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Study on antibiotic resistance and resistance genes of methicillin-resistant Staphylococcus aureus in children

Xia WU ; Chuanqing WANG ; Xiufeng YAN ; Aimin WANG ; Leiyan HE ; Zuhuang MI ; Hui YU

Chinese Journal of Infectious Diseases.2013;31(11):641-645. doi:10.3760/cma.j.issn.1000-6680.2013.11.001

Objective The aim of this study was to study the antibiotic resistance and resistance genes of methicillin-resistant Staphylococcus aureus (MRSA) in children from Shanghai area,and to determine the relationship between phenotypic and genotypic resistance profiles.Methods In this study,a total of 37 MRSA strains isolated from clinical specimens of hospitalized patients in Children's Hospital of Fudan University from March 2009 to November 2011 were collected.The mecA,ermA,ermB,ermC,aac (6') /aph (2),aph (3')-Ⅲ,ant (4',4),and qacA genes were detected by polymerase chain reaction (PCR).Resistance to antibiotics was detected by agar dilution tests.The data analysis was done by chi square test.Results Among the 37 MRSA isolates,all (100.0 ％) were mecA gene positive,9 (24.3％) were ermB gene positive,none was ermA/C gene positive,21 (56.8％) were aac (6')/aph (2) gene positive,10 (27.0％) were aph (3')-Ⅲ gene positive,6 (16.2％) were ant(4',4) gene positive,and 9 were qacA gene positive (24.3％).The positive rate of aac(6')/aph(2) in hospital acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) was significantly higher than that of community acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) (85.7％ vs18.8％,x2=60.340,P=0.000).Among the 37 MRSAisolates,37 (100.0％) were resistant to penicillin,ampicillin-sulbactam,cefazolin,cefoxitin and cefuroxime.The 37 isolates were all susceptible to teicoplanin,vancomycin,and linezolid.The resistant rates to gentamicin,erythromycin,clindamycin,sulfamethoxazole,fosfomycin,rifampicin,and levofloxacin were 51.4％ (19/37),81.1％ (30/37),51.4％ (19/37),16.2％ (6/37),27.0％ (10/37),37.8％ (14/37) and 54.0％ (20/37),respectively.Compared with CA-MRSA,HAMRSA isolates had significantly higher resistance rates to gentamicin (12.5％ vs 81.0％; x2 =17.033,P=0.000),levofloxacin (31.2％ vs 71.4％; x2 =5.903,P=0.017),and rifampin (6.2％ vs 61.9％; x2=11.959,P=0.001).The rate of gentamicin resistance in aac(6')/aph(2) gene carrying strains was significantly higher than strains not carrying the gene (x2 =29.757,P=0.000).Conclusions MRSA in children carry a variety of drug-resistant genes,showed multi-drug resistance.HA-MRSA carries more resistance genes,and has higher rates resistance to antimicrobials than CA-MRSA.

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Study of plasmid-mediated quinolone resistance determinants in Citrobacter freundii

Yibo SHAO ; Xu LI ; Lifen HU ; Qinxiu XIE

Chinese Journal of Infectious Diseases.2013;31(9):513-518. doi:10.3760/cma.j.issn.1000-6680.2013.09.001

