ABSTRACT
Objective To evaluate the effect of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)on the detection of carbapenemase producing enterobacteriaceae. Methods A total of 21 carbapenem non-susceptible enterobacteriaceaeclinical strains were collectedfrom the Second Affiliated Hospital of Soochow University during January to May, 2018, including 11 strains of Klebsiella pneumonia, 3 strains of Klebsiella oxytoca, 3 strains of Enterobacter cloacae, and 4 strains of Escherichia coli. All the isolates were incubated with 0.5g/L meropenem solution for 2 hours. The supernatant was centrifuged and collected for MALDI-TOF MS detection. The characteristic peaks were captured to determin whether the strain was producing carbapenemase or not. And then, the results were compared with PCR results by Kappastatistical analysis. Results The PCR results showed that all the strains were positive for carbapenmase genes, among them 15 isolates were encoding KPC genes, 6 isolates encoding GES genes, 2 isolates encoding NDM genes, 1 isolate encoding VIM genes, 4 isolates encoding GIM and 1 isolate encoding SIM. And the strains could carry one or more carbapem-resistant determinants. MALDI-TOF MS showed that meropenem were hydrolyzed by 21 isolates and a characteristic drug hydrolysis peak appeared at 199 m/z, as a result of carbapenemase produced by enterobacteriaceae. The assay of MALDI-TOF MS was highly consistentwith PCR results. Conclusions The investigation showed that MALDI-TOF MS can directly detect the carbapenemase by capture the characteristic drug hydrolysis peak.
ABSTRACT
Objective To investigate the identification of staphylococcus aureus lineage ST59 using the combined detection of delta hemolysin allelic variant G10S(HldG10S) and beta hemolysin(β-toxin). Methods Perspective study.A total of 82 non-duplicate clinical staphylococcus aureus were collected from November 2017 to April 2018 in the department of Clinical laboratory, the Third Xiangya Hospital of Central South University, China.The strains were routinely identified by MALDI-TOF MS and the mass spectra were obtained. According to the m/z expression intensity of delta hemolysin(Hld), all strains were divided into three groups:HldG10S (3036±2.0)m/z, Hld (3006±2.0)m/z and ND [no (3036±2.0)m/z and no (3006±2.0)m/z]. The distribution of ST59 in the three groups was detected by MLST. Reverse synergic hemolysis test was used to determine theβ-toxin phenotype. And the sensitivity, specificity and accuracy of HldG10S,β-toxin and the combined detection of HldG10S and Hld to identify ST59 were compared. Results Among the 82 strains, 21 strains expressed HldG10S toxin, accounting for 25.6％. 39 strains expressed Hld toxin, accounting for 47.6％.22 strains did not express HldG10S and Hld toxin, accounting for 26.8％. In HldG10S group,16 strains were ST59, accounting for 76.19％(16/21).ST59 was not found in both Hld and ND groups. All 16 strains of ST59 in HldG10S group producedβ-toxin, while none of the 5 strains of non-ST59 producedβ-toxin. The specificity(100％) and accuracy(100％) of the combined detection was significantly higher than that of HldG10S andβ-toxin single detection of specificity(92.4％, 77.3％) and accuracy(80.5％, 81.7％) (χ2=19.472, P<0.001;χ2=17.792, P<0.001). Conclusion The combined detection of HldG10S andβ-toxin can preliminarily and rapidly identify ST59, which can assist the routine monitoring of the change trend of staphylococcus aureus epidemic.
ABSTRACT
Objective@#To evaluate the ability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in identifying the rmpA2-carrying hypervirulentKlebsiella pneumoniae.@*Methods@#A total of 57nonduplicateKlebsiella pneumoniae isolates were collected from the Second Affiliated Hospital of Zhejiang University and Henan Provincial People′s Hospital. Virulence gene rmpA2 and capsule K serotype-specific genes were detected by PCR, multilocus sequence typing (MLST) was performed for molecular typing, and string test was conducted to identify the hypermucoviscous phenotype. The ClinProTools software was used for peak analysis. Four standard algorithms, including support vector machine (SVM), genetic algorithm (GA), supervised neural network (SNN), and quick classifier (QC), were tested for their power to differentiate between rmpA2-positive and rmpA2-negative strains.@*Results@#Among the 57 Klebsiella pneumoniae isolates, 28 isolates belonged to sequence type (ST) 11 and carried the virulence gene rmpA2, of which 5 isolates were positive for string test; while the other 29 isolates were not detected rmpA2, and MLST divided them into five STs including ST11 (n=23), ST15 (n=3), ST1 (n=1), ST76 (n=1), and ST473 (n=1). The results of four standard algorithms were similar, while the sensitivity and specificity of SVM was the highest at 93.23% and 100% respectively. Analysis results of ClinProTools software suggested that the specific peaks for differentiating the rmpA2-positive and rmpA2-negative strains were 7 168.9 and 7 280.76, however, the peak intensity of themwere variant.@*Conclusion@#The sensitivity and specificity ofMALDI-TOF MS for rapid identification of rmpA2-carrying hypervirulentKlebsiella pneumoniae werehigher than 90% and there was a difference on the peak intensity of two specific peaks, which needs further study.(Chin J Lab Med, 2018, 41: 571-675)
ABSTRACT
Objective To establish and optimize a loop-mediated isothermal amplification (LAMP) method for the rapid detection of Escherichia coli and its microbial toxin.Methods The LAMP reaction system and reaction conditions were determined by optimizing LAMP reaction,and the optimized LAMP system was used for the detection.Results Primers targeting shiga toxin (stx) gene and O157 antigen gene rfbe were designed.The established and optimized LAMP amplification system contained 1.2 mmol/L dNTPs,10 mmol/L MgSO4,0.4 mol/L betaine,1 μl 10 × Bst DNA polymerase Buffer,8 U Bst DNA polymerase fragment,2 μl DNA template,and the ratio of inner-primer (FIP and BIP) and outerprimer (F3 and B3) were 8∶ 1.Time and temperature for LAMP was 60 min,60 ℃.The sensitivity was 103 times higher than polymerase chain reaction (PCR),reached 5 × 101 CFU/ml.When LAMP was applied to 19 reference strains,102 EHEC strains,the specification was 100％ while identification rate of rfbe,stx1 and stx2 gene reached 100％,95.2％,92.9％.Conclusions The LAMP method showed a promising prospect for the rapid detection of common nosocomial pathogens microbial toxin.
