Dissemination mechanism of blaKPC-2 among carbapenem-resistant Klebsiella pneumoniae strains / 中华微生物学和免疫学杂志

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is highly prevalent and poses a great health challenge due to the lack of effective treatments. Klebsiella pneumoniae carbapenemase-2 (KPC-2), encoded by blaKPC-2 gene, is one of the major contributors to carbapenem resistance in CRKP. In China and other Asian regions, Tn1721 and plasmid IncFⅡ are the main vectors for blaKPC-2 transfer between Kpn ST11 strains, which lack clustered regularly interspaced short palindromic repeats (CRISPR) and restriction-modification (R-M) systems. The structure of transposons has a significant impact on the transposition frequency of blaKPC-2, which may be related to the different transposition patterns of transposons. The prevalence advantage of blaKPC-2 in Kpn ST11 strains is highly associated with the immune deficiency in Kpn ST11. By acquiring a re-engineered CRISPR-Cas3 system via conjugation, the high-risk IncFⅡ plasmid can be successfully cleaved and ST11 CRKP can regain antibiotic sensitivity, which provides a promising approach for clinical treatment and prevention of CRKP.

Application of molecular diagnostic technology in detection of antimicrobial resistance / 中华检验医学杂志

Antimicrobial resistance is a serious problem for anti-infective treatment. Molecular technology can quickly, sensitively and accurately detect the mechanism of drug resistance of bacteria, improving the efficacy of anti-infection treatment and the level of infection control. The construction of quality assurance system is a guarantee for the effective application of molecular diagnostic technology in the detection of bacterial resistance. However, due to the complex mechanism of drug resistance, coupled with genetic mutations and other factors, there are problems such as false negatives, false positives, and inconsistency between mechanisms and phenotypes, there are certain restrictions on the application of molecular detection technology. With the development of molecular technology and deepening of people′s understanding of the drug resistance mechanism, the application of molecular diagnostic technology in the detection of bacterial resistance will be more widespread.

Antimicrobial resistance profile of clinical isolates in hospitals across China: report from the CHINET Surveillance Program, 2017 / 中国感染与化疗杂志

Objective To investigate the antimicrobial resistance profile of the clinical isolates collected from selected hospitals across China. Methods Twenty-nine general hospitals and five children's hospitals were involved in this program. Antimicrobial susceptibility testing was carried out according to a unified protocol using Kirby-Bauer method or automated systems. Results were interpreted according to CLSI 2017 breakpoints. Results A total of 190 610 clinical isolates were collected from January to December 2017, of which gram negative organisms accounted for 70.8％ (134 951/190 610) and gram positive cocci 29.2％ (55 649/190 610). The prevalence of methicillin-resistant strains was 35.3％ in S. aureus (MRSA) and 80.3％ in coagulase negative Staphylococcus (MRCNS) on average. MR strains showed much higher resistance rates to most of the other antimicrobial agents than MS strains. However, 91.6％ of MRSA strains were still susceptible to trimethoprim-sulfamethoxazole, while 86.2％ of MRCNS strains were susceptible to rifampin. No staphylococcal strains were found resistant to vancomycin. E. faecalis strains showed much lower resistance rates to most of the drugs tested (except chloramphenicol) than E. faecium. Vancomycin-resistant Enterococcus (VRE) was identified in both E. faecalis and E. faecium. The identified VRE strains were mainly vanA, vanB or vanM type based on phenotype or genotype. The proportion of PSSP or PRSP strains in the non-meningitis S.pneumoniae strains isolated from children decreased but the proportion of PISP strains increased when compared to the data of 2016. Enterobacteriaceae strains were still highly susceptible to carbapenems. Overall, less than 10％ of these strains (excluding Klebsiella spp.) were resistant to carbapenems. The prevalence of imipenem-resistant K. pneumoniae increased from 3.0％ in 2005 to 20.9％ in 2017, and meropenem-resistant K. pneumoniae increased from 2.9％ in 2005 to 24.0％ in 2017, more than 8-fold increase. About 66.7％ and 69.3％ of Acinetobacter (A. baumannii accounts for 91.5％) strains were resistant to imipenem and meropenem, respectively. Compared with the data of year 2016, P. aeruginosa strains showed decreasing resistance rate to carbapenems. Conclusions Bacterial resistance is still on the rise. It is necessary to strengthen hospital infection control and stewardship of antimicrobial agents. The communication between laboratorians and clinicians should be further improved in addition to surveillance of bacterial resistance.