Objectives This study was conducted to detect and analyze the presence of plasmidmediated quinolone resistance (PMQR) determinants [qnr,aac-(6′)-Ib-cr and qepA] among clinical isolates of Citrobacter freundii strains isolated from patients in Anhui,China,and to understand the susceptibility of PMQR positive strains to commonly used antimicrobial agents.Methods During the year 2009,31 Citrobacter strains were collected from the First Affiliated Hospital of Anhui Medical University.Polymerase chain reaction (PCR) was used to detect PMQR genes.Amplicons were purified,sequenced and compared with data from the GenBank.Conjugation experiments were conducted to determine whether the qnr-carrying plasmids were self-transferable.The susceptibility of the positive isolates and transconjugants were tested by agar dilution method according to Clinical and Laboratory Standards Institute (CLSI) guidelines.The minimum inhibitory concentrations (MIC) of ciprofloxacin and levofloxacin were determined by E-test strips.Results Among the 31 Citrobacter strains,the qnr genes were detected in 8 isolates (25.8％),among which,6 carried qnrB.Aac-(6′)-Ib-cr and qepA were not identified in these isolates.The qnr genes were transferred from four clinical isolates to their transconjugants.Sequence analysis identified one novel qnrB variant (qnrB24).The resistant rate of qnr-positive clinical isolates to quinolone was 87.5 ％.Most of them were also resistant to various other antibiotics,including cefotaxime (75.0 ％),amikacin (7.5 ％),ceftazidime (62.5 ％),cefapime (37.5 ％),and gentamycin (87.5 ％).All qnr positive strains were susceptible to imipenem.MIC of all transconjugants showed reduced susceptibility to fluoroquinolones,with MIC increased by 10-23 folds.Conclusions Our study shows that qnr gene has occurred in Citrobacter freundii isolates from Anhui Province,China.QnrB is most prevalent in these isolates.Most qnr positive isolates are resistant to commonly used antimicrobial agents.

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Influences of plumbagin on phosphatidylinositol 3-kinase/protein kinase B signaling pathway in rats with hepatic fibrosis

Xiaowei CHEN ; Weiwei ZHANG ; Jinxia CHEN ; Youcai ZHANG ; Yongping CHEN ; Ji LI

Chinese Journal of Infectious Diseases.2013;31(9):519-523. doi:10.3760/cma.j.issn.1000-6680.2013.09.002

Objective To explore the influences of plumbagin on phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (Akt) signaling pathway in rats with carbon tetrachloride induced liver fibrosis.Methods Forty male SD rats were assigned to control group,model group,2 mg/kg plumbagin treated group and 3 mg/kg plumbagin treated group,with 10 rats in each group.Rats of the control group were injected with 0.9％ NaCl solution (2 mL/kg) intraperitoneally,while rats of the other three groups were injected with 60％ carbon tetrachloride/peanut oil (2 mL/kg,3 times a week for 6 weeks) intraperitoneally.Since the third week after modeling,rats of plumbagin treated groups were treated with intraperitoneal injection of plumbagin at a dose of 2 mg/kg and 3 mg/kg (twice a week for 4 weeks),respectively.Serum levels of alanine aminotransferase (ALT),aspartate aminotransferase (AST),albumin (Alb) were monitored routinely.Hyaluronic acid (HA) and laminin (LN) were measured by radioimmunoassay after 6 weeks; protein expression of liver PI3K,Akt and phosphorylated Akt (p-Akt) were evaluated by Western blotting and immunohistochemistry,respectively.Comparison of means among groups was performed by univariate analysis of variance.Results The hepatic fibrosis model was successfully established after 6 weeks.Serum levels of ALT,AST,HA and LN of model group were significantly higher than those of control group (all P＜0.05).Serum levels of ALT,AST,HA and LN of 2 mg/kg plumbagin treated group and 3 mg/kg plumbagin treated group were significantly lower than those of model group,and the differences were statistically significant (all P＜0.05).The protein expressions of PI3K and Akt in each group were comparable (all P＞0.05).The p-Akt protein was mainly expressed in nucleus of hepatocytes.The levels of p-Akt protein in control group,model group,2 mg/kg plumbagin treated group and 3 mg/kg plumbagin treated group were 0.0821±0.0003,0.7374±0.0037,0.3679 ±0.0332 and 0.1327±0.0561,respectively,and that in model group was significantly higher than control group (t =851.302,P＜0.05),but those in plumbagin treated groups were both lower than model group (t=71.858 and 28.363,all P＜0.05).Conclusions Plumbagin presents anti-fibrotic effects in the liver,by down-regulating the expression of p-Akt during the development of fibrosis,which might be one of the antifibrotic mechanisms.

