A rapid and sensitive method of loop-mediated isothermal amplification(LAMP) was established to detect Pseudomonas aeruginosa(P.aeruginosa).Three pairs of LAMP primers(inner,outer and ring primers) were designed according to the gbca gene of P.aeruginosa.Since adding hydroxy naphthol blue(HNB) to the reaction system, a positive reaction was indicated by a colorchange before and after the reaction,and was verified by agarose gellectrophoresis.Both LAMP and PCR were applied to detect clinical specimens, the sensitivity and specificity of the detection method were evaluated,and were compared with those of conventional PCR.A LAMP method for detecting P.aeruginosa was successfully established.The LAMP method showed specificity for P.aeruginosa without other bacteria amplification.The established LAMP method in this study enables rapid,sensitive and specific detection of P.aeruginosa,and can be applied for grass roots and small scale laboratories as well as field surveillance.

Objective To research the resistant characteristics, genotype and prevalence of vancomycin-resistant Enterococci( VRE) . Methods K-B disc diffusion method was used to determine the susceptibility, minimum inhibi-tory concentration ( MIC) for vancomycin of VRE was detected by E-test method;VRE was then subjected to PCR for resistance related genes;6 strains sequencing results of PCR product were contrastively analyzed the amino acid sequence by BLAST. Results 6 VREFm strains were found from 193 strains enterococci;6 VREFm strains were completely resistant to high unit gentamicin, ampicillin, ciprofloxacin, teicoplanin,but were sensitive to linezolid and macrodantin,MIC for vancomycin was more than 256 mg/L;Genotypes were vanA;Amino acids in vanA gene have changed with Asn83→Asp( AAC→GAC) . Conclusion VRE in our hospital is mostly multi-drug resistant , which bringS difficulty in clinical treatment for its infection. So the hospital should strengthen the prevention and monitoring, to prevent the spread of VRE strains in the hospital and popular.

Objective To investigate acquired carbapenemases and prevalence of carbapenem- resistant gram-negative bacill.Methods The antimicrobial susceptibility was determined by agar dilution method.Metallo-B-lactamase(MBLs)were screened by EDTA-disk synergy tesL The encoding genes of MBLs were amplified by PCR followed by sequencing.Strain homology Was investigated by pulsed-field gel electronphoresis(PFGE).Results In 141 carbapenem-resistant Pseudomonas aeruginosa(P.aeruginosa), there were three resistant patterns which were imipenem(IMP)-resistant+meropenem(MEM)-resistant (66.7%),IMP-resistant+MEM-sensitive(32.6%),and IMP-resistant+MEM-sensitive(0.7%).AⅡthe carbapenem-resistan Acinetobacter baumannii(A.baumannii),Acinetobacter lwoffi(A.lwoffi),Citrobactor freundii(C.freundii),Klebsielta pneumoniac(K.pneumoniac)and Serratia were resistant to imipenem and meropenem.Four strains of 141 P.aeruginosa were positive by EDTA-disk synergy test,and they produced VIM-2-type Metallo-B-1actamase.Of 34 carbapenem-resistan A.baumannii,30 strains produced OXA-tllrpe.Among them, OXA23, OXA24 and OXA66 accounted for 79.4%,38.2% and 67.6%,respectively.And 22 of 34 strains(64.7%)produced multiple OXA-carbapenemases.All 7 strains A.lwoffi produced OXA-23-type carbapenemases.A11 11 strains C.freundii,5 strains k pneumoniac and 1 strains Serratia produced KPC-2-type carbapencmases.And 6 of 11 strains C.freundii produced new subtype IMP-8.Of 15 PFGE type in 34 strains A.baumannii,14 strains belonged to A-type,7 strains belonged to B-type.Seven A.lwoffi strains distilbuted in difierent PFGE type.Four strains of P.aeruginosa producing VIM-2.type Metallo-8-lactamase did not have the same PFGE type.Eleven C.freundii strains had the same PFGE type.Five k pneumoniae strains had the sanle PFGE type.Conclusions Drug resistance to 12 common antibiotics in carbapenem-resistant gram-negative bacill was higher than non-carbapenem-resistant gram-negative.The former produced many kinds of carbaponemases and the strains prducing carbapenemases were prevalent in the C.freundii, A.baumanii, and k pneumoniae.

