Objective Using 16S rRNA gene sequencing as the gold standard method,to compare the performance of two matrix-assisted laser desorption ionization time of flight mass spectrometry system (MALDI Biotyper and VITEK MS) for identifying clinical isolates of Streptococcus spp.Methods One hundred and sixty two clinical Streptococcus isolates were collected at the Second Affiliated Hospital of Zhejiang University,from April to June,2014,and confirmed by 16S rRNA gene sequencing analysis.MALDI Biotyper and VITEK MS mass spectrometry system were used for identification and further evaluated by performance respectively.Results Of all the isolates tested,155 (155/162,95.68％) Streptococcus isolates were accurately identified to species level by MALDI Biotyper.Besides,MALDI Biotyper identified three Streptococcus mitis group as S.pneumoniae and one S.parasanguinis as S.australis.Another three S.pneumonia isolates were not identified accurately (values ＜ 1.7).Although 156 (156/162,96.30％) isolates were accurately identified to species level (including subspecies) by VITEK MS system,two S.pneumoniae as S.mitis/S.oralis and one S.euinus as S.infantarius ssp.infantarius were misidentified.The two systems showed a 100％ (51/51) accuracy in identifying all S.pyogenes and S.agalactiae isolates,and an accuracy higher than 85％ for S.pneumoniae.Conclusions Both systems showed potent identification ability for Streptococcus spp.,VITEK MS system showed more clinical significance in accurately identifying some subspecies.Mass spectrometry system can be used as a rapid identification method for Streptococcs spp.in clinical practice.

Objective To evaluate the performance and clinical utility of Verigene gram-negative blood culture ( BC-GN ) assay for a rapid identification of gram-negative bacteria and resistance genes . Methods Non-repetitive blood culture samples containing gram-negative bacteria were collected from inpa-tients in the Second Affiliated Hospital of Zhejiang University School of Medicine from June to October , 2013 .BC-GN assay was performed to identify the species and genetic resistance determinants of gram -nega-tive bacteria directly from the positive blood culture bottles .VITEK MS and the VITEK 2 Compact were used for species identification and antimicrobial susceptibility test , the results of which were considered as gold standards.The resistance genes were further validated by PCR amplification and sequencing analysis .A comparison of the results and time between the BC-GN assay and routine methods was conducted . Results The detection range of BC-GN assay almost covered all of the common gram-negative bacteria .BC-GN assay showed an advantage of high accuracy in the identification of Escherichia coli (13/13), Klebsiella pneumoniae (19/24), Klebsiella oxytoca (9/9), Pseudomonas aeruginosa (39/39), Serratia marcescens (4/5), Enterobacter spp.(6/8), Citrobacter spp.(11/11), Proteus spp.(6/6) and Acinetobacter spp. (24/24) with an overall accuracy of 94.24%for the identification of mono-microbial blood culture samples . Moreover , BC-GN assay accurately identified all of the bacteria and resistance genes from the two multi -mi-crobial samples .Species identification and resistance profiles could be 42 hours earlier available by using BC-GN assay than those by using routine methods .Conclusion BC-GN assay could simultaneously and ac-curately identify bacteria and resistance determinants from blood cultures within 2 hours.More time for clini-cally effective therapy could be achieved by using BC-GN assay for the reduction of mortality associated with bloodstream infection .

Objective To investigate the antimicrobial activity of fosfomycin combined with other antibiotics against carbapenem-resistant enterobacteriaceae(CRE).Methods A total of 233 non-repititive CRE isolates were collected from January 2010 to December 2014 from 4 hospitals, including Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou Traditional Chinese Medicine Hospital, Zhejiang Provincial People's Hospital and Second Hospital of Jiaxing.Antimicrobial susceptibility of fosfomycin, imipenem, meropenem, cefepime, ceftazidime, ceftriaxone, cefperazone-sulbactam, piperacillin-tazobactam, ciprofloxacin, amikacin, tobramycin, polymyxin B and tigecycline were determined by agar dilution method.Synergistic effect between fosfomycin and other antibiotics, including meropenem, cefepime, ceftazidime, cefperazone-sulbactam, ciprofloxacin, amikacin, tobramycin and tigecycline against 30 CRE isolates was determined by chequerboard assay.Chi-Square test was used for statistical analysis, and the difference was statistical significant when P<0.05.Results An overall 45.1%(105/233) of 233 CRE isolates were resistant to fosfomycin.Among which, Klebsiella spp.possessed the highest resistance rate (61.9%,73/118), followed by Enterobacter spp.(50%,15/30), Serratia marcescens (25%,7/28), and Escherichia coli (8.2%,4/49).Fosfomycin in combination with tigecyclin showed best activity against CRE isolates with a synergy rate of 76.7%(23/30).Fosfomycin and aminoglycosides also presented good activity against CRE isolates with synergy rate of 53.3%(16/30) to 70.0%(21/30).Synergism was observed only in 30%(9/30) of CRE isolates for the combination of fosfomycin and ciprofloxacin.As for the combination of fosfomycin and β-lactam antibiotics, even less synergism was observed ( 0%-3.3%) ( 1/30 ).No antagonism was demonstrates among all of the combinations.Conclusions Fosfomycin demonstrates certain in vitro activity against CRE isolates.A combination of fosfomycin and tigecyclin or aminoglycosides shows good activity, which suggests a new strategy in the campaign against serious infections caused by CRE.