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Gene cloning and expression of the Tp0453 antigen immuno-dominant epitope fragment of Treponema pallidum and its potential use in serodiagnosis of syphilis

Xianling WANG ; Fukun WANG ; Keran JIA ; Shiying LI ; Wei LI ; Fei TANG

Chinese Journal of Infectious Diseases.2013;31(9):524-528. doi:10.3760/cma.j.issn.1000-6680.2013.09.003

Objective To clone and express the Tp0453 antigen immuno-dominant epitope fragment of Treponema pallidum (Tp) in Escherichia coli,in an effort to develop serological tests with increased specificity for the diagnosis of syphilis.Methods The gene encoding Tp0453 recombinant outer membrane protein fragment was amplified by polymerase chain reaction (PCR),and inserted into expression vector pQE30 after T-A cloning,then confirmed by restriction map.The constructed recombinant plasmid pQE30-Tp0453 was transformed to E.coli M15 for expression induced by isopropyl β-D-1-thiogalactopyranoside.The expressed product was identified by Western blot,and purified by Ni2+-NTA agarose column chromatography.A double antigen sandwich enzymelinked immunosorbent assays (ELISA) was established by using the recombinant Tp0453 protein to test sera from 48 patients with positive Treponema pallidum particle agglutination test (TPPA),and 40 negative sera as control.Results The PCR amplicon of the target gene was about 490 bp.The recombinant plasmid pQE30-Tp0453 was correctly constructed and successfully expressed in E.coli M15.The expressed product,with a relative molecular of about 21 000,existed in a form of inclusion body,accounting for about 18％ of total somatic protein,and reached a purity of more than 95％ after purification.Western blot showed specific reaction of the expressed protein with Tp positive serum.The ELISA tests with the 88 clinical samples yielded a sensitivity of 97.9％ (47/48),and specificity of 100.0 ％ (40/40).The consistency of results between the ELISA test and the TPPA test was 98.9 ％ (87/88).Conclusion The expressed Tp0453 fragment has showed good immunoreactivity with serum from patients with syphilis,providing the foundation of further development of serological diagnostic kit with increased specificity for the diagnosis of TP infection.

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Impact of hepatitis B virus infection on efficacy of combined antiretroviral therapy in patients with acquired immunodeficiency syndrome

Rongrong YANG ; Xien GUI ; Yong XIONG ; Pingzheng MO ; Shicheng GAO ; Yuping RONG ; Yajun YAN

Chinese Journal of Infectious Diseases.2013;31(9):533-537. doi:10.3760/cma.j.issn.1000-6680.2013.09.005

Objective To investigate the influence of hepatitis B virus (HBV) infection on efficacy of combined antiretroviral therapy (cART) in patients with acquired immunodeficiency syndrome (AIDS).Methods Seventy-eight subjects with human immunodeficiency virus (HIV)/HBV co-infection and 156 subjects with HIV mono-infection were included.CD4+ T cell count,HIV viral load,HBV-markers and liver functions were routinely tested.The differences in survival rate,as well as immunological and virological responses between the two groups (HIV/HBV co-infection group and HIV mono-infection group) during cART were compared.Categorical data were compared by Chisquare test,measurement data were compared by t test,and measurement data with abnormal distribution were compared by Mann-Whitney test.Results At month 42 of cART,HIV RNA levels and CD4+ T cell counts of the two groups were comparable.However,at month 48,54 and 60 of cART,the immunological and virological responses of HIV/HBV co-infection group were less favorable than those of HIV mono-infection group.At each time point of month 12,24,36,48 and 60 of cART,3 out of 13 subjects with HIV/HBV co-infection maintained hepatitis B e antigen (HBeAg)loss; the HBeAg seroconversion rates were 32.1％ (9/28),50.0％ (14/28),53.6％ (15/28),64.3％ (18/28) and 71.4％ (20/28),respectively (x2 =10.189,P=0.037) ; HBV DNA negative rates were 95.1％ (39/41),82.9％ (34/41),68.3％ (28/41),43.9％ (18/41) and 43.9％ (18/41),respectively (x2 =29.982,P=0.000); liver dysfunction rate was 32.1 ％ (25/78),51.4％ (38/74),33.8％ (22/65),47.9％ (23/48) and 6.7％ (3/45),respectively (x2 =28.053,P=0.000).Mortalities in HIV/HBV co-infected and HIV mono-infected individuals were 24.4％ (19/78) and 5.1 ％ (8/156),respectively (x2 =18.841,P＜0.01).Sixteen out of the 19 deaths (84.2 ％) in HIV/ HBV co-infected subjects died of end stage liver diseases.Conclusions HBV co-infection diminishes the long term efficacy of cART.End stage liver diseases are the primary cause of death in HIV/HBV co-infected subjects during cART.

