide A ，Z-ligustilide and E-ligustilide may be the differential components that affect the quality of L. sinense from different habitats ，the contents of the first 3 components in L. sinense from Ruichang are the highest ，and the content of E-ligustilide in samples from De’an is the highest.

Objective:To analyze the improvement of patients with basilar invagination and atlantoaxial dislocation that treated by anterior or posterior surgery.Methods:50 patients with basilar invagination and atlantoaxial dislocation that underwent simple anterior or posterior surgery from 2009 to 2021 were included. There were 34 females and 16 males with a mean age of 45.04 years (range, 18-65 years). All patients underwent thin- slice CT scan of the neck. Preoperative and postoperative measurements of atlantoaxial joint distance, atlantoaxial joint angle, atlantoaxial joint displacement, Claus' Height, atlas-dens interval, space available for the cord, cervicomedullary angle, C 0-C 2 angle, and C 2-C 7 angle were measured. Then, the data were analyzed by independent sample t test. Results:25 patients (7 males, 18 females) were included in the anterior surgery group, and 25 patients (9 males, 16 females) were included in the posterior surgery group. The mean age of the two groups was 45.24±9.86 years and 44.72±14.06 years, respectively, showing no statistical difference. The mean last follow-up time of the anterior and posterior surgery group was 6.48±3.14 months and 7.04±2.87 months, respectively. The odontoid distance, atlas-dens interval, space available for the cord and cervicomedullary angle in 2 groups were significantly improved after surgery ( P<0.05), while there were no significant differences in the above parameters between 2 groups ( P>0.05). In the anterior surgery group, the distance and angle of atlantoaxial joint were increased, and the atlantoaxial joint displacement decreased significantly. While in the posterior surgery group, only the atlantoaxial joint space increased ( P<0.05). The C 0-C 2 angle was significantly increased and the C 2-C 7 angle was significantly decreased in the anterior surgery group ( P<0.05), but there was no significant difference in these parameters in the posterior surgery group ( P>0.05). In addition, there was no significant difference in parameters between the two groups at the last follow-up compared with those immediately after surgery. Conclusion:Both anterior and posterior surgery can improve the compression of the spinal cord in patients with basilar invagination and atlantoaxial dislocation. Anterior surgery may be more adequate for the extension and reduction of the atlantoaxial joint space, however, excessive enlargement of the lordosis angle in upper cervical may lead to the reduction of the lordosis in lower cervical.

Objective To investigate the resistance profile of Klebsiella pneumoniae isolates in Huashan Hospital, Fudan University. Methods The MICs of fluoroquinolones were determined by agar dilution method against 112 clinical strains of K. pneumoniae. Multilocus sequence typing (MLST) were applied to 48 K. pneumoniae strains. The characteristic sequence type (ST) associated with antibiotic resistance was identified by PCR. Results Lower percentage (＜40％) of K. pneumoniae strains were susceptible to fluoroquinolones. Majority (86.2％) of ciprofloxacin non-susceptible K. pneumoniae strains belonged to CC1 (ST11), ST494 or CC4 (ST15 and ST655), indicating the potential of clonal dissemination. ST494 (18.8％) was the second commonest sequence type, next only to ST11. ST494 strains harbored the genes encoding beta-lactamases, oqxAB, qnrD, aac-(6')-lb-cr and armA and had a single point mutation in gyrA. Therefore, ST494 strains were highly resistant to cephalosporins, fluoroquinolones and aminoglycosides and 22％ of the strains were resistant to carbapenems. However, all the ST494 strains were susceptible to tigecycline and tetracycline. Conclusions ST11 and ST494 are the commonest STs of K. pneumoniae conferring multidrug resistance in this hospital. These STs may contribute to the high resistance rates of K. pneumoniae to fluoroquinolones. The susceptibility of ST494 strains to tigecycline and tetracycline allows us to consider the promising potential of such drugs in managing K. pneumoniae infections.