Objective To investigate the molecular background of the New Delhi-metallo-1 (NDM-1)-producing Morganella morganii.Methods Two carbapenem-resistant M.morganii named 1 and 2 were isolated in the Second Hospital of Jiaxing,Zhejiang on October 4th and 29th,respectively.Antimicrobial susceptibility was determined by agar dilution method.Pulsed-field gel electrophoresis (PFGE) was performed to analyse the homololgy of isolates.Amplification with specific primers,DNA sequencing,conjugation experiments and genetic environment analysis were conducted to investigate the molecular mechanisms of resistance.Results The two M.morganii isolates were resistant to carbapenem and fluoroquinolones,while susceptible to aztreonam.PFGE analysis indicated that the two isolates were distinguishable.Amplification and DNA sequencing confirmed the coexistence of blaNDM-1,blasHv-12,qnrS1 and aac(6')-Ib-cr in both isolates.Transconjugants were detected with blaNDM.1 and qnrS1 simultaneously.Genetic environment analysis demonstrated that the blaNDM-1-bleMBL-trpF-dsbC-cutA1 structure was in consistence with those from known blaNDM-1-carrying Klebsiella pneumoniae.Conclusion The blaNDM-1 in M.morganii isolates possiblely obtained from K.pneumoniae through translatable plasmids.

Objective To investigate the expression level of lipoprotein lipase (LPL) mRNA in chronic lymphocytic leukemia (CLL) patients and evaluate the prognostic value of LPL in CLL Methods Quantitative real-time RT-PCR (qRT-PCR) was performed in 62 CLL patients, 10 normal controls using Taqman probe system. Association between LPL and other known prognostic factors, such as IgVH mutation status, ZAP-70 and CD38 expression, was determined using the Spearman correlation analysis. ROC curve was used to determine the cut-off value of LPL expression level, the positive and negative predictive value of IgVH mutation status. Results The correlation coefficients of the standard curves in qRT-PCR were not less than 0.990. The coefficients of variation (CV) of interrun assay and intramn assay were < 5%, and the sensitivity can reached 102 copies/μg RNA. The median LPL mRNA expression level was 0.006 0 (0-0.737 0) in 62 CLL patients, whereas in 10 normal controls LPL mRNA expression level was extremely low with the median level of 0 (0-0.000 4). The expression levels of LPL in three CLL samples after miniMACS-sorted CD19 positive B cells were 0.036 0, 0.075 0 and 0.197 0, which were similar to the levels before miniMACS-sorted (0.024 0, 0.074 0 and 0.225 0). LFL expression was significantly associated with IgVH mutation status (r=0.45, P<0.05) . LPL expression level in IgVH unmutated patients [0.006 0 (0.000 7-0.110 0)] was significantly higher than the level in IgVH mutated patients [0.002 0(0.000 2-0.027 0)] (U=96.5, P<0.05). LPL expression was also significantly associated with ZAP-70 (r=0.38, P<0.05), CD38 expressions (r=0.43, P<0.05). According to ROC curve, the cut-off of LPL mRNA expression level was 0.036, with a 66.7% specificity, a 72.4% sensitivity, a 51.8% positive predictive value (IgVH unmutated), and a 83.3% negative predictive value (IgVH mutated) for IgVH mutation status. Conclusions The qRT-PCR assay is reliable and sensitive. LPL mRNA expression significantly correlates with IgVH mutation status, ZAP-70 and CD38 expression, and could be a predictive marker of IgVH mutation status. Our data confirms a role for LPL as a novel prognostic indicator in CLL.