ABSTRACT
Objective To investigate the effect of artesunate (ASN) on the expression of Heme oxygenase-1 (HO-1) in THP-1 cells induced by the early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) antigens of Mycobacterium tuberculosis and to investigate its possible mechanism.Methods THP-1 ceils were cultured in vitro.The effects of ESAT-6 and CFP-10 on cell viability were detected by methyl thiazolyl tetrazolium (MTT) assay.THP-1 cells were pre-treated with or without ASN prior to incubation with or without ESAT-6 and CFP-10,the mRNA expression of HO-1 was detected by real time quantitative polymerase chain reaction (RT-qPCR) and Toll-like receptor 2 (TLR2) level was measured by Western blot.Results MTF assay showed that ESAT-6 and CFP-10 were non-toxic to cells in the range of 0-5 μg/ml.Compared with the control group,5 μg/ml ESAT-6 and 5 μg/ml CFP-10 could significantly increased the mRNA expression of HO-1 (P ＜ 0.05).In addition,20 μg/ml ASN could significantly enhance the mRNA expression of HO-1 induced by ESAT-6 and CFP-10,and inhibit the expression of TLR2 induced by ESAT-6.Conclusions ASN in combination with ESAT-6 or CFP-10,may have potential value in treatment of pathogen-associated inflammatory diseases.
ABSTRACT
Objective To investigate the effect and mechanism of CRM197, the heparin binding-epidermal growth factor-like growth factor (HB-EGF) inhibitor, on the reverse of the resistance ofovarian cancer to paclitaxel. Methods (1)The effect of CRM197 on the 50% inhibitory concentrations (IC50) of human ovarian carcinoma cell line A2780 and paclitaxel-resistant ovarian carcinoma cell line A2780/Taxol was tested by methyl thiazolyl tetrazolium (MTT) assay. Western blot was used to detect the effect of CRM197 on the expression of HB-EGF,epidermal growth factor receptor (EGFR) and plasma membrane glycoprotein (P-gp) protein in A2780 and A2780/Taxol cells. Real-time PCR was used to examine the MDR1 mRNA expression in these cells. (2) A2780/Taxol cells were divided into 4 groups, including the cells transfected with empty vector and saline treatment (empty vector group), MDR1 small interference RNA (siRNA)vector and saline treatment (MDR1 siRNA group),empty vector and CRM197 treatment (empty vector+CRM197 group) and MDR1 siRNA vector and CRM197 treatment (MDR1 siRNA+CRM197 group), respectively. Flow cytometry was used to detecte the effect of intracellular rhodomine 123 (Rh123) accumulation, and caspase-3 activity assay was used to test the effect of apoptosis in four groups of A2780/Taxol cells. (3) In experiments in vivo, A2780/Taxol cells were inoculated to nude mouse subcutaneously to determine the EGFR and P-gp protein expression following CRM197 treatment by immunohistochemistry. Results (1)In vitro,MTT examination showed that the IC50 of A2780/Taxol cells to paclitaxel in A2780/Taxol+CRM197 group [(6.4±0.3)μmol/L] was significantly lower than the IC50 in A2780/Taxol group [(34.1± 0.5)μmol/L,P<0.01], and the reveral fold of CRM197 was 5.3. The expression level of HB-EGF protein in A2780/Taxol+CRM197 group (1.44 ± 0.29) was significantly lower than HB-EGF protein in A2780/Taxol group (2.72 ± 0.32),respectively (P<0.05). The expression level of EGFR protein (0.71 ± 0.25) and P-gp protein (0.82±0.19) in A2780/Taxol+CRM197 group was significantly lower than EGFR protein (1.87±0.31) and P-gp protein (1.84 ± 0.27) of A2780/Taxol group (P<0.05). Compared with A2780/Taxol group(1.78 ± 0.27), MDR1 mRNA was significantly down-regulated in A2780/Taxol+CRM197 group (0.79 ± 0.13,P<0.05). (2) The fluorescence intensity of Rh123 of the A2780/Taxol cells in empty vector group, MDR1 siRNA group,empty vector+CRM197 group, MDR1 siRNA+CRM197 group was 33.4±1.6, 56.3±3.3, 43.5± 3.1,100.4 ± 7.4, and the pNA of the A2780/Taxol cells was(11.4 ± 1.2),(52.8 ± 0.9),(71.2 ± 3.6),(82.7 ± 3.8)μmol/L. The expression levels in MDR1 siRNA+CRM197 group were both higher than the expression levels in empty vector+CRM197 group, and the expression levels in empty vector+CRM197 group, MDR1 siRNA group were both higher than the expression levels in empty vector group (P<0.05). (3) In vivo, the expression scores of EGFR protein in A2780/Taxol+CRM197 tumors (4.4 ± 1.4) were lower than that in A2780/Taxol tumors (10.2 ± 3.1,P<0.05). The expression scores of P-gp protein in A2780/Taxol+CRM197 tumors (3.8 ± 1.1) were lower than that in A2780/Taxol tumors (8.8 ± 2.7,P<0.05). Conclusion CRM197 reverses the resistance of ovarian cancer to paclitaxel by increasing caspase-3 activity to advance apoptosis via EGFR/MDR1/P-gp pathway.