Clinical retrospective analysis of Klebsiella pneumoniae bloodstream infection / 中华传染病杂志

Objective To explore the clinical characteristics,drug resistance and prognosis of Klebsiella pneumoniae bloodstream infection (KP-BSI),and to analyze the risk factors of death and drug resistance.Methods The clinical data of hospitalized patients with KP-BSI from April 2015 to April 2017 in Huashan Hospital were retrospectively analyzed.Continuous variables were compared using t test.Categorical variables were compared using x2 test or Fisher exact test.The independent risk factors for death were determined by logistic regression model.Results The majority of the 74 patients with KP-BSI were male (67.6％) and elderly patients (78.4％).Nosocomial infection occurred in 58 cases (78.4％) and a total of 24 (32.4％) cases died.The patients were widely distributed in various departments of the hospital.The first was the Department of Infectious Diseases (29.7％),followed by the intensive care unit (23.0％).The patients were often complicated with various underlying diseases and the most common was pulmonary infection (56.8％).There were 45 (60.8％) multiple drug resistance (MDR) strains and 29 (39.2％) Carbapenems resistant Klebsiella pneumoniae (CRKP) strains.There were significant differences of nosocomial infections (x2 =4.655,P =0.031),deep venous catheters (x2 =5.432,P-0.02),and invasive mechanical ventilation (x2 =7.630,P =0.006) between MDR and non-MDR patients.Deep venous catheters (x2 =5.923,P=0.015),invasive mechanical ventilation (x2 =16.845,P=0.000),other catheters (x2 =4.009,P=0.045) and surgery (x2 =3.910,P=0.048) were all significantly different between CRKP and non-CRKP patients.APACHE Ⅱ scores were performed in all patients.The average APACHE Ⅱ score was 8.74-±5.32 of the 50 cases (67.6％) in the survival group and that was 16.46 ± 6.62 of the 24 cases (32.4％) in the death group.The APACHE Ⅱ score in the survival group was significantly lower than that in the death group.The difference was statistically significant (t=5.091,P=0.000).APACHE Ⅱ ≥15 was the independent factor of death (B =-2.708,P=0.000).Conclusions The situation of drug-resistant KP-BSI is severe in the clinic.According to the clinical data,nosocomial infections,invasive mechanical ventilation and deep venous catheters may be the risk factors for MDR bloodstream infection.Deep venous catheters,invasive mechanical ventilation,other catheters and surgery may be the risk factors for bloodstream infection with CRKP.APACHE Ⅱ ≥15 is the independent risk factor for death.The evaluation of APACHE Ⅱ score may predict the prognosis of patients with bloodstream infection.

Differentiation of Pc and P2 variants in class 1 integron by high-resolution melting analysis / 中华检验医学杂志