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Impact of hepatitis B virus infection on semen parameters and sperm function

Jiujia ZHENG ; Peiyu WANG ; Qingxing LI ; Xu YANG ; Qianjin FEI ; Xuefeng HUANG

Chinese Journal of Infectious Diseases.2013;31(9):543-547. doi:10.3760/cma.j.issn.1000-6680.2013.09.007

Objective To investigate the impact of hepatitis B virus (HBV) infection on semen parameters,sperm DNA integrity,acrosin activity and sperm-nucleoprotein transition.Methods Semen samples from 527 subjects including 273 hepatitis B surface antigen (HBsAg) positive and 254 HBsAg negative,who sought medical attention and received in-vitro feritilization in reproductive medicine center of First Hospital of Wenzhou Medical University from Jan 2011 to Oct 2012 were collected.Semen parameters,sperm DNA fragmentation index (DFI),sperm-nucleoprotein transition and acrosin activity of both HBsAg-positive and HBsAg-negative subjects were analyzed.Results Semen parameters of both groups were within the normal range,but sperm concentration and percentage of forward moving sperms of HBsAg positive group were significantly lower than those of HBsAg negative group (P=0.000),while percentage of static sperms of HBsAg positive group were significantly higher than that of HBsAg negative group (P =0.000).DFI in HBsAg positive and negative group were (17.85 ± 0.70) ％ and (11.85 ± 0.50) ％,respectively,which was significantly different (t=6.951,P=0.000).Percentage of sperms with normal morphology in both groups were within the normal range,but sperms with neck and tail deformity in the HBsAg positive group was significantly higer than those in HBsAg negative group (all P＜0.05).Acrosin activity of sperms in HBsAg positive group was significantly lower than that in HBsAg negative group (t=3.756,P=0.000).Linear regression analysis indicated that serum HBsAg level was reversely correlated with sperm concentration (r=-0.140,P =0.021),but positively correlated to DFI (r =0.151,P =0.014).Conclusions HBV infection not only affects the routine semen parameters and sperm morphology,but also compromises sperm function including impaired DFI and acrosin activity.However,the impact of anti-HBV agents on sperm quality and male fertility requires further research.

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Nineteen cases of biliary ascariasis diagnosed and managed by endoscopic retrograde cholangiopancreatography

Zhuo YANG ; Feng GAO ; Yunfeng ZHAO ; Shuren MA ; Ning ZHANG ; Zhaojie GONG

Chinese Journal of Infectious Diseases.2013;31(9):548-551. doi:10.3760/cma.j.issn.1000-6680.2013.09.008

Objective To investigate the role of endoscopic retrograde cholangiopancreatography (ERCP) in the diagnosis and treatment of adult biliary ascariasis.Methods Clinical data of 19 cases of adult biliary ascariasis diagnosed and treated with ERCP during Jan 2005 to Dec 2012 were retrospectively analyzed.Results Among the 19 adult patients with biliary ascariasis,11 were male,and the mean age was (62.7± 10.8) years.Eight cases lived in the countryside,7 cases in the integration of urban and rural area,and 4 cases in urban area.All cases had histories of eating raw fruits and vegetables.Living worms were found in 9 cases.Four cases were complicated with common bile duct stones.Petrification of residual dead worms in the common bile duct was found in 10 cases.All ascariasis and residual bodies were removed successfully by ERCP.There were no postoperative complications except for 3 cases of hyperamylasemia.Conclusions Adult biliary ascariasis commonly occurs in the elderly,and is frequently complicated with common bile duct stones.ERCP is efficacious in treating biliary ascariasis.