ABSTRACT
Objective To investigate antibiotics resistant characteristics and carbapenemases genotype of Acinetobacter baumannii in Intensive Care Unit (ICU),so as to provide theoretical basis for clinical prevention and treatment.Methods Retrospective study was made on 90 non-duplicated clinical isolates of Acinetobacter baumannii,which were collected From January 2013 to January 2014 in three tertiary hospitals of Qingdao.All strains were identified by VITEK2 automated microbiology analyzer;K-B method was used to do drug susceptibility test;polymerase chain reaction (PCR) was used to amplify the OXA-23,OXA-24,OXA-51,OXA-58,KPC-2,VIM,IMP genes,and the positive products of genes were sequenced;the chi-square test was used to compare the difference of the resistance rates.Results The detection rate of multi-drug resistant A.baumannii (MDRAB)and Pan-drug resistant A.baumannii (PDRAB)was 61.11% (55/90) and 17.78% (16/90).In the 32 strains of imipenem-resistant Acinetobacter baumannii,the resistant rates to Cefoperazone/sulbactam,Polymyxin B was lower,while the resistant rates to other drugs tested were more than 85%.The difference of the resistance rates to 9 drugs between imipenem resistant group and Imipenem sensitive group were statistically significant (P≤0.05).PCR result showed: 32 strains detected OXA-51 gene,28 strains detected OXA-23 gene,and 3 strains detected VIM gene,the detection rates of which were 100%,87.50% and 9.38% respectively.All strains were not detected OXA-24,OXA-58,KPC-2 and IMP genes.The sequenced results were absolutely homology with the corresponding genes in genbank.Conclusions The resistance of A.baumannii in ICU is serious in this region,especially imipenem-resistant A.baumannii,which were nearly no-sensitive to most of the drugs commonly used in clinical.The gene existence of carbapenemase and carbapenemase producing is one of the main resistance mechanism of Acinetobacter baumannii to carbapenem antibiotics.OXA-23 was the major genotypes in this region.
ABSTRACT
Objective To investigate the drug resistant genes and genotypes of carbapenem-resistant Klebsiella pneumoniae in Tianjin First Center Hospital. Methods A total of 52 strains of carbapenem-non-susceptible Klebsiella pneumoniae were collected from 2012 to 2015. The MICs of antimicrobial drugs were detected using agar dilution methods. Phenotypes of carbapenemases were screened using modified Hodge test. Drug resistant genes were detected by multiplex-PCR assay. Multilocus sequence typing ( MLST) was used to determine the genotypes and homology of these carbapenem-resistant Klebsiella pneumoniae strains. Results Susceptibility of antimicrobial agents indicated that all these strains with multiple drug resistance. The resistance rate to piperacillin/tazobactam, ceftriaxone, ceftazidime, cefepime, aztreonam imipenem,meropenem was 100%( 52/52 ) . The resistance rate of ST11 type to amikacin was 93. 5%( 43/46), ciprofloxacin was 97. 8%(45/46), levofloxacin was 97. 8%(45/46), compound sulfamethoxazole was 17. 4%(8/46), tigecycline was 0. The resistance rate of ST101 type to amikacin was 3/3, ciprofloxacin was 2/3, levofloxacin was 3/3, compound sulfamethoxazole was 3/3, tigecycline was 0. The resistance rate of ST709 type to amikacin was 1/1, ciprofloxacin was 1/1, levofloxacin was 1/1, compound sulfamethoxazole was 1/1, tigecycline was 0. The resistance rate of ST1393 type to amikacin was 1/1, ciprofloxacin was 1/1, levofloxacin was 1/1, compound sulfamethoxazole was 1/1, tigecycline was 0. The resistance rate of ST2068 type to amikacin was 1/1, ciprofloxacin was 1/1, levofloxacin was 1/1, compound sulfamethoxazole was 1/1, tigecycline was 0. PCR results showed that 43 isolates were blaKPC-2 positive and 5 isolates were blaOXA-48 positive, 1 isolate was blaDNM-1 positive. There were 46 strains of ST11 type. The 43 strains of Klebsiella pneumoniae producing KPC-2 type carbapenemase were all ST11. While among 5 strains of Klebsiella pneumoniae carrying OXA-48 carbapenem resistant gene, 3 strains were ST101, 1 was ST709, 1 was ST1393. One strain of Klebsiella pneumoniae harboring DNM-1 type carbapenemase was ST2068. Conclusions Drug resistant genes of carbapenem-resistant Klebsiella pneumoniae were KPC-2 dominant, OXA-48 and DNM-1 were sporadical;the genotype was mainly ST11 by MLST in the hospital. The research provided effective basic and reference for the hospital infection t control.