Objective To investigate the distribution and antibiotic resistance of the pathogens isolated from blood culture . Methods Identification and antimicrobial susceptibility testing were performed with MicroScan WalkAway 96 PLUS or VITEK 2 compact .WHONET 5 .6 software was used for analysis of the susceptibility data according to CLSI 2013 breakpoints .IBM SPSS 20 .0 was employed to compare the resistance rates between groups . Results Gram-positive bacteria , gram-negative bacteria and fungi accounted for 57 .8% ,36 .0% and 6 .2% of the 503 nonduplicate isolates ,respectively .The most common isolates included coagulase-negative Staphylococcus , Escherichia coli , Enterococcus spp ., Staphylococcus aureus and K lebsiella pneumoniae . The prevalence of methicillin-resistant S . aureus (MRSA ) and coagulase-negative Staphylococcus (MRSCN) was 32 .3% and 71 .7% respectively .The coagulase-negative Staphylococcus isolates from ICU patients showed higher resistance rates to many antibiotics than those non-ICU strains (P<0 .05) .E .coli and K .pneumoniae strains showed high percentage of resistance to cephalosporins , but relatively low resistance to piperacillin-tazobactam , imipenem and amikacin .A .baumannii isolates were highly resistant to most antimicrobial agents . Candida species were less resistant to antifungal agents .Conclusions The pathogens isolated from blood culture are diverse .The resistance profile is quite different among various pathogens .The distribution and antibiotic resistance should be actively monitored for the pathogens isolated from blood culture in order to facilitate the rational use of antimicrobial agents .

Objective To determine the risk factors of nosocomial infections caused by Enterococcus,and to develop effective protective measures.Methods A retrospective study was conducted among 193 patients who had been infected by Enterococcus from Jan.2011 to Dec.2011 in the First Affiliated Hospital of Anhui Medical University.Clinical data were collected and related risk factors for enterococcal infection were screened.Results The risk factors for enterococcal infection included biliary tract surgery (OR=1.264,95％CI:1.141-1.401,P =0.000),drainage tube intubation (OR=1.619,95％CI:0.301-4.190,P=0.010),urinary tract intubation (OR=2.001,95％CI:1.739-3.803,P 0.000),uses of third generation cephalosporin (OR=2.085,95％CI:1.181-3.682,P=0.011) andcarbapenems (OR=1.473,95％CI:1.060-2.023,P =0.024).Furthermore,biliary tract surgery (OR=4.861,95％CI:3.029-7.802,P=0.000) and urinary tract intubation (OR=2.737,95％CI:1.764-4.246,P=0.002) were risk factors for both Enterococcus faecalis and Enterococcus faecium infection,while drainage tube intubation (CR =1.861,95％CI:1.702 1.911,P=0.038),uses of the third generation cephalosporin (OR=1.122,95％CI:1.022-1.224,P=0.016) and carbapenems (OR=3.051,95％CI:1.185-7.855,P=0.021) were independent risk factors for Enterococcus faecium infection.Conclusions Surveillance

Objective To investigate the distribution and drug resistance of pathogens isolated from urinary tract infections.Methods A total of 6 262 midstream urine samples were collected from patients in the First Alfiliated Hospital of Anhui Medical University during April 2012 and March 2013.MicroScan WalkAway 96 Plus or Vitek 2 Compact system was applied in bacteria identification and drug sensitivity test.WHONET 5.6 was adopted to analyze drug resistance,and IBM SPSS 20.0 was applied to compare resistance rates between isolates from outpatients and hospitalized patients.Results A total of 1 426 strains were isolated,in which 370 strains were gram-positive coccus (25.9％),942 strains were gram-negative bacilli (66.1％) and 114 strains were fungi (8.0％).Escherichia coli and Klebsiella pneumoniae,Enterococcus faecium and Enterococcus faecalis were the top two among gram-negative bacilli and grampositive coccus,respectively.The prevalence of extended-spectrum β-lactamases (ESBLs)-producing Escherichia coli,Klebsiella pneumoniae and Proteus mirabilis were 60.5％,51.0％ and 30.3％,respectively; About 73.3％ of Staphylococcus aureus strains and 86.7％ of coagulase-negative Staphylococcus strains were methicillin-resistant.Candida albicans and Candida glabrata were the two most prevalent fungi in urinary tract infections,and they were sensitive to most antifungal agents.Conclusion Gram-negative bacilli,especially Escherichia coli are the most prevalent pathogen in urinary tract infections,and strains are highly resistant to most antibacterial agents,suggesting that antimicrobial resistance monitoring system is needed.

Objective To investigate the loss of outer membrane protein D2 (OprD2) gene in imipenem-resistant Pseudomonas aeruginosa.Methods The minimal inhibitory concentrations (MIC) of 10 antimicrobial agents against 80 strains of imipenem-resistant clinical isolates were determined by agar dilution method.PCR was used to detect OprD2 gene.Results PCR results showed that 44 of the 80 isolates of imipenem-resistant P.aeruginosa were positive for OprD2 gene.There was significant difference (P