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Inhibition of silent information regulator 1 induces glucose metabolism disorders of hepatocytes and enhances hepatitis C virus replication

Lijie SUN ; Yonghua ZHAO ; Shuchen LI ; Jianwu YU ; Peng KANG ; Wei LIU

Chinese Journal of Infectious Diseases.2013;31(10):577-584. doi:10.3760/cma.j.issn.1000-6680.2013.10.001

Objective The aim of this study was to investigate the effect of hepatitis C virus (HCV) replication on expression of silent information regulator 1 (SIRT1) and glucose metabolism of hepatocytes using Huh 7.5 cells harboring HCV replicon.Methods The level of reactive oxygen species (ROS),value of nicotinamide adenine dinucleotide (NAD+)/reduced form of nicotinamide adenine dinucleotide (NADH) was detected by flow cytometry and chromatometry.The activity,mRNA expression,and protein level of SIRT1 were detected by a scintillation counter,real-time fluorescence quantitative polymerase chain reaction (RT-PCR),and Western blot,respectively.Glucose uptake by hepatocytes and gluconeogenesis were detected using radioactive isotope method and glucose oxidase method.The mRNA levels of SIRT1 downstream glucose-metabolism genes were measured by RT-PCR.Measurement date were compared by t test.Results In replicon cells,the level of ROS (3.8±0.5 vs 1.0±0.2; t=12.736,P＜0.01) was increased and the value of NAD+/NADH (0.03±0.01 vs 0.12±0.03; t=6.971,P＜0.01) decreased compared with Huh 7.5 cells.The activity (0.3±0.1 vs 1.0±0.2; t=7.668,P＜0.01),mRNA expression(0.4±0.1 vs 1.0± 0.3; t=4.648,P＜0.01) and protein level(0.3±0.1 vs 0.8±0.2; t=5.941,P＜0.01) of SIRT1 were reduced.Inhibition of SIRT1 not only increased insulin receptor substrate-1 (IRS-1) phosphorylation (0.7±0.2 vs 0.4±0.1; t=3.286,P＜0.01),decreased protein kinase B (Akt) phosphorylation (0.3 ± 0.1 vs 0.6 ± 0.2; t=3.286,P＜0.01),down regulated cell surface expression of glucose transporler 2 (GLUT2,0.4±0.1 vs 1.0 ± 0.2; t =6.573,P＜0.01) and suppressed cellular glucose uptake (count per minute:4600±500 vs 21 000±4600; t=8.682,P＜0.01); but also decreased phosphorylation of forkhead box O1 (FoxO1,0.2＝0.1 vs 0.5±0.1; t=5.196,P＜ 0.01),up-regulated phosphoenolpyruvate carboxykinase (PEPCK,2.8±0.6 vs 1.0±0.3; t=6.573,P＜0.01) and glucose 6-phosphatase (2.6±0.5 vs 1.0±0.2; t=7.278,P＜0.01) genes,and promoted glucose production (2.5±0.5 vs 1.0±0.2; t=5.543,P＜0.01).Conclusions HCV replication decreases NAD+/NADH ratio,which might down-regulate the activity and the expression of SIRT1,leading to changes in the expression profile of glucose metabolism related genes and causing glucose metabolism disorders of hepatocytes by a decrease in glucose uptake and an increase in glucose production,and promotes HCV replication.

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Expression profiles of microRNA molecules in different stages of chronic hepatitis B virus infection and its clinical significance

Tongjing XING ; Hongtao XU ; Wenqing YU ; Bian WANG ; Jing ZHANG

Chinese Journal of Infectious Diseases.2013;31(10):586-592. doi:10.3760/cma.j.issn.1000-6680.2013.10.003