Objective To investigate the prevalence and drug resistance of clinical Klebsiella vari-icola ( K. variicola ) isolates and to illuminate the mechanism of drug resistance in carbapenem-resistant strains. Methods Clinical K. variicola isolates were identified with matrix-assisted laser desorption/ioniza-tion time-of-flight mass spectrometry ( MALDI-TOF MS ) . The antimicrobial susceptibility profile of these strains was determined using broth microdilution. Resistance genes carried by carbapenem-resistant K. vari-icola strains were detected by PCR with specific primers. Multilocus sequence typing ( MLST) was used for molecular typing. A pan-drug resistant strain which was isolated from cerebrospinal fluid sample was ana-lyzed with whole genome sequencing ( WGS) . Results Twenty-six isolates were identified as K. variicola by MALDI-TOF MS. Results of the antimicrobial susceptibility test showed that there were 15. 4％ (4/26) re-sistant to carbapenem and 11. 5％ (3/26) unsusceptible to tigecycline. These strains were highly suscepti-ble to amikacin and gentamicin, which accounted for 96. 2％ (25/26). As for the third-and fourth-genera-tion cephalosporins, the resistance rate was 23. 1％ (6/26). All of the four carbapenem-resistant isolates carried the resistance genes of blaIMP-4 , qnrA/B and blaTEM , and one of them was also positive for blaNDM-1 gene. The fosfomycin resistance gene, fosA, was detected in three of them. Molecular typing analysis indica-ted these isolates belonged to two sequence types ( ST) of ST357 ( three strains) and ST1737 ( one strain) . Two plasmids were obtained from the pan-drug resistant strain by WGS, including IncFⅡ/FIB( k) type plas-mid (160 kb) that was highly homologous to LMG 23571 plasmid (GenBank: CP013986. 1) and IncHⅠ1B/FIB type plasmid (260 kb) sharing high homology with pIMP4 LL34 (GenBank: CP025964. 1). Be-sides the resistance genes mentioned above, the two plasmids also carried a variety of other genes that media-ted the resistance to aminoglycosides (strB, strA, armA, aac3-Ⅱd, aadA2), macrolides (msrE, mphE), chloramphenicol (catA2), sulfonamides (sulⅠ) tigecycline (tetA variant) and trimethoprim (dfrA16). However, no virulence genes were detected. Conclusions In general, the resistance profile of K. variicola was similar to that of Klebsiella pneumoniae, but the differences were that carbapenem-resistant K. variicola strains mainly belonged to ST357 and the leading causes of resistance were carrying the genes encoding IMP-4 and NDM-1 metalβ-lactamases. WGC analysis revealed that the pan-drug resistant K. variicola strain carried multiple drug resistance genes without virulence determinants, which might be resulted from the evo-lution of drug resistance.

Objective:To evaluate the clinical value of NG-Test Carba5 for rapid detection of carbapenemases produced by carbapenem-resistant Enterobacteriaceae (CRE) strains. Methods:A total of 1 210 CRE strains were collected during 2018-2022 from 77 hospitals in 21 provinces of China and were subjected to NG-Test Carba5 for rapid detection of carbapenemase. The whole genome sequencing (WGS) analysis was referenced as the gold standard method.Results:Overall, the NG-Test Carba5 demonstrated excellent performance in detection of five kinds of carbapenemases [Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-β-lactamase (NDM), imipenemase metallo-β-lactamase (IMP), Verona integron-encoded metallo-beta-lactamase (VIM) and oxacillinase-48-type carbapenemases(OXA-48)] from CRE strains, with a sensitivity of 98.47% (1 161/1 179), specificity of 100% (31/31), and positive predictive value of 100% (1 161/1 161). The sensitivity for detection of NDM, IMP, OXA and VIM reached 100% (307/307), and 97.70% (763/781) for KPC. For 11 strains carrying blaKPC-25, blaKPC-78, or blaKPC-93, NG-Test Carba5 reported positive KPC detection (11/11). For strains carrying blaKPC-33 and blaKPC-77, however, NG-Test Carba5 delivered negative results. Additionally, for those strains co-producing two or three kinds of carbapenemases, NG-Test Carba5 was able to report all of the targets with a sensitivity of 100% (91/91). Conclusions:NG-Test Carba5 showed excellent performance in rapid and accurate detection of carbapenemases from CRE strains. Nonetheless, for those strains with negative results, some other phenotypic and genotypic methods should be implemented alongside to avoid missing targets.

Objective:Evaluate the application of Fourier transform infrared spectroscopy in the identification of homology of carbapenem-resistant Escherichia coli(CREC). Methods:A total of 26 carbapenem-resistant Escherichia coli strains were isolated from 9 provinces in China in 2018. The 900-1 200 cm -1 was selected as a spectral region for the Euclidean distance calculating and average linkage clustering between all isolates.The single nucleotide polymorphism (SNP) was analyzed by whole genome sequencing (WGS). Results:Twenty-six CREC strains were divided into 14 infrared spectros copy(IR) types by FTIR. The same IR type belonged to the same sequence type type.Compared with cluster analysis based on WGS, the consistency of FTIR cluster analysis was 92.3% (24/26).Conclusions:FTIR presented excellent performance in identification of homology of CREC.Besides, with the advantages of simple operation and rapid acquisition of results, FTIR may be a useful tool in clinical labs.