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ABSTRACT:To understand the status of contamination and antimicrobial resistance of Staphylococcus aureus (S .aureus) isolated from raw cow milk ,600 samples were collected from five different provinces in China .After isolation and identification of S .aureus from these samples ,157 S .aureus isolates were obtained .Furthermore ,their resistant phenotype and resistant gene were analyzed by MIC detection ,as well as nine drug‐resistance genes determination and analysis .The result revealed that most of the S .aureus isolates contained multiple antibiotic resistant genes ,and all of the isolates were resistant to penicillin . Of 98 .73% isolates were resistant to amoxicillin .Conversely ,all of the isolates were sensitive to enrofloxacin and vancomycin . Moreover ,there were 29 MRSA positive strains among which 22 strains carrying femA gene .In conclusion ,much more atten‐tion should be paid to the drug‐resistance capabilities of field S .aureus isolates in raw cow milk ,and further monitoring of drug resistant S .aureus strains in milk should also be strengthened so as to ensure milk safety .
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Staphylococcus aureus is one of the major causes of clinical infections and increasing mortality due to multi-drug resistance. In this study, eight drug-resistant genes, beta-lactamase, metallo-beta-lactamase, vanB, mecA, norA, qacA, qacB and qacC of S. aureus strain Mu50 (vancomycin resistant) were studied to predict the evolutionary conserved functional site residues in their protein sequences. It was found that in beta-lactamase, Tyr, Gly, Thr, Asn and in metallo-beta-lactamase, Thr, His, Gly, Leu, Arg and Asp residues were highly conserved. In vanB, Gly, His and Asp residues were highly conserved. Whereas in mecA, His, Val, Phe, Gln, Lys and in norA, Ser, Leu and Ala residues showed conservedness at moderate level. In the multi-drug efflux pump (corresponding to qacA, qacB and qacC), Gly residue was found to be highly conserved. The homology clustering of target proteins through SCI-PHY algorithm and homologues identified through PSI-BLAST were compared to identify the degree of conservation of functional residues. The phylogenetic motifs identified using homologues of target proteins were validated through domain search to locate their site and functionality in the protein sequences. Interactome analysis was performed to understand the possible mode of interaction of target proteins with their functional partners.
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DNA Barcoding, Taxonomic/methods , Drug Resistance, Multiple, Bacterial/genetics , Staphylococcus aureus/genetics , beta-Lactamases/genetics , Protein Structure, TertiaryABSTRACT
@#BACKGROUND: The virulent factors ofEscherichia coli (E.coli) play an important role in the process of pathopoiesis. The study aimed to compare drug-resistant genes and virulence genes between extended spectrum β-lactamases (ESBLs)-producingE.coli and non-ESBLs-producing E.coli to provide a reference for physicians in management of hospital infection. METHODS: From October 2010 to August 2011, 96 drug-resistant strains ofE.coli isolated were colected from the specimens in Qingdao Municipal Hospital, Qingdao, China. These bacteria strains were divided into a ESBLs-producing group and a non-ESBLs-producing group. Drug sensitivity tests were performed using the Kirby-Bauer (K-B) method. Disinfectant gene, qacEΔ1-sull and 8 virulence genes (CNF2, hlyA, eaeA, VT1, est, bfpA, elt, and CNF1) were tested by polymerase chain reaction (PCR). RESULTS: Among the 96E.coli isolates, the ESBLs-producingE.coli comprised 46 (47.9％) strains and the non-ESBLs-producingE.coli consisted of 50 (52.1％) strains. The detection rates of multiple drug-resistant strain, qacEΔ1-sull, CNF2, hlyA, eaeA,VT1, est, bfpA, elt, and CNF1 in 46 ESBLs-producingE.coli isolates were 89.1％, 76.1％, 6.5％, 69.6％, 69.6％, 89.1％, 10.9％, 26.1％, 8.7％, and 19.6％, respectively. In the non-ESBLs-producingE.coli strains, the positive rates of multiple drug-resistant strain, qacEΔ1-sull, CNF2, hlyA, eaeA, VT1, est, bfpA, elt, and CNF1 were 62.0％, 80.0％, 16.0％, 28.0％, 64.0％, 38.0％, 6.0％, 34.0％, 10.0％, and 24.0％, respectively. The difference in the detection rates of multiple drug-resistant strain, hlyA and VT1 between the ESBLs-producingE.coli strains and the non-ESBLs-producingE.coli strains was statistically significant (P<0.05). CONCLUSION: The positive rate of multiple drug-resistant strains is higher in the ESBLs-producing strains than in the non-ESBLs-producing strains. The expression of some virulence genes hlyA and VT1 varies between the ESBLs-producing strains and the non-ESBLs-producing strains. Increased awareness of clinicians and enhanced testing by laboratories are required to reduce treatment failures and prevent the spread of multiple drug-resistant strains.