Objective To explore the expression profiles and their clinical significance of microRNA (miRNA) molecules in different stages of chronic hepatitis B virus (HBV) infection.Methods The miRNA molecule expressions of 12 patients with chronic hepatitis B,12 chronic HBV carriers,12 inactive hepatitis B surface antigen (HBsAg) carriers,and 9 healthy controls were screened using miRNA microarray.The miRNA profiles were validated by the real time fluorescence quantitative polymerase chain reaction (PCR).The t-test was used for comparison between twogroups,whereas one-way ANOVA and SNK-q tests were used for multiple comparisons.Mann-Whitney and Kruskal Wallis H tests were used for comparison of two or more groups of data with skewed distribution.The receiver operation characteristic (ROC) curve was constructed to evaluate the diagnostic significance of miRNA molecules.Results Compared with the healthy controls,significant differences in the expression profiles of miRNA molecules were found in peripheral blood mononuclear cells of chronic HBV carriers (2 molecules up-regulated,and 18 down regulated) and chronic hepatitis B patients (33 molecules up-regulated and 19 down-regulated).No significant difference was found between inactive HBsAg carriers (2 molecules up regulated,and 3 down-regulated) and controls.The results of six miRNA molecules detected by real-time fluorescence quantitative PCR were basically consistent with the results detected by microarray.The area under ROC curve of the six miRNA molecules of hsa miR-4711-3p,hsa-miR-3191 5p,hsa-miR-5704-5p,hsa-miR 548ah-5p,hsa-miR-146a-5p and hsa-miR-29b-3p in distinguishing immune tolerance and clearance of chronic HBV infection were 0.994,0.984,0.967,1.000,1.000 and 0.996,respectively.Conclusions The different expression profiles of miRNA molecules could be used to distinguish the different stages of chronic HBV infection,and are closely related with the immune tolerance and activation in chronic HBV infection.

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Assessment of indocyanine green clearance test combined with the model for end-stage liver disease in the prediction of short-term prognosis for liver failure

Hongling FENG ; Qian LI ; Lin WANG ; Wanyou YU ; Guiyu YUAN ; Wukui CAO ; Jiming YANG

Chinese Journal of Infectious Diseases.2013;31(10):593-597. doi:10.3760/cma.j.issn.1000-6680.2013.10.004

Objective To investigate the efficacy of the indocyanine green (ICG) clearance test (ICGR15) combined with the model for end-stage liver disease (MELD) for assessing the short-term prognosis of patients with liver failure.Methods Eighty patients with liver failure were analyzed retrospectively.ICGR15 and relevant clinical data within 24 hours of diagnosis were analyzed.Meanwhile,the MELD score and King's College Hospital (KCH) were evaluated.All findings were tested for correlation with 3-month mortality.Quantitative data were analyzed with analysis of variance and Student's t-test.Count data were analyzed with chi-square test.Correlation analysis was performed with Pearson's coefficient test.Results Among 80 patients with liver failure,39 patients survived and 41 died.The mortality rate of all patients was 51.2％.The serum total bilirubin,creainine concentrations,ICGR15,MELD scores and patient number in accordance with KCH criteria of surviving patients were (288.0±109.1) μmol/L,(63.3±24.4) μmol/L,(48.1±10.2)％,20.6±4.4,and 6 cases,respectively,which were lower than those in dead patients [(340.7 ± 108.2) μmol/L,(98.8 ± 59.1) μmol/L,(60.2 ± 10.6) ％,26.9 ± 7.1 and 19 cases,respectively] (P =0.033,P＝ 0.001,P＝ 0.000,P＝ 0.000 and P =0.003,respectively).There was no significant difference of ICGR15 among four types of liver failure.A positive correlation was observed between ICGR15 and MELD score (r=0.289,P=0.009).The ICGR15-MELD model was created by subjecting ICGR15 and MELD scores to Logistic regression analysis.The following ICGR15-MELD model,Logit (P) =0.105 × ICGR15 + 0.178 × MELD score-9.734,was constructed by Logistic regression analysis.The area under the receiver operating characteristic (ROC) curve was 0.860 and the cut offpoint of 0.3 had sensitivity of 85.40％ and specificity of 74.40％.The area under the curve of the ICGR15-MELD model was significantly higher than those of ICGR15 (0.791),MELD score (0.770) and KCH criteria (0.655).Conclusions ICGR15 and MELD scores perform better than the KCH criteria in predicting the prognosis of liver failure.The ICGR15-MELD model is superior to ICGR15,MELD score,and KCH in predicting the short term prognosis of patients with liver failure.

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