Objective To develop a simple high-resolution melting ( HRM) analysis method for differentiation of Pc and P2 variants in class 1 integron.Methods DNA fragments containing Pc and P2 variants were amplified from plasmids pACW ( PcW ) and pACWP2 ( PcW-P2 ) respectively , then these purified PCR products and P 2 promoters were analyed full-length amplicon by HRM .Eight DNA fragments containing different Pc promoters were amplified and site-specific mutated from plasmids pACS ( PcS ) , pACH2 ( PcH2 ) , pACH1 ( PcH1 ) , pACW ( PcW ) , genomic DNA of Klebsiellar pneumonia HS07-68 (PcWTGN-10)and HS05-1792(PcH2TGN-10)respectively.The purified PCR products and eight Pc variants were characterized by HRM analyses of an unlabeled probe and full-length amplicon.This assay was applied to the differentiate Pc and P2 variants in 109 class 1 integrons from 95 urine clinical Escherichia coli isolates in Huashan Hospital during 2004 -2007.The differentiation results were compared with that determined by direct sequencing .Results P2 promoter with a significant higher melting temperature ( Tm ) can be identified by HRM analysis clearly .P2 promoters were identified in 2 class 1 integrons and consistent with direct sequencing results .Eight Pc variants were classified into three groups: PcS, PcSTGN-10 , PcW, PcWTGN-10, PcH1, PcH1TGN-10.Using direct HRM analysis.PcH2, PcH2TGN-10 were classified into four groups:PcS, PcH1, PcH2, PcW, PcSTGN-10 , PcH1TGN-10 , PcH2TGN-10 , PcWTGN-10 according to the melting curves of the unlabeled probe .Combined the HRM analyses of the whole amplicon and unlabeled probe , the eight Pc variants can be differentiated from each other .Five different Pc variants, PcS, PcW, PcH1, PcH2TGN-10 and PcWTGN-10 , were identified and consistent with direct sequencing results .Conclusions This developed a simple Pc and P 2 variants differentiation method via simultaneous HRM analyses of an unlabeled probe and full-length amplicon .This method is cost-effective and accurate , could be used in differentiation of Pc and P2 variants of class 1 integrons in clinical isolates .

Surveillance of antibiotic resistance among the bacterial strains isolated from hospitals in Shanghai: results of 2016 / 中国感染与化疗杂志

Objective To investigate the resistance profile of clinical bacterial isolates to antibiotics in Shanghai during 2016.Methods Antimicobial susceptibility testing was carried out for the clinical isolates collected from 47 hospitals according to a unified protocol using Kirby-Bauer method or automated systems.The participating hospitals included 28 tertiary hospitals and 19 secondary hospitals across Shanghai.Results were analyzed according to CLSI 2016 breakpoints.Results A total of 122 548 clinical isolates were collected,including 35 522 (29.0％) strains of gram positive cocci and 87 026 (71.0％) strains of gram negative bacilli.Overall,28.9％ of the isolates were from secondary hospitals and 71.1％ from tertiary hospitals.Gram positive and gram negative isolates accounted for 25.8％ and 74.2％ in secondary hospitals,30.3％ and 69.7％ in tertiary hospitals,respectively.The overall prevalence of MRSA in Staphylococcus aureus was 48.7％ and 77.2％ of MRCNS in coagulase-negative Staphylococcus.The average prevalence of MRSA and MRCNS was 55.9％ and 73.3％ in secondary hospitals,45.9％ and 78.6％ in tertiary hospitals.No strains were found resistant to vancomycin in Staphylococcus.About 77.4％ of the 1 111 strains of non-meningitis S.pneumoniae isolated from children were penicillin-susceptible (PSSP),13.2％ were penicillin-intermediate (PISP) and 9.4％ were penicillinresistant (PRSP).The prevalence of PSSP,PISE and PRSP was 97.8％,2.2％,and 0 in secondary hospitals,76.5％,13.7％,and 9.8％ in tertiary hospitals.Of the 285 strains isolated from adults,94.0％,4.2％ and 1.8％ were PSSP,P1SP and PRSP,respectively.The prevalence of PSSP,PISP and PRSP among the isolates from adults was 93.7％,5.3％,and 1.0％ in secondary hospitals,94.2％,3.7％,and 2.1％ in tertiary hospitals.Overall,37 strains of vacomycin-resistant E.feacium (14 from secondary hospitals and 23 from tertiary hospitals) and 25 strains of vacomycin-resistant E.feacalis (all from tertiary hospitals) were identified.PCR and sequencing analysis indicated that most of these resistant strains were vanA type.The overall prevalence of ESBLs-producing srains was 52.2％ in E.coli,30.9％ in Klebsiella pneumoniae and 29.8％ in Proteus mirabilis.Specifically,the corresponding prevalence of such strains was 55.1％,33.6％ and 34.0％ in secondary hospitals,51.0％,29.7％ and 28.0％ in tertiary hospitals,respectively.Enterobacteriaceae strains were still highly susceptible to carbapenem antibiotics.Overall,8.9％ and 9.1％ of the Enterobacteriaceae strains were resistant to imipenem and meropenem,respectively.The figure was 6.6％ and 7.1％ in secondary hospitals,9.9％ and 10.0％ in tertiary hospitals.Extensively drug-resistant strains were identified in A.baumannii,K.pneumoniae,P.aeruginosa,and E.coli,specifically,223,63,10,and 4 strains in secondary hospitals;224,201,22,and 9 strains in tertiary hospitals.Conclusions Antibicotic resistance is still very serious in the common clinical strains,which poses a critical threat to healthcare facilities.This issue should be taken seriously and effective infection control measures must be put in place.