ABSTRACT
Objective To elucidate the resistance mechanisms of clinical colistin-resistant Klebsiella pneumoniae and Escherichia coli isolates in China.Methods A total of 964 K.pneumoniae and 1 389 E. coli isolates were retrospectively collected from national surveillance programs from 2011 to 2014 in China. Antimicrobial susceptibility testing was determined by the microdilution method.The PCR amplification followed by sequencing was used to detect the mcr-1 gene and colistin-resistance genes, including mgrB, pmrB and phoQ.Real-time quantitative PCR was performed to examine the relative transcriptional levels of pmrB, pmrC, pmrD, pmrK and pmrE genes in K.pneumoniae, and pmrA, pmrB, pmrC, phoP and phoQ genes in E.coli.Conjugation experiment was used to detect the transferability of the resistance plasmid carrying the mcr-1 gene.Statistical analyses were performed using IBM SPSS Statistics (version 16.0) and a P value <0.05 was considered statistically significant. Results The colistin-resistant rates of K. pneumoniae and E.coli were 0.62% ( 6/964 ) and 1.66% ( 23/1 389 ) , respectively.No amino acids substitutions were identified in mgrB genes among colistin-resistant isolates.Among six colistin-resistant K. pneumoniae isolates, five isolates were identified to have point mutations in pmrB gene, but no point substitution was detected in phoQ gene.One to four point mutations had been found in pmrB and phoQ genes in colistin-resistant E.coli isolates, respectively.The expression level of pmrB, pmrC, pmrD, pmrK and pmrE genes showed no significant difference between colistin-resistant and colistin-susceptible isolates [pmrB, (1.04 ±1.12) vs.(0.94 ±0.67), P=0.945; pmrC, (1.39 ±2.01) vs.(0.16 ±0.27), P=0.101;pmrD, (1.59 ±2.43) vs.(0.88 ±0.34),P=0.445;pmrK, (0.64 ±0.62) vs.(0.04 ±0.10), P=0.051;pmrE, (3 492 833 388.83 ±8 478 977 986.85) vs.(20 771 428.93 ±38 000 732.85), P=0.445].However, the transcriptional level of pmrB genes in colistin-resistant group was 9.5-fold higher than that of the colistin-susceptible group in E.coli isolates.Four in six colistin-resistant K.pneumoniae isolates possessed mcr-1 gene, whereas all of the colistin-resistant E. coli had the mcr-1 gene. The conjugation verified the transferability rate of the plasmid carrying mcr-1 gene was 5.78 ×10-6 , and the MIC value of colistin of the conjugant increased 21-fold than the recipient strain.Conclusions Plasmid-mediated mcr-1 gene was the major reason for colistin resistance in clinical isolates of K.pneumoniae and E.coli. Some other resistance mechanisms such as transcriptional up-regulated pmrB gene also involved in colistin resistance.
ABSTRACT
Objective The expression of agr and sigB regulation system in Staphylococcus aureus with different infection types were assessed by analyzing the characteristics of m /z value generated by MALDI-TOF-MS.Methods A total of 50 isolates with specific genotypes were collected from Tianjin First Center Hospital during Jun 2013 to Feb 2014 for retrospective study .The pattern profiles of these isolates were obtained by MALDI-TOF-MS with RUO model, and the m/z value was also analysed to evaluate the expression the agr and sigB regulation system .The phylogenetic tree based on mass spectrum peak feature was constructed using SARAMIS software .Results A total of 50 strains of Staphylococcus aureus were divided into two groups: acute infection and chronic persistent and recurrent infection .The expression of delta toxin in acute infection and in chronic infection was 99.2 ±4.1 and 60.5 ±10.1 ( t =16.83, P<0.05), respectively.The regulation of stress proteins of sigB system was enhanced in chronic persistent and recurrent infections , and the expression intensities of SAS 030, SAS049 and SA0772 were 27.1 ±14.7, 54.8 ±21.5 and 51.6 ±19.2, respectively; while in acute infections , those were 4.9 ±1.9, 12.4 ±2.8 and 15.7 ±6.9, respectively.The t values between the two groups were -6.88 (P<0.05),-8.98 (P<0.05) and -1.87 (P<0.05), respecitively.The expression of phenol-soluble modulins (PSMs) was inconsistent , and the relative strength of PSMα3 was 100%in the colony variants small strains .Conclusions Different types of the Staphylococcus aureus infections could be evaluated through the assessment of the agr and sigB regulation system .The m/z value obtained by MALDI-TOF mass spectrometry is a marker for the expression of agr and sigB regulation system .The application of this technology needs further development .