Objective To study the relationship between efflux pump MexAB‐OprM and carbapenem resistance of Pseudomonas aerginosa strains .Methods The minimum inhibitory concentrations of imipenem and meropenem were determined by agar dilution method for 75 strains of P .aerginosa in the absence or presence of MC207110 to screen the phenotypes of active efflux pump .Reverse transcriptase‐polymerase chain reaction (RT‐PCR) method was used to determine the mRNA expression level of mexA which encodes the membrane fusion protein in active efflux pump MexAB‐OprM and the reference (housekeeping) gene rpoD .PCR method was used to amplify the regulatory genes mexR ,nalC ,and nalD of active efflux pump MexAB‐OprM in the strains overexpressing the efflux pump . The PCR products were subject to DNA sequencing and BLAST analysis . Results Of the 75 P .aeruginosa strains ,13 (17 .3% ) were positive for efflux pump MexAB‐OprM .Overexpression of the efflux pump was identified in 10 of the 13 strains and associated with positive regulatory genes mexR ,nalC and nalD .A Gly71→Glu mutation in nalC was found in 9 strains ,and a Ser209→Arg mutation in nalC was identified in 8 strains .Only one strain had a Thr158→Ile mutation in nalD .Eight strains had mutation in mexR .Conclusions Overexpression of multidrug efflux pump MexAB‐OprM plays an important role in carbapenem resistance of P .aeruginosa .High level expression of MexAB‐OprM is related to the mutations of its regulatory genes .

Objective To investigate the distribution and the antibiotic resistance profile of clinical isolates in the First Affiliate Hospital of Anhui Medical University during 2015.Methods Antimicrobial susceptibility testing was carried out according to a unified protocol using Kirby-Bauer method and automated systems.Results A total of 5 524 nonduplicate clinical isolates were collected during 2015,including gram-negative bacteria (3 882,70.3 ％),gram-positive bacteria (1 642,29.7 ％).The prevalence of methicillin-resistant isolates in Staphylococcus aureus (MRSA) and coagulase-negative Staphylococcus (MRCNS) was 57.6％ and 83.0 ％,respectively.All staphylococcal isolates were susceptible to vancomycin and linezolid.E.faecalis and E.faecium accounted for 46.1％ and 46.8 ％ of enterococcal isolates.Some E.faecalis and E.faecium strains were nonsusceptible to vancomycin or linezolid.The prevalence of extended-spectrum lactamases (ESBLs) positive strains was 62.0 ％ in E.coli,32.2 ％ in Klebsiella and 18.8 ％ in Proteus mirabilis.Enterobacteriaceae strains were still highly susceptible to carbapenem antibiotics,evidenced by lower resistance rate of Enterobacteriaceae strains to ertapenem,imipenetn and meropenem (a11＜22 ％).Conclusions It seems that antibiotic resistance still poses a serious threat to clinical antimicrobial therapy.More attention should be paid to resistance surveillance and rational use of antibiotics.

Objective To investigate the profile and antibiotic resistance of bacteria in patients with ascites infection in intensive care unit (ICU) patients in order to provide a reference for rational clinical use of antibiotics. Methods A retrospective analysis was conducted. The bacteria isolated from ascetic fluid patients admitted from January 1st, 2004 to October 31st, 2015 to ICU of the First Affiliated Hospital of Anhui Medical University were identified, and their susceptibility to antibiotics was analyzed. Patients, who were admitted from January 1st, 2004 to December 31st, 2009 were assigned to group A, and patients admitted afterwards were assigned to group B. Results A total of 637 specimens of ascetic fluid were examined, with 185 positive culture (29.0%) during the 12 years, and 203 strains of bacteria were found. Among them 126 strains (62.1%) of gram-negative bacteria (G-), 54 (26.6%) of gram-positive bacteria (G+) and 23 (11.3%) strains of fungi were found. Compared the result of group B with that of group A, the proportion of G- bacteria was increased [71.2% (99/139) vs. 44.2% (27/64)], and that of G+ decreased [17.3% (24/139) vs. 46.9% (30/64)] in group B. The difference was statistically significant (χ2 = 20.34, P = 0.001). The main pathogenic bacteria were G-, and Enterobacteriaceae was the most common pathogenic bacteria in intra-abdominal infection of ICU patients. The isolation rate of Escherichia coli and Klebsiella pneumoniae(35.7%, 10.3%) ranked in the first and third in G- bacteria, respectively. The resistant rate of Escherichia coli against penicillin and third generation cephalosporin were > 95.0% and > 73.3%, and it showed a sensitive rate of 70% to β-lactam/inhibitor, amikacin and minocycline, and a higher sensitivity to carbapenems and tigecycline (11.1%, 0). Forty-eight strains of non-fermentation bacteria were found with a rate of 23.7%. The positive rates of Acinetobacter baumannii in groups A and B were 7.8% (5/64) and 23.7% (33/139), respectively, and they ranked first among non-fermentation bacteria. Twenty strains (62.5%) multidrug-resistant Acinetobacter baumannii were found. Acinetobacter baumannii showed a resistance rate of 84.6% to cefoperazone/sulbactam, 35.3% to minocycline, and 53.3% to tigecycline. Candida albicans was the most commonly isolated fungus in intra-abdominal infections (87.5%). No strains resistant to common antifungal drugs were isolated. Conclusions G- bacteria was the main pathogen in intra-abdominal infection in patients with ascites. Non-fermenters showed an increasing trend of producing infection, and the proportion of multidrug-resistant Acinetobacter baumannii infection increased year by year, and more attention should be taken by attending doctors.