Objective To investigate the profile of antimicrobial susceptibility of the Mycoplasma pneumoniae (Mpn)strains isolated from pediatric patients with respiratory tract infection.Methods Antimicrobial susceptibility testing was conducted with a total of 112 Mpn clinical strains by broth microdilution method.Sequence analysis of full 23S rRNA genes was performed for all Mpn strains.Results One hundred and twelve Mpn strains were isolated from January 2009 to March 2011. Of these clinical isolates,98 (87.5%)were resistant to erythromycin and azithromycin.All macrolide-resistant Mpn strains harbored an A2063G or A2064G transition mutation in domain V of 23S rRNA genes.Mpn isolates were still very susceptible to the tetracyclines and fluoroquinolones tested.Conclusions The Mpn strains from pediatric patients are highly resistant to macrolides.The mechanism of macrolide resistance may be associated withthe transition mutation on 23S rRNA gene.

Objective Through the exploration of virtual simulation surgery to find a way to treat lumbar spinal metastases. Methods Based on 64 row spiral CT continuous 2-dimensional images of lumbar segments, normal lumbar vertebral, destruction of disease, abdominal aorta and kidneys were reconstructed by the Mimics software. 3D visualization structure was contemplated by anterior lesions clear, titanium mesh of bone cement support, and posterior pedicle screw fixation. Results The three-dimensional reconstruction distinctly displayed the structures of lumbar and its adjacent organs, and the entire virtual simulation surgery was intuitive. Conclusion The application of virtual simulation surgery ensures more accurate 3D model of lumbar establishment and its adjacent organs, and it provides an objective basis for in-dividualized treatment programs.

Objective To screen fosfomycin-resistant genes in the clinical isolates of Enterococcus faecium Efm-HS0661 and verify their functions. MethodsAntimicrobial susceptibility and conjugation experiments were carried out to determine if the antimicrobial resistance in clinical strain was transferable.By Solexa high-throughput sequencing,the genes conferring fosfomycin resistance were screened. The function of resistance gene was identified by cloning.ResultsThe clinical isolates of Enterococcus faecium Efm-HS0661 were resistant to glycopeptide antibiotics and fosfomycin, and the fosfomycin resistance was found to be transferred by conjugation. Within the 2414 bp nucleotide sequence obtained by high-throughput sequencing, fosB, a plasmid-mediated fosfomycin resistance gene was found. The fosB gene was 420 bp in length, which shared 99. 8％ amino acid identity with other fosB from Staphylococcus spp. The minimal inhibitory concentration (MIC) of DH5α transformant containing fosB gene against fosfomycin was higher than that of DHSa transformant without fosB gene. ConclusionsThe high-throughput sequencing can be used to screen unknown resistance genes in clinical isolates. The plasmidmediated resistance gene fosB can confer fosfomycin resistance in Enterococcus faecium.

Objective To develop a method for rapid detection of Mycoplasma pneumoniae and its macrolide resistance mutation. Methods The primers and cycling probe sets were designed to detect two single nucleotide mutation, A2063G and A2064G, in the 23s rRNA gene of Mycoplasma pneumoniae. By using recombinant plasmids containing 23s rRNA gene fragments, 102 Mycoplasma pneumoniae clinical isolates from 2005 to 2008, and 136 nasopharyngeal suction specimens from pediatric patients with low respiratory tract infections in Shanghai Children's Hospital from November to December in 2009 were investigated to determine the specificity and the sensitivity of the CycleavePCR method. PCR amplification and sequence analysis of 23S rRNA genes were performed for all Mycoplasma pneumoniae strains and Mycoplasma pneumoniae positive specimens to confirm the results of the CycleavePCR method. Results Of 102 clinical isolates, 83 was resistant to erythromycin and sequence results show that all macrolide-resistant Mycoplasma pneumoniae strains harbored an A2063G ( 82/83 ) or A2064G ( 1/83 ) transition mutation in 23S rRNA genes. Twelve was Mycoplasma pneumoniae detected positive by CycleavePCR in 136nasopharyngeal suction specimens. The CycleavePCR results were consistent with those of routine PCR and sequencing. There was no signal production from other bacterial species. Sensitivity and specificity were 100%. The detection limit of the CycleavePCR was 10 plasmid copies per reaction. Experiment can be done within 1.5 h. Conclusion A novel method is developed to detect erythromycin-resistant strains harboring A2063G and A2064G transition mutation in the 23s rRNA gene using CycleavePCR.