ABSTRACT
The worldwide spread of carbapenem-resistant Gram-negative bacteria has become a major threat to public health.Being different from other resistance mechanism,most of the carbapenemase-encoding genes are located on mobile genetic elements.It speeds up the emerging of carbapenem-resistant strains and impose severe challenges to clinical treatment and infection control.The Carba NP test is a rapid,reliable and economical method for detection of carbapenemases.In particular,the method has superior specificity to Ambler classes A and B carbapenemases.Application of this rapid carbapenemase-detection assay will benefit clinical practice,antibiotic stewardship and prevention for the development of nosocomial resistant bacteria.
ABSTRACT
Objective To understand the drug resistance and antibiotic resistance mechanism ofβ-lactam antibiotics of invasive Streptococcus pneumoniae isolated from Shanghai Children′s Hospital, provides the reference for the rational use of antimicrobial agents.Methods This study is based on the research of the mechanism of drug resistance.62 isolates of invasive Streptococcus pneumoniae were collected from Shanghai Children′s Hospital from January 2005 to December 2011.Minimum inhibitory concentrations ( MIC) of strains to 9 antimicrobial agents were determined by E-test method.The penicillin binding protein coding genes pbp2x, pbp2b, and pbp1a of Streptococcus pneumoniae were amplified by PCR.Then, the correlation between the gene mutation andβ-lactam antibiotics resistant level were analyzed.The murM gene of Streptococcus pneumoniae was amplified by PCR and the correlation of mutation and β-lactam antibiotics resistant level was analyzed.Results Out of 62 strains of invasive Streptococcus pneumoniae from children, the detection rate of penicillin resistant Streptococcus pneumoniae was 43.6% (27/62).Between penicillin intermediate Streptococcus pneumoniae ( PISP ) ( 100%, 25/25 ) and penicillin sensitive Streptococcus pneumoniae (PSSP) (3/10), the difference of gene mutation rate near the pbp2b conserved sequence was statistically significant (χ2 =21.875, P<0.01).The same situation occurred between penicillin resistant Streptococcus pneumoniae (PRSP)(100%, 27/27)and PSSP (3/10) (χ2 =23.310, P<0.01).Also the difference of gene mutation rate of PISP (84%, 21/25) vs PSSP (0) and PSSP (0) vs PRSP (85.2%, 23/27) near or in the pbp2x conserved sequence were statistically significant (χ2 =21.000, P <0.01;χ2 =22.513,P<0.01).The difference of gene mutation rate near the pbp1a conserved sequence and Insertion sequence, which were statistically significant, occurred between PISP and PSSP (χ2 =13.22,P<0.01), between PRSP and PSSP (χ2 =37.000,P<0.01), between PISP and PRSP (χ2 =10.211,P=0.001). MurM gene mutation rate was statistically significant different between the 2 group penicillin MIC≥8 mg/L or ceftriaxone MIC≥2 mg/L group (95.8%, 23/24) and penicillin MIC<8 mg/L or ceftriaxone MIC<2 mg/L group (0) (χ2 =56.2,P =0.002 6).Conclusions The resistance phenomenon of invasive Streptococcus pneumoniae in Shanghai Children′s Hospital is serious.The gene mutations of pbps and murM play a role in amide in the beta of antibiotic resistance, and there is a certain correlation with the antibiotic resistance level.