Mechanisms of carbapenem and fosfomycin resistance in an Escherichia coli strain isolated from bloodstream infection / 中国感染与化疗杂志

Objective This study was designed to determine the mechanisms of carbapenem and fosfomycin resistance in an Escherichia coli strain isolated from bloodstream infection in Huashan Hospital,Shanghai in 2010 and the mode of transmission of resistance genes.Methods The Escherichia coli isolate was characterized by antibiotic susceptibility testing,multilocus sequence typing (MLST),molecular identification of resistance genes,plasmid typing and the resistant genetic environment analysis.Results It was found that the isolate was resistant to carbapenem,fosfomycin and produced extended-spectrum β-1actamases.MLST genotyping showed it belonged to ST46.The carbapenem-resistant gene blaKPC-2 and fosfomycin resistant genefosA3 co-located on the same conjugative plasmid (～ 70 kb).The β-lactamases gene blaTEM and blaCTX-M located on another conjugative plasmid (～ 150 kb).PCR mapping showed that blaKPC-2 gene located in the structure Tn1721-blaKPC-2-Tn3 andfosA3 gene located between two IS26 elements.Conclusions This Escherichia coli strain isolated from bloodstream infection carried multiple antibiotic resistant genes,including blaKPC-2,fosA3,blaTEM,and blaCTX-M.More attention should be paid to the mechanisms of antibiotic resistance and transmission of resistance genes in Escherichia coli isolates for better control of hospital infections.

Analysis of distribution characteristics of clinical isolates of nontuberculous mycobacteria in a general hospital / 中华传染病杂志

Objective To investigate the current prevalence of nontuberculous mycobacteria (NTM) in Shanghai,and to study the distribution characteristics of NTM clinical isolates,which may help to improve the diagnostic level of NTM and provide guidance for effective prevention and treatment of NTM infection.Methods Culture-positive isolates of clinical mycobacteria were collected from 2008 to 2013 in Huashan Hospital affiliated to Fudan University.All isolates were heat inactivated,and the genomic DNA was extracted and the species were identified by comprehensive comparative analysis of 16S rDNA,hsp65 and rpoB target genes sequencing.Results From January 2008 to December 2013,the overall mycobacterial culture-positive rate was 4.1 ％ (411/10 015).After excluding the repeated isolates,a total of 253 culture-positive mycobacteria isolates were collected for the species identification.By genes sequencing analysis,140 isolates were identified as mycobacterium tuberculosis complex (MTBc),102 NTM and 11 Nocardias,accounting for 55.3％,40.3％ and 4.4％,respectively.Positive rate of NTM isolates had an increasing trend from 25.0％ in 2008 to 42.7％ in 2013,reaching a highest rate of 54.9％ in 2012.In further analysis of 102 NTM isolates,16 species were identified.Among them,28 were M.abscesses,18 strains of M.marinum,17 strains of M.avium-intracellulare complex and 10 strains of M.fortuitum,accounted for 27.5％,17.6％,16.7％ and 9.8％,respectively.Conclusions Both of the isolation number and isolation rate of NTM in the general hospital are increasing.NTM related cases are also increasing in recent years,which mainly caused by M.abscess,M.marinum,M.aviumintracellulare complex and M.fortuitum.