Objective To learn the current in vitro antimicrobial susceptibility of Mycoplasma pneu-moniae in Shanghai and to understand the mechanisms of resistance to macrolides. Methods M. pneumoniae was isolated from pediatric patients with low respiratory tract infections(RTI) using broth and PPLO agar medi-um. PCR amplification and sequence analysis of P1 adhesion gene were performed to identify all M. pneumoniae strains. Susceptibility testing was carried out for macrolides, tetracyclines and fluoroquinolones using broth mi-crodilution method with SP4 broth. PCR amplification and sequence analysis of 23S rRNA genes were performed for all M. pneumoniae strains. P1 gene PCR-RFLP typing was performed to subtype the M. pneumoniae strains. Results One hundred and two M. pneumoniae strains were isolated in Shanghai from Oct 2005 to Dec 2008. All M. pneumoniae isolates were susceptible to the tetracyclines and fluoroquinolones tested. Of 102 clinical isolates, 83(81.4%) was resistant to erytbromycin and all 83 erythromycin-resistant strains had MIC>128 mg/L. An increasing trend of resistance rates were showed: 16.7% (1/6) in 2005, 76.5% (13/17) in 2006, 100.0% (24/24) in 2007 and 81.8% (45/55) in 2008. All macrolide-resistant M. pneumoniae strains harbored an A2063G transition mutation in domain V of 23S rRNA genes. The P1 gene RFLP type 1 is predominant (85.3%, 87/102) in M. pneumoniae clinical isolates. Conclusion The macrolide resistance rate of M. pneu-moniae is very high in Shanghai. The mechanism of macrolide resistance is associated with transition mutation on the 23S rRNA gene.

Objective To investigate acquired carbapenemases and prevalence of carbapenem- resistant gram-negative bacill.Methods The antimicrobial susceptibility was determined by agar dilution method.Metallo-B-lactamase(MBLs)were screened by EDTA-disk synergy tesL The encoding genes of MBLs were amplified by PCR followed by sequencing.Strain homology Was investigated by pulsed-field gel electronphoresis(PFGE).Results In 141 carbapenem-resistant Pseudomonas aeruginosa(P.aeruginosa), there were three resistant patterns which were imipenem(IMP)-resistant+meropenem(MEM)-resistant (66.7%),IMP-resistant+MEM-sensitive(32.6%),and IMP-resistant+MEM-sensitive(0.7%).AⅡthe carbapenem-resistan Acinetobacter baumannii(A.baumannii),Acinetobacter lwoffi(A.lwoffi),Citrobactor freundii(C.freundii),Klebsielta pneumoniac(K.pneumoniac)and Serratia were resistant to imipenem and meropenem.Four strains of 141 P.aeruginosa were positive by EDTA-disk synergy test,and they produced VIM-2-type Metallo-B-1actamase.Of 34 carbapenem-resistan A.baumannii,30 strains produced OXA-tllrpe.Among them, OXA23, OXA24 and OXA66 accounted for 79.4%,38.2% and 67.6%,respectively.And 22 of 34 strains(64.7%)produced multiple OXA-carbapenemases.All 7 strains A.lwoffi produced OXA-23-type carbapenemases.A11 11 strains C.freundii,5 strains k pneumoniac and 1 strains Serratia produced KPC-2-type carbapencmases.And 6 of 11 strains C.freundii produced new subtype IMP-8.Of 15 PFGE type in 34 strains A.baumannii,14 strains belonged to A-type,7 strains belonged to B-type.Seven A.lwoffi strains distilbuted in difierent PFGE type.Four strains of P.aeruginosa producing VIM-2.type Metallo-8-lactamase did not have the same PFGE type.Eleven C.freundii strains had the same PFGE type.Five k pneumoniae strains had the sanle PFGE type.Conclusions Drug resistance to 12 common antibiotics in carbapenem-resistant gram-negative bacill was higher than non-carbapenem-resistant gram-negative.The former produced many kinds of carbaponemases and the strains prducing carbapenemases were prevalent in the C.freundii, A.baumanii, and k pneumoniae.