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Objective:To investigate the multilocus sequence typing feature of the virulence-associated genes of Staphylococcus aureus(S. aureus) separated from the clinical specimens of a multi-center cohort children in Guangzhou area. Methods:A total number of 412 Staphylococcus aureus strains isolated from 2 059 non-repeated fecal specimens of children by three groups′ researchers in Guangzhou Women and Children′s Medical Center from August 2018 to November 2018. While collecting specimens, patient clinical information is also properly collected and preserved. After extracting the DNA of the strain, the virulence-associated genes were detected by polymerase chain reaction (PCR), including the staphylococcal enterotoxin (SE) genes ( sea, seb, sec, sed, see) and the Panton-Valentine leucocidin-encoding gene ( pvl).The multi-locus sequence typing (MLST) method was performed to reveal the MLST feature of these genes and the statistical difference were examined by the the χ 2 test. Results:Among the 412 isolates of S. aureus, 256 strains (256/412, 62.1%) contains at least one SE gene. Among the enterotoxin gens, the sec (125/412, 30.3%), seb(98/412, 23.8%)and sea (66/412, 16.0%)genes were the three most prevalent members of SEs. The frequency of pvl gene in Staphylococcus aureus was 18.7%(77/412).Among them, the frequency of Staphylococcus aureus sea gene isolated from patients with gastroenteritis (58/319, 18.2%) was significantly higher than that from the non-gastroenteritis group (8/93, 8.6%)(χ2=4.912, P=0.027). The frequency of Staphylococcus aureus pvl gene isolated from the patients with pneumonia (8/21, 38.1%) was greater than that from the non-pneumonia group (6/47, 12.8%)(χ2=4.252, P=0.039). In addition, the virulence-associated gene of S. aureus was closely related to the specific ST type, 82.4% (28/34) of ST6 carried sea gene, all ST338 and ST59 carried seb gene, 96% (48/50) ST45 carried sec gene, and the pvl gene carrying rate of ST338 was 5/5. Conclusions:The SEA toxin produced by ST6 Staphylococcus aureus may be closely related to the diagnosis of gastroenteritis in children. The frequency of pvl virulence gene in Staphylococcus aureus in children with community-acquired pneumonia was higher than that in the non-pneumonia group, and closely related to the CC59.
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Objective To explore the sample type and drug resistance characteristics of Streptococcus pneu-monia(Spn)isolated from pediatric patients in Guangzhou district,and their age distribution to offer instruc-tions for prevention and clinical treatment.Methods Spn isolates were cultured and identified according to the national standard procedure for clinical laboratory operation,followed by analysis of sample type and age dis-tribution of pediatric patients with positive isolates of Spn in Guangzhou Women and Children′s Medical Cen-ter from 2013 Jan 1st to 2015 Dec 31st,drug resistance status was determined by MIC test.Results Totally, 1 243 strains of Spn were isolated,which were mainly from pediatric patients under 1 year old(42.80%).Spn isolates were mainly isolated from respiratory tract(72.81%),ear secretions(15.37%),blood(5.63%),cere-brospinal fluid(3.06%)and hydrothorax(2.01%).For all Spn isolates,the resistance rate to erythromycin, tetracycline and sulfamethoxazole was especially high as 94.93%,85.76%,73.53% respectively,with relative high resistance to penicillin G(24.70%),amoxicillin(39.59%),ceftriaxone(24.05%),meropenem(22.85%) and cefotaxime(19.89%),low resistance to quinolone antibiotics(<10.00%),and no resistance to vancomycin and linezolid.Conclusion The major age group of children with Spn infection is infants under one year old in Guangzhou,clinicians should be serious about the high resistant rate of Spn to erythromycin,tetracycline and sulfamethoxazole,the significantly increased resistant rate to penicillin,amoxicillin and ceftriaxone.Clinicians should choose antibiotics rationally according to the characteristics of drug sensitivity for better treatment.
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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.