ABSTRACT
Objective To investigate the molecule phenotype, epidemiology, and resistance genes of the New Delhi metallo- β-lactamase-1 ( NDM-1 ) producing Klebsiella pneumoniae ( K. pneumoniae ) . Methods Retrospective study was made on one hundred and ten non-repetitive carbepenem-resistant K. pneumoniae clinical isolated strains, which were collected from January 2011 to December 2012 in our hospital. The minimal inhibitory concentrations ( MICs ) of antibiotics were tested by the GN13 cards of BioMerieux Company. Modified Hodge test were used for the detection of carbapenemases. The blaNDM-1 encoding gene and linkage of ISAba125-NDM were detected by PCR method. The purified PCR products were cloned and sequenced. The homology of the K. pneumoniae were analyzed by the multilocus sequence typing ( MLST ) . Plasmid conjugation experiment and curing method were used to study the transfer of bacterial resistance. The Fisher′s exact probability test was used to compare the data. Results 13% NDM-1-producing K. pneumoniae were detected and confirmed as blaNDM-1 by sequencing (14/110). The resistance rates of the 14 NDM-1-producing K. pneumoniae strains to imipenem, meropenem, ertapenem, ciprofloxacin, levofloxacin, amikacin, and aztreonam were 14/14, 14/14, 13/14, 10/14, 9/14, 5/14, and 11/14. Meanwhile, the positive rate of ISAba125-NDM linkage of those 14 NDM-1-producing K. pneumoniae strains was 14/14. The E. coli J53 transconjugants, whose MICs of imipenem, meropenem, and ertapenem were increased by 4 to 64 times, were blaNDM-1 gene and ISAba125-NDM linkage positive. In addition, it was showed that the blaNDM-1 gene and ISAba125-NDM linkage were located on a plasmid with a size of approximately 65 000 bp. Conclusions The NDM-1 producing K. pneumoniae strains in this study were resistant to many commonly used antibiotics, however, the resistance rate to aminoglycoside and aztreonam were relatively low. The carbapenemase-resistant genotype spread by blaNDM-1 carried plasmid. Attention should be paid to its easily transmissible feature among the strains in clinic. The insertion sequence ISAba125 may be involved in the blaNDM-1 gene mediated carbapenemase-resistant genotype.
ABSTRACT
Objective To examine the expression of heparin binding-epidermal growth factor-like growth factor (HB-EGF) in paclitaxel-resistant ovarian cancer and elucidate the relationship between HB-EGF and the resistance of ovarian cancer to paclitaxel. Methods The human ovarian carcinoma cell line A2780 and the paclitaxel-resistant human ovarian carcinoma cell line A2780/Taxol were cultured in vitro. Western blot was used to dectect the expression of HB-EGF protein in A2780 and A2780/Taxol groups. The A2780 cells were treated with cross-reacting material 197 (CRM197 and A2780 + CRM197 group) or dimethyl sulphoxide (DMSO;A2780 group), while the A2780/Taxol cells were treated with CRM197 (A2780/Taxol+CRM197 group) or DMSO (A2780/Taxol group). The effects of CRM197 on growth and proliferation was tested by methyl thiazolyl tetrazolium( MTT) and the results were showed as absorbance (A).The effects of CRM197 on cell cycles was tested by flow cytometry, while the effects of CRM197 on apoptosis was examined by caspase-3 activity assay and the results were showed as p-nitroaniline(pNa). In animal experiment, four groups of cells were inoculated to BALB/c nude mouse subcutaneously to observe tumor formation ability following CRM197 treatment. Immunohistochemistry was used to determine the expression of HB-EGF protein in A2780 and A2780/Taxol group. Results The expression level of HB-EGF protein in A2780/Taxol group (2.11±0.41) was significantly higher than that of A2780 group (0.75±0.20;P<0.01). The inhibition effect of CRM197 on the cell growth of A2780+CRM197 and A2780/Taxol+CRM197 group was accompanied by the acceleration of CRM197 concentration(P<0.01). When CRM197≥1 μg/ml, the inhibition effect of CRM197 on the cell growth of A2780/Taxol+CRM197 group was significantly higher than that in A2780/Taxol group(P<0.05). In cell cycle experiment, CRM197 induced the cell-cycle arrest at the G0/G1 phase in A2780+CRM197 cells[(67 ± 4)%] compared with A2780 cells[(54 ± 6)%;P<0.01], while CRM197 significantly induced the cell-cycle arrest at the G0/G1 phase in A2780/Taxol+CRM197 cells [(72± 4)%] compared with A2780/Taxol cells [(24±8)%;P<0.01]. CRM197 treatment in A2780+CRM197 group [(40 ± 6)μmol/L] led to the acceleration of the caspase-3 activity when compared to A2780 group [(6 ± 6)μmol/L;P<0.01], while CRM197 treatment in A2780/Taxol+CRM197 group [(66 ± 12)μmol/L] led to significant acceleration of the caspase-3 activity when compared to A2780 group [(9 ± 6)μmol/L;P<0.01]. In experiments in vivo, the expression scores of HB-EGF protein in A2780/Taxol tumors(10.8 ± 3.3) were higher than that in A2780 tumors (5.0±2.2;P<0.01). The tumor size and tumor weight of the A2780/Taxol+CRM197 group were both higher than those of the A2780+CRM197 group [(546 ± 85) mm3 vs (1 355 ± 119) mm3,(0.56 ± 0.09) g vs (1.31 ± 0.27) g; all P<0.01]. The CRM197 inhibition rate of the A2780+CRM197 and A2780/Taxol + CRM197 group were 43% and 68% respectively, showed that CRM197 significantly suppressed the growth of A2780/Taxol xenografts in vivo(P<0.01). Conclusions HB-EGF is over-expressed in paclitaxel-resistant ovarian cancer and may be contributes to drug resistance. Inhibition of HB-EGF expression potently enhances apoptosis and inhibit the growth of paclitaxel-resistant ovarian cancer, shedding light on the HB-EGF-targeted therapy options for chemoresistant ovarian cancer patients.