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ObjectiveTo analyze the homology of Citrobacter freundii strains and detect multidrug resistant genes.MethodsThe minimum inhibition concentration was tested with standard agar dilution method and the the homology of C.freundii strains was measured with pulsed field gel electrophoresis (PFGE) and the PCR was used to perform the drug resistant genes such as β-lactamase,disappearing of outer membrane channel protein.ResultsThe four strains ( No.1,2,4,5 ) showed nineteen banding patterns with PFGE.PCR experiment showed that there were 2 strains (No.1,4) with blaCTX-M,3 strains (No.2,3,5 ) with blaDHA,1 strains(No.3) with blaACT/MIR,and 4 strains(No.1,2,4,5) with blaKPC-2.PCR analysis comfirmed that No.2 and No.4 disappearing of OmpF and No.3 disappearing both of OmpC and OmpF.The expression levels of the chromosomal ampC gene of No.3 isolates was 106.7 times higher than the negative isolates.ConclusionThere is high homology in carbapenem-resistant isolates and the mechanism of drug resistance is complex including blaKPC,AmpC,ESBLs,disappearing of outer membrane channel protein.The spreading of drug resistance result from above genes.
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Objective To study the structures of the plasmids of Klebsiella pneumoniae KF3 at the genome metagenome level througth with whole plasmid DNA sequencing, to analyze the functional genes carried by plasmid and to identify the correlation of resistance and pathogenicity between the plasmids and the host strains. Methods The alkaline lysis method was used to extract plasmids. We constructed the small insert pUC18 library and the large insert Forsmid library, sequenced and used the Phred / Phrap / Consed package to assemble these sequences and gained a complete sequence. The open reading frame(ORFs) were predicted by the Glimmer software and annotated, analyzed the functions of these genes. Results We successfully constructed the pUC18 library and the Fosmid libraries for the plasmid DNA and obtained three circular double-stranded DNA plasmids: pKF3-70 (69 477 bp), pKF3-90 (91 327 bp) and pKF3-147 ( 147 416 bp). There were drug resistant genes, conjugative transfer genes and mobile DNA elements identified on three plasmids. Conclusion The three plasmids of KF3 could be transferred among different strains. It would lead to the dissemination of the resistant genes.
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OBJECTIVE To investigate the antibiotics resistance of Escherichia coli(ECO) and distrubtion of aminoglycoside-modifying enzymes and 16S RNA methylase genes in ICU.METHODS The samples of 20 ECO isolates were collected from Dec 2007 to Jun 2008 of patients in ICU.To determine the sensitivity to the 30 antibacterials K-B method was used and aminoglycoside-modifying enzymes and 16S RNA methylase genes were analyzed by polymerase chain reaction(PCR).RESULTS In among the 20 ECO isolates,8 strains carried aac(3)-Ⅱ(40%),3 carried aac(6′)-Ⅰb(15%) and ant(3″)-Ⅰ(15%),1 be found aph(3′)-Ⅰ(10%)and 13 be found aadA4/5 aminoglycoside-modifying enzymes genes,no strain carried 16S RNA methylase genes.CONCLUSIONS aac(3)-Ⅱ、aac(6′)-Ⅰb,ant(3″)-Ⅰ,aph(3′)-Ⅰ and aadA4/5 aminoglycoside-modifying enzymes genes exist in ECO widely,they should be the main cause inducing the high rate of drug-resistance to aminoglycosides.
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OBJECTIVE To investigate the antibiotic resistance of multi-resistant Acinetobacter baumannii(ABA) and distribution of aminoglycoside-modifying enzymes and 16S rRNA methylase genes in ICU in Yinzhou People′s Hospital in Ningbo. METHODS The samples of 20 ABA isolates were collected from Oct 2007 to Jul 2008 in ICU.K-B method was used to determine the sensitivity to 32 antibacterials and the aminoglycoside-modifying enzymes and 16S rRNA methylase genes were analyzed by polymerase chain reaction(PCR). RESULTS From 20 ABA isolates,8 strains carried aminoglycoside-modifying enzymes genes aac(3)-Ⅰ,aac(3)-Ⅱ,aac(6′)-Ⅰb,and ant(3″)-Ⅰ,their positive rate was 10%,15%,30% and 25%,respectively;no strain carried 16S rRNA methylase genes. CONCLUSIONS The antibiotics resistance of A.baumannii is very serious in Yinzhou People′s Hospital in Ningbo.Aminoglycoside-modifying enzymes genes aac(3)-Ⅰ,aac(3)-Ⅱ,aac(6′)-Ⅰb and ant(3″)-Ⅰ exist in multi-resistant A.baumannii widely.They would be the main causes of high drug-resistantce to aminoglycosides.