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Objective To investigate the infection and epidemiological characteristics of group A rotavirus (RV-A)and adenovirus in children with diarrhea in Guangzhou. Methods The colloidal gold technique was used to detect RV.A and adenovirus antigen in 2,171 stool samples from children with diarrhea in Guangzhou Women and Children′s Medical Center from January to December 2015,and the data were statistically analyzed. Results Among the 2,171 patients,the positive rate of RV-A infection was 17.96%and that of adenovirus infection 8.66%, and the co-infection rate of both virus was 3.45%. The positive rates between different genders were not significantly different(P > 0.05);the infectious time peak of RV-A was January(40.78%),followed by December(39.24%) and February(32.61%)and that of adenovirus infection was July(15.89%)and May(15.79%). The infectious peak of RV-A and adenovirus was December(7.29%),followed by January(7.01%). The peak age of infection ranged from 1y to 3y. Conclusion RV-A and adenovirus are the main pathogens of children diarrhea ,and the onset of virus infection has obvious seasonal change.
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Objective To understand the molecular epidemiology of penicillin resistance Streptococcus pneumonia (PNSP) isolated from children in Guangzhou area to provide the experimental basis for clinical prevention and control of Streptococcus pneumonia infectious diseases.Methods Specific primers were designed according to Genebank,penicillin binding protein(PBP) genes PBP1A,PBP1B,PBP2A,PBP2B,PBP2X,PBP3 were amplified by PCR.The sequencing analysis was performed.The PCR products were digested by Hinf I,and the restriction fragment length polymorphism (RFLP) was analyzed.Results DNA of PNSP was successfully extracted,the PCR results showed that in 50 strains of PNSP,the positive rates of bacterial strains containing PBP1A,PBP1B,PBP2A,PBP2B,PBP2X and PBP3 were 48.9%,64.4%,71.1%,31.1%,40.0% and 31.1% respectively.The sequencing showed that their homologies with known sequences in GenBank were 99%,98%,100%,97%,95% and 100% respectively.Using RFLP in Hinf I showed that PBP1A,PBP1B,PBP2A and PBP3 only had one kind of genotype,PBP2B and PBP2X had two kinds of genotypes,the positive rates were 71.4%,28.6%,66.7% and 33.3% respectively.Conclusion The gene distribution of PNSP strains among children in Guangzhou is dominated by PBP2A,PBP1B and PBP1A,there are two subtypes in PBP2B,PBP2X when digested by Hinf I,in which the predominant subtype >65%.
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Objective To study genotyping and molecular epidemiology distribution of GBS pathogenic strains of GBS positive pregnant women in Guangzhou,for GBS pathogenic strains of rapid molecular diagnosis and epidemiological surveillance pro-vide certain theoretical basis and method.Methods In the Guangzhou area,used multi stage stratified sampling method col-lecting GBS positive pregnant women’s reproductive tract specimens from January to December 2015,drug sensitivity quality control standard strains:Streptococcus pneumoniae (ATCC49619)and Staphylococcus aureus (ATCC25923),took culture of bacterial,strain,identification,DNA extraction,PCR,gene detection method,through the relevant software for data analy-sis,analyzed GBS strains of gene and molecular epidemiology.Results In the study,collected 2 812 samples of secretions,af-ter identification of strains isolated from 178 strains of pathogenic GBS strains,the detection rate was 6.33%.GBS patho-genic strains to linezolid vancomycin,penicillin,nitrfurantion and other antimicrobial drug resistance rate was 0,GBS parho-genic strains to ampicillin,ciprfloxacin moxifloxacin and levofloxacintesistant parts,the restance rates were 1.1%,16.9%, 18.0% and 22.5%,but GBS pathogenic strains to erythromycin,clindamycin tetracydine antibiotics showed a high resistance rate,the resistance rates were 50.6%,47.8%(of which 20 cases of erythromycin induced clindamycin resistance accouted for 23.5%)and 73.0%.Among them,65 strains of GBS detected the mreA gene,56 strains of GBS detected the ermB gene,36 strains of GBS detected the mefA gene,28 strains of GBS detected the mefE gene,5 strains of GBS detected the ermA gene, ermC gene was not detected in the gene.Among them,carried five multidrug resistance gene of 3 strains (1.6 9%)and 4 kinds of resistant gene carried with 15 strains (8.43%),carried three resistance genes of 19 strains (10.67%),2 kinds of resistant gene carrying a 25 strains (14.04%),carried the resistance gene of 5 strains (2.81%),did not carry resistance gene of 1 strain (0.56%).The nucleotide sequences of the five drug resistance genes were 100%,and no gene mutation oc-curred.Conclusion The main GBS disease resistant gene was mreA,ermA,ermB,mrfA,mefE and its nucleotide sequence homology was 100%.The clinical need to strengthen the detection of resistant gene and molecular level and guide clinical more scientific and rational drug use.