ABSTRACT
Objective To detect the hypermucoviscosity phenotype , capsular serotype and virulence gene of Klebsiella pneumonia (K.pneumonia) from various kinds of clinical specimens and understand the characteristics of different K.pneumonia causing infections.Methods A retrospective study was conducted through collection of 178 K.pneumonia isolates from blood, sputum, bronchoalveolar lavage fluid , urine, normally sterilized fluid , puncture fluid from liver abscess and other abscesses between January 2010 and December 2012 in General Hospital of Chinese PLA.String test was carried out for detection of hypermucoviscosity phenotype.Capsular serotype and virulence gene ( rmpA) were checked by polymerase chain reaction.Analysis was made according to the hypermucoviscosity , capsular serotype , rmpA gene , as well as the sources of K.pneumoniae.Statistic data was analyzed by contingency table analysis and χ2 test.Results Eighty-three out of 178 ( 46.6%) strains of K.pneumonia were hypermucoviscous with positive string test, the positive rate of virulence gene rmpA was 92.8%(77/83).K1/K2/K57 capsular serotypes were the predominant serotypes in the group of puncture fluid from liver abscess and other abscesses (75.0%,27/36)than the group of blood (32.4%,12/37), urine(21.7%,5/23) and normally sterilized fluid(25.0%,5/20), and also more than in the group of sputum , bronchoalveolar lavage fluid (50.0%,22/44),χ2 =21.19,P<0.01.The positive rate of string test in the group of puncture fluid from liver abscess and other abscesses (77.8%, 28/36)was significantly higher than the group of blood (29.7%, 11/37), urine (30.4%, 7/23), or normally sterilized fluid (25.0%, 5/20),χ2 =27.90,P<0.01.The positive rate of rmpA gene in the isolates from puncture fluid of liver abscess and other abscesses was higher than other groups.Conclusions As the pathogens of various kinds of infections , mainly abscess and respiratory infection, hypermucoviscous strains of K.pneumonia were of great clinical significance.Capsular serotype K57, as well as K1 and K2, possessed hypermucoviscosity and hypervirulence in China.
ABSTRACT
Objective To investigate the effect of reserpine,an efflux pumps inhibitor,on the activities of fluoroquinolones (FQNs) against Enterococci,and the distribution of efflux pump genes emeA and its correlation with the resistance of Enterococci.To elucidate the relationship between FQN resistance in Enterococci and active efflux.Methods One hundred isolates of enterococci were identified by VITEK microbe automatic system.The antibacterial agent susceptibility tests were performed by the disc diffusion method (K-B) in accordance with the CLSI standards.The minimum inhibitory concentration (MIC) of each FQN was tested by the agar dilution method,and the MIC changes were detected after adding reserpine.The distribution of emeA in 100 isolates of Enterococci was determined by PCR.Thex2 test was used to compare the differences of statistical results.Results After reserpine was used,three-FQN resistance in Enterococci was reduced.Ciprofloxacin,gatifloxacin and levofloxacin resistance was reduced from 42％ (42/100) to 28％ (28/100),from 30％ (30/100) to 17％ (17/100),and from 33％ (33/100) to 23％ (23/100),respectively.The positive rate of emeA gene in 100 strains of Enterococci was 55％ (55/100).There were 45 positive strains(72.6％) in 62 E.faecalis and 10 positive strains (26.4％) in 38 E.faecium.The positive rate of emeA gene in the resistant strains against ciprofloxacin,gatifloxacin and levofloxacin was 73.8％ (31/42),76.7％ (23/30),75.8％ (25/33),respectively,and the positive rate of emeA gene in the susceptible strains against above 3 antibacterials were 41.4％ (24/58),45.7％ (32/70),44.8％ (30/67).Efflux pump genes emeA in resistant strains is higher than the sensitive strains,with the statistically significant difference(x2 =13.02,8.13 and 8.57,P ＜ 0.005).Conclusions Reserpine could inhibit the active efflux of in FON Enterococci and reduce the MIC for drug-resistant strains in vitro.Multidrug-resistant efflux pump gene emeA was relevant to antimicrobial drug resistance in Enterococci.