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OBJECTIVE To investigate the prevalence of drug-resistant genes in seventeen strains of all-resistant Acinetobacter baumannii.METHODS Microdilute tests were performed to detect the susceptibility of 17 A.baumannii strains to 16 kinds of antimicrobial agents and antimicrobial-resistant genes were detected by PCR methods.RESULTS Seventeen A.baumannii strains showed all-drug resistance.Genes of TEM-1,OXA-23,OXA-27,gyrA and AmpC were detected in all 17 strains of A.baumannii.The positive rates of aacC31 and PER-1 genes were 11.8% and 52.9%,respectively.CONCLUSIONS A.baumannii with multi-resistant genes of our hospital carries TEM-1,OXA-23,OXA-27,gyrA,AmpC,aacC1 and PER-1.
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OBJECTIVE To study the molecular mechanism of the drug-resistance of Gram-negative bacilli to the third generation of cephalosporins.METHODS MICs of 13 ?-lactams to the eleven Gram-negative bacilli clinical isolates were detected with the standard agar dilution technique.K-B disc confirmatory method was conducted to determine the ESBLs phenotype of the eleven isolates.The ESBLs encoding genes were analyzed by using PCR.RESULTS The eleven isolates were all resistant to the third generation of cephalosporins(MIC≥64 ?g/ml).Disk confirmatory test showed that 10 isolates produced ESBLs.The hydrolytic activity of the ESBLs from the 10 isolates to cefoperazone and cefamandole was very high.However,the hydrolytic activity of the ESBLs from the 10 isolates to ceftazidime was very low.CONCLUSIONS The enzyme activities and the genes of extended-spectrum ?-lactamases from 10 Gram-negative bacilli clinical isolates are preliminarily analyzed.These results provide the basis for further study on the molecular mechanism of the drug-resistence of Gram-negative bacilli.
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OBJECTIVE To study the epidemiology of drug-resistant and antiseptic-resistant genes of(productive)(enzyme) in meticillin-resistant coagulase negative Staphylococcus(MRCNS).METHODS mecA Gene of the(drug-)resistant ?-lactam,aac(6′)/aph(2″) and aph(3′)-Ⅲ genes of aminoglycoside-modifying enzyme(AME) and(ermA/B/C) genes of erythromycin methyltransferase were detected by polymerase chain reaction(PCR) in 40 MRCNS strains.RESULTS From them there were 39 strains with mecA gene,32 strains with aac(6′)/aph(2″) gene,15 strains with aph(3′) -Ⅲ gene,30 strains with ermA/B/C gene,2 strains with tetM and 6 strains with qacA/B gene.There were 26 strains(65.0%) simultaneously with mecA,aac(6′)/aph(2″)and(or) aph(3′)-Ⅲ and ermA/B/C genes in MRCNS.CONCLUSIONS The drug-resistant rate is higher in MRCNS.There are more than half tested strains simultaneously with 3 to 4 drug-resistant genes.
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OBJECTIVE To investigate the condition of drug-resistant genes in MRSA and MSSA. METHODS The drug-resistant genes mecA,ermA/B/C,aac(6′)/aph(2″),aph(3′)-Ⅲ,ant(4′,4″) and tetM of MRSA and MSSA were detected by polymerase chain reaction(PCR). RESULTS The 5 kinds of drug-resistant genes,such as mecA,ermA/B/C,aac(6′)/aph(2″),(aph(3′)-Ⅲ) and tetM were positive in MRSA. CONCLUSIONS MRSA is a multi-resistant pathogen.
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OBJECTIVE To investigate Staphylococcus aureus(SAU)and coagulase-negative Staphylococcus(CNS)with continuously isolated virulence gene and drug-resistant genes status.METHODS We collected 100 SAU strains and 27 CNS strains,virulence gene(pvl)and drug-resistant genes(mecA,aac(6')/aph(2″),aph(3')-Ⅲ,ant(4',4″),ermA/B/C,msrA,tetM and qacA/B)were detected by PCR.RESULTS The positive rates of pvl,mecA,aac(6')/aph(2″),aph(3')-Ⅲ,ant(4',4″),ermA/B/C,msrA,tetM and qacA/B in MRSA were 11.6%,100.0%,94.2%,69.2%,2.9%,85.5%,85.5% and 46.4%.The positive rates of pvl,mecA,aac(6')/aph(2″),aph(3')-Ⅲ,ant(4',4″),ermA/B/C,msrA,tetM and qacA/B in MSSA were 74.2%,0%,9.7%,3.2%,0%,51.6%,9.7% and 0%,respectively.CONCLUSIONS There are very high positive percentages of mecA,aac(6')/aph(2″),aph(3')-Ⅲ,ermA/B/C,msrA,tetM and qacA/B genes in MRSA,and very high positive percentages of pvl and ermA/B/C genes in MSSA.