CHINET surveillance of bacterial resistance across China: report of the results in 2016 / 中国感染与化疗杂志

Objective To investigate the susceptibility profile of clinical isolates collected from hospitals across China.Methods Twenty-six general hospitals and four children's hospitals were involved in this program.Antimicrobial susceptibility testing was carried out according to a unified protocol using Kirby-Bauer method or automated systems.Results were analyzed according to CLSI 2016 breakpoints.Results A total of 153 059 clinical isolates were collected from Junuary to December 2016,of which gram-negative organisms and gram-positive cocci accounted for 71.6％ and 28.4％,respectively.The overall prevalence of methicillin-resistant strains was 38.4％ in S.aureus (MRSA) and 77.6％ in coagulase negative staphylococcus (MRCNS),respectively.The resistance rates of methicillin-resistant strains to most of other antimicrobial agents were much higher than those of methicillin-susceptible strains.However,92.3％ of the MRSA strains were still sensitive to trimethoprim-sulfamethoxazole,while 86.5％ of the MRCNS strains were susceptible to rifampin.No staphylococcal strains were found resistant to vancomycin or teicoplanin.The resistance rates of E.faecalis strains to most drugs tested (except chloramphenicol) were much lower than those of E.faecium.A few strains of both species were resistant to vancomycin.Vancomycin resistant E.faecalis and E.faecium strains were mainly VanA,VanB or VanM type based on their phenotype or genotype.Regarding the non-meningitis S.pneumoniae strains,the prevalence of PSSP or PISP strains isolated from children was higher than that isolated in 2015,but the prevalence of PRSP strains decreased.However,the prevalence of PISP and PRSP strains isolated from adults was lower than that isolated in 2015.The prevalence of ESBLs-producing strains was 45.2％ in E.coli,25.2％ in Klebsiella spp.(K.pneumoniae and K.oxytoca) and 16.5％ in Proteus mirabilis isolates on average.ESBLs-producing Enterobacteriaceae strains were more resistant than non-ESBLs-producing strains in terms of antibiotic resistance rate.The strains of Enterobacteriaceae were still highly susceptible to carbapenems.Overall,less than 10％ of these strains were resistant to carbapenems.About 68.6％ and 71.4％ ofAcinetobacter spp.(A.baumannii accounts for 90.6％) strains were resistant to imipenem and meropenem,respectively.The prevalence of extensively-drug resistant strains in P.aeruginosa was higher than that in 2015.Conclusions Bacterial resistance to commonly used antibiotics is still on the rise.It is necessary to strengthen hospital infection control and management of clinical use of antimicrobial agents,and maintain good practice in surveillance of bacterial resistance.

Changing susceptibility ofKlebsiella strains in hospitals across China：data from the CHINET Antimicrobial Resistance Surveillance Program, 2005-2014 / 中国感染与化疗杂志