ABSTRACT
Objective To investigate the correlation between expression of Panton-Valentine leukocidin gene and accessory gene regulator among different clinical isolates of Staphylococcus aureus.Methods All non-duplicate Staphylococcus aureus clinical isolates were isolated from various clinical specimens of the patients at 4 hospitals from January 2003 to December 2010.Panton-Valentine leukocidin genes among Staphylococcus aureus clinical isolates were detected by PCR and DNA sequencing.The expressions of lukS-PV and agrA were determined by real-time PCR.Results Ninty-six S.aureus isolates including 58 hospital-acquired and 28 community-acquired isolates were positive for PVL genes,among which 54 from blood,33 from pus and 9 from sputum.Ten isolates cannot be classified due to lack of information.Sixty-seven and 29 PVL-positive isolates were isolated from the specimens of adults and children.The median relative quantities of lukSmRNA of the isolates from pus and blood were 1.500 and 0.818.The quantity of lukSmRNA among the isolates from pus was significantly higher than that from blood (U =634,P =0.025).The median relative quantities of lukSmRNA of the isolates from children and adults were 1.292 and 0.540,respectively.The quantity of lukSmRNA among the isolates from children was significantly higher than that from adults (U =660,P =0.013).The median relative quantities of lukSmRNA among community-acquired and hospital-acquired isolates were 1.034 and 0.536,respectively.The quantity of lukSmRNA among community-acquired isolates was significantly higher than that from hospital-acquired isolates (U =338,P =0.012).The correlation coefficients between lukSmRNA and agrAmRNA of total isolates,pus isolates and blood isolates were 0.592 (P ＜ 0.01),0.810 (P ＜ 0.0l) and 0.543 (P ＜0.01),respectively.While the correlation coefficients of those among the isolates from children and adults were 0.804 (P ＜ 0.01) and 0.476 (P ＜ 0.01).The correlation coefficients of those among the isolates from community-acquired and hospital-acquired isolates were 0.767 (P ＜ 0.01) and 0.556 (P＜0.01).Conclusions The quantity of lukSmRNA of Staphylococcus aureus isolates from pus was significantly higher than that from blood.The agr may have positive regulation effect on the expression of lukS/F-PV,especially among the isolates from pus and children.(Chin J Lab Med,2013,36:313-317)
ABSTRACT
Objective To construct recombinant plasmid containing CFP-10 gene of Mycobacterium tuberculosis(MTB).Methods The gene fragment of CFP-10 was amplified by PCR from Mycobacterium tuberculosis H37Rv genomic DNA and cloned to pET-32a(+) vector.The recombinant plasmid pET-32a-CFP-10 was transformed into E.coli BL21 (DE3) and induced by IPTG.Results CFP-10 gene fragment was amplified from genomic DNA of Mycobacterium tuberculosis H37Rv strain,and thepET-32a(+) prokaryotic recombinant plasmid was constructed successfully.The recombinant protein was expressed with the induction of IPTG.Conclusions The prokaryotic expression vector for CFP-10 was successfully constructed and the recombinant protein was highly expressed in E.coli BL21 (DE3),which lays a foundation for its subsequent immunological function study.
ABSTRACT
Objective To construct the recombinant plasmid of protein CFP10-MPT48-TB8.4 of Mycobacterium tuberculosis and to investigate the diagnosis potential of this fusion protein in tuberculosis serodiagnosis.Methods The recombinant fusion protein CFP10-MPT48-TB8.4 was expressed, and identified by Western blot.The ELSIA based on the purified fusion protein was done,and used for screening in 230 cases of clinical serum samples including pulmonary tuberculosis patients ( n =150 ),pulmonary disease patients other than tuberculosis (n =70) and health controls (n =103 ).The test result was analyzed by Medcale11.5 software.Results The fusion protein CFP10-MPT48-TB8.4 was successfully expressed with a purity over 95％.Specific immunogenicity of the recombinant protein was confirmed by Western blot.The overall sensitivity and specificity obtained of ELISA were 56.7％ (85/150) and 90.8％ ( 157/173 ),respectively.The specificity was 85.7 ％ (60/70) in non-tuberculosis group and 94.2％ (97/103 ) in healthy group,respectively.Conclusion The recombinant protein of CFP10-MPT48-TB8.4 has a high sensitivity and specificity and may be a potential candidate antigen in tuberculosis serodiagnosis.
ABSTRACT
Objective To evaluate the detection of culture filtrate protein 10 (CFP10) and 6000 early secretory antigenic target (ESAT-6) in cerebrospinal fluid to be used in diagnosing tuberculous meningitis. Methods Dot enzyme linked immunosorbent assay ( Dot ELISA) method that was improved by applying concentrated cerebrospinal fluid was used to detect CFP10 and ESAT-6 in cerebrospinal fluid to analyze small protein antigen secreted by M. tuberculosis. Cerebrospinal fluid of 111 subjects were collected,in which 58 specimens were clinically diagnosed as tuberculous meningitis and 53 as non-tuberculous.CFP10 and ESAT-6 were detected in cerebrospinal fluid using Dot ELISA method and the results were analyzed. Results The sensitivities of detecting CFP10 and ESAT-6 antigen were 93.1% and 91.4% respectively, and the specificities were 92. 5% and 94. 3% respectively. The sensitivities and specificities are generally higher compared with the other methods of detecting M. tuberculosis or materials of M. tuberculosis by acid-fast staining or mycobacterium tuberculosis culture and polymerase chain reaction.Conclusions Using Dot ELISA method to detect CFP10 and ESAT-6 in cerebrospinal fluid to diagnose tuberculous meningitis has a high sensitivity and specificity. Our study provided the evidence of detecting the specific antigen of M. tuberculosis to be used in diagnosing tuberculosis.