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Objective To clone and express Staphylococcus aureus drug resistance adenylyltransferase gene in E .coli BL21 ,and to make the foundation for its function research .Methods Primers were designed on the basis of adenylyltransferase gene in gen‐bank ,PCR was used to amplify adenylyltransferase gene using Staphylococcus aureus genomic DNA as template .The obtained PCR production was attatched with pGEX‐4t‐1(+ ) plasmid ,and transformed into E .coli BL21 (DE3) .The recombinant plasmid was di‐gested by double enzyme digestion and identified by gene sequence .The recombinant protein was induced to expression by IPTG and identified by Western‐blotting .Results Using Staphylococcus aureus genome as a template ,the target fragment about 800 bp was successful amplified .After enzyme‐cutting and DNA‐sequencing ,the target fragment showed that the ORF begin with ATG ,end with TAG ,783 bp in length ,the predicted isoelectric point and molecular weight were 7 .75 and 29 × 103 ,and it was homology 99%homology with the reported sequence gene in genbank .SDS‐PAGE and Western‐blot showed the molecular weight of recombinant fusion protein was about 55 × 103 .Conclusion Adenylyltransferase gene of Staphylococcus aureus was successfully cloned and ex‐pressed in E .coli as a fusion protein ,which makes the foundation for the research of its function .
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Objective To perform the amplification ,sequencing and prokaryotic expression of APH (3′′)‐Ⅰ and AAC (2′)‐Ⅰgenes from the clinically isolated gzch810 strain(SM gzch810)of Stenotrophomonas maltophilia to provide the basic materials for the next step functional test .Methods The SM gzch810 genome chromosome was extracted ,the APH (3′′)‐Ⅰ ,AAC (2′)‐Ⅰ whole genes were amplified by PCR and sequenced after being cloned into pMD18‐T vector .The recombination were subcloned into pGEX‐4T‐1 vector and the expression of the recombinant APH (3′′)‐Ⅰ and AAC (2′)‐Ⅰ were analyzed by SDS‐PAGE .Results The 800bp and 550bp DNA fragments of APH(3′′)‐Ⅰ ,AAC(2′)‐Ⅰ gene were amplified from SM gzch810 by PCR and sequenced ;the sequence comparison analysis showed that DNA and amino acid sequence identities of APH (3′′)‐Ⅰand AAC (2′)‐Ⅰ genes with other strains were 91% and 95% respectively .The sequence of APH (3′′)‐Ⅰand AAC(2′)‐Ⅰ of SM gzch810 were submitted to GenBank(accession number :HQ315852 and HQ315853);two major protein bands corresponding to the expected recombinant GST‐TP fusion proteins (56 × 103 and 46 × 103 respectively) were identified by SDS‐PAGE .Conclusion APH(3′′)‐Ⅰand AAC(2′)‐Ⅰgene of SM gzch810 are successfully cloned and expressed ,which lays a good foundation for further detecting corresponding antibi‐otic resistance and functional evaluation of above two kinds of recombinant E .coli .