Objective To evaluate the changing pattern of antibiotic resistance inKlebsiella strains isolated from the patients in 19 hospitals across China based on the data from CHINET Antimicrobial Resistance Surveillance Program during the period from 2005 through 2014.Methods Kirby-Bauer disk diffusion and automated susceptibility testing methods were used to test the susceptibility ofKlebsiella isolates to the commonly used antibiotics. The results were interpreted according to the criteria of the Clinical and Laboratory Standards Institute (CLSI) Performance Standards for Antimicrobial Susceptibility Testing (CLSI-2014).Results A total of 61 406Klebsiella strains were identified between 2005 and 2014, includingK. pneumoniae (56 281 strains), K. oxytoca(4 779),Klebsiella pneumoniae subsp.Ozaenae (300) and otherKlebsiella species (46). Most (89.0%, 54 664/61 406) of theKlebsiella strains were isolated from inpatients, and 60.0% (36 835/61 406) were from respiratory tract speciems. About 16.7% (10 248/61 406) of the strains were isolated from pediatric patients aged 0-17 years and 83.3% (51 158/61 406) from adult patients. The prevalence ofKlebsiella spp. increased with time from 10.1% in 2005 to 14.3% in 2014. Based on the surveillance data during the 10-year period, we found a marked increase of resistance to imipenem (2.9% to 10.5%) and meropenem (2.8% to 13.4%) inKlebsiella spp. The prevalence of ESBLs-producing isolates inK. pneumoniae andK. oxytoca decreased from 39.0% in 2005 to 30.1% in 2014. The resistance to amikacin, ceftazidime, ciprolfoxacin, pipracillin-tazobactam and cefoperazone-sulbactam was on decline. The resistance rate to cefotaxime remained high about 49.5%. Carbapenem resistantance was identiifed in 5 796 (9.4%) of the isolates, including 5 492 strains ofK. pneumoniae and 280 strains ofK. oxytoca. Overall, 4 740 (7.8%) strains were identiifed as extensively-drug resistant (XDR), including 4 520 strains ofK. pneumoniae and 202 strains ofK. oxytoca. The carbapenem-resistant strains showed high (>60%) resistance rate to majority of the antimicrobial agents tested, but relatively low resistance to tigecycline (16.8%), amikacin (54.4%), and trimethoprim-sulfamethoxazole (55.5%).Conclusions During the 10-year period from 2005 through 2014, carbapenem resistance amongKlebsiella isolates has increased dramatically in the hospitals across China. The level of resistance to other antibiotics remains stable.

CHINET 2014 surveillance of bacterial resistance in China / 中国感染与化疗杂志

Objective To investigate the susceptibility and resistance of clinical isolates from hospitals in several regions of China .Methods Fifteen general hospitals and two children′s hospitals were involved in this program . Antimicrobial susceptibility testing was carried out according to a unified protocol using Kirby‐Bauer method or automated systems .Results were analyzed according to CLSI 2014 breakpoints .Results A total of 78 955 clinical isolates were collected from January to December 2014 ,of which gram negative organisms and gram positive cocci accounted for 72 .6% and 27 .4% ,respectively . Methicillin‐resistant strains in S .aureus(MRSA)and coagulase negative Staphylococcus(MRCNS)accounted for an average of 44 .6% and 83 .0 % ,respectively .The resistance rates of methicillin‐resistant strains to β‐lactams and other antimicrobial agents were much higher than those of methicillin‐susceptible strains .However ,92 .0% of MRSA strains were still susceptible to trimethoprim‐sulfamethoxazole ,while 85 .6% of MRCNS strains were susceptible to rifampin .No staphylococcal strains were found resistant to vancomycin ,teicoplanin or linezolid .In Enterococcus spp .,the resistance rates of E .f aecalis strains to most tested drugs (except chloramphenicol) were much lower than those of E . f aecium .Some strains of both species were resistant to vancomycin .Vancomycin resistant strains of E . f aecalis and E . f aecium were mainly V anA ,V anB or V anM type based on their phenotype or genotype .Regarding non‐meningitis S .pneumoniae strains ,the prevalence of penicillin‐susceptible S .pneumoniae strains isolated from both adults and children were higher than those isolated in 2013 ,but the prevalence of penicillin‐intermediate S . pneumoniae or penicillin‐resistant S . pneumoniae strains decreased . The prevalence of ESBLs producingstrainswas55.8% in E.coliand29.9% in Klebsiellaspp.(K.pneumoniaeand K.oxytoca)and24.0% in Proteus mirabilis isolates on average . ESBLs‐producing Enterobacteriaceae strains were more resistant than non‐ESBLs‐producing strains in terms of antibiotic resistance rates . The strains of Enterobacteriaceae were still highly susceptible to carbapenems .Overall less than 10 % of these strains were resistant to carbapenems . About 62 .4% and 66 .7% of Acinetobacter spp .(A .baumannii accounts for 93 .0 % ) strains were resistant to imipenem and meropenem ,respectively . Compared with the data of year 2013 ,extensively‐drug resistant strains in K . pneumoniae and A .baumannii increased . Conclusions The antibiotic resistance of clinical bacterial isolates is growing .The disseminated multi‐drug or pan‐drug resistant strains in a special region poses a serious threat to clinical practice and implies the importance of strengthening infection control .