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Objective To explore the main pathogenic bacteria and antibiotic resistance patterns in children with bacterial diar‐rhea from Guangzhou region .Methods Regular bacterial culture of stool samples from children with suspicious bacterial diarrhea was performed to isolate the pathogen during 2011 to 2012 ,followed by the analysis of its composition and serum type ,ward distri‐bution characteristics and drug resistance to 12 antimicrobacterial drugs .Results 416 strains of pathogenic bacteria were isolated from diarrhea children during 2011-2012 ,in which salmonella ,enteropathogenic E .coli ,Campylobacter jejuni and Candida albicans isolates accounted for 53 .61% ,37 .98% ,5 .29% and 1 .68% respectively .Drug resistance rate of the main strains to 12 antimicrobi‐al agents was 85 .25% to ampicillin ,54 .28% to compound sulfamethoxazole ,44 .70% to cefotaxime ,42 .53% to ceftriaxone , 40 .66% to chloramphenicol ,23 .55% to ceftazidime ,23 .36% to aztreonam ,14 .88% to ciprofloxacin ,8 .07% to cefepime ,7 .99% to cefperazone/sulbactam ,7 .42% to piperacillin/tazobactam respectively ,and no resistance to imipenem was detected .Conclusion The pathogenic bacteria causing diarrhea mainly includes salmonella ,pathogenic e .coli ,campylobacter jejuni in children from guang‐zhou region ,the top five sensitive antimicrobial reagents for the main strains includes imipenem ,piperacillin/tazobactam ,cefpera‐zone/sulbactam ,cefepime and ciprofloxacin .
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Objective To conduct the amplification,cloning,bioinformatics analysis,prokaryotic expression and purification of enterovirus 71 VP1 gene segment and to initially confirm the biological activity of the recombinant expression product.Methods A pair of specific primers was designed according to GenBank EV71 sequence,viral RNA as a template was extracted from the throat swab specimens in the EV71 patients.EV71 VP1 gene was amplified by RT-PCR.After enzyme digestion,the expression vector pET28a was inserted.The prokaryotic expression vector of pET28a-EV71 VP1 was constructed.Then the E.coli DH5a transforma-tion was performed.IPTG was adopted for induction expression.The expression results were analyzed by using SDS-PAGE and Western blot.The bioinformatics analysis of the sequenced results was performed by the software.Expressed protein was purified and the plates were coated,ELISA was used to test the VP1 specific IgG antibody in serum samples of EV71 positive and COX A16-positive patients.Results The BLAST alignment showed that the homology of the objective gene EV71 VP1 was 99% com-pared with other strains(JQ766207.1)in GenBank.EV71 VP1 protein was about 32×103 ,which mainly existed in the form of in-clusion body.The bioinformatics analysis showed that EV71 VP1 protein was a hydrophilic protein,without transmembrane region and N-terminal signal peptide sequence,the tertiary structure existed.The ELISA results showed that the specific IgG OD value in EV71-positive patients was(2.425±0.521),OD value in COX A16 positive patients was(1.205 ±0.314),the normal control OD value was(0.353±0.128).The sensitivity and specificity of EV71 VP1 protein detection were 84% and 88% respectively.Conclu-sion The pET28a-EV71 VP1 expression vector is successfully constructed;the preliminary analysis on the serum of the infected patients by ELISA shows that the obtained objective protein has higher sensitivity and specificity,which is initially confirmed to have biological activity and can be further used for the related study on EV71 diagnosis and vaccine.
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Objective To evaluate the therapeutic effects of capsule Huoluoshugan on schistosomal hepatocirrhosis.Methods Ninety-six patients with schistosomal hepatocirrhosis were selected and divided randomly into three groups:Huoluoshugan group,interferon-?(IFN-?)group and control group,and treated with capsule Huoluoshugan,IFN-? and routine therapy,respectively.The course of treatment was 6 months in all groups.Before and after the treatment,the symptoms,physical signs,indexes of liver function and hepatic fibrosis,and changes of B-type ultrasonic images were detected for all the patients.Results The indexes of liver function,hepatic fibrosis and B-type ultrasonic image of the patients in Huoluoshugan group improved obviously compared with the control group after the treatment(P0.05).In addition,there was no obvious adverse reactions during the treatment in the patients of Huoluoshugan group.Conclusion Capsule Huoluoshugan can reduce the indexes of hepatic fibrosis and improve the liver function in patients with schistosomal hepatocirrhosis.