The relationship between the personality and the mindfulness level of the nursing college students / 中国实用护理杂志

Objective To explore the prevalence of the personality and mindfulness level of the nursing college students and investigate their correlation.Methods 180 nursing college students were investigated by the scale of NEO and FFMQ.Results The scores of personality trait were in the moderate level.The correlation between the score of the adaptability items and the mindfulness was negatively correlated,the moral sense items,sociability items and agreeableness items and the mindfulness was positively correlated.Conclusions The personality of nursing college students is closely related with their mindfulness level.To perfect the personality of the nursing college students can improve their mindfulness level.

CHINET 2013 surveillance of bacterial resistance in China / 中国感染与化疗杂志

Objective To investigate the susceptibility and resistance of clinical isolates collected from hospitals in several regions of China . Methods Fourteen general hospitals and two children ’ s hospitals were involved in this program . Antimicrobial susceptibility testing was carried out according to a unified protocol using Kirby-Bauer method or automated Systems .Results were analyzed according to CLSI 2013 breakpoints .Results A total of 84 572 clinical isolates were collected from January to December 2013 ,of which gram negative organisms and gram positive cocci accounted for 73 .0% and 27 .0%respectively .Methicillin-resistant strains in S .aureus (MRSA) and coagulase negative Staphylococcus (MRCNS) accounted for an average of 45 .2% and 73 .5% respectively .The resistance rates of methicillin-resistant strains to β-lactams and other antimicrobial agents were much higher than those of methicillin-susceptible strains .However ,92 .2% of MRSA strains were still susceptible to trimethoprim-sulfamethoxazole while 87 .4% of MRCNS strains were susceptible to rifampin . No staphylococcal strains were found resistant to vancomycin ,teicoplanin or linezolid .In Enterococcus spp .,the resistance rates of E . f aecalis strains to most tested drugs (except chloramphenicol) were much lower than those of E . f aecium .Some strains of both species were resistant to vancomycin .Vancomycin-resistant strains of E . f aecalis and E . f aecium were mainly VanA type based on their phenotype .Regarding non-meningitis S . pneumoniae strains ,the prevalence of penicillin-susceptible S . pneumoniae and penicillin-intermediate S . pneumoniae strains isolated from both adults and children were lower than those isolated in 2012 ,but the prevalence of penicillin-resistant S .pneumoniae strains increased .The prevalence of ESBLs producing strains was 54 .0% in E .coli ,31 .8% in Klebsiella spp .(K .pneumoniae and K .oxytoca) and 16 .5% in Proteus mirabilis isolates on average . ESBLs-producing Enterobacteriaceae strains were more resistant than non-ESBLs-producing strains in terms of antibiotic resistance rates .The strains of Enterobacteriaceae were still highly susceptible to carbapenems .Overall less than 7 .0% of these strains were resistant to carbapenems .About 62 .8% and 59 .4% of Acinetobacter spp .(A .baumannii accounts for 89 .2% ) strains were resistant to imipenem and meropenem ,respectively .Compared with the data of year 2012 , extensively-drug resistant strains in K . pneumoniae and A . baumannii decreased .Conclusions The antibiotic resistance of clinical bacterial isolates is growing in 2013 .The disseminated multi-drug or pan-drug resistant strains in a special region poses a serious threat to clinical practice and implies the importance of strengthening infection control .

Preparation and primary application of antiserum to aminoacy1-3″-adenylyltransferase in integron / 中华检验医学杂志

ObjectiveTo prepare antiserum specific to aminoacy1-3″-adenylyltransferase [ AAD (3″) ],and to explore the application value of the prepared antiserum in detecting the expression levels of aadA2 gene that downstream of 8 different promoters (PcS,PcH2,PcH1,PcW,PcS-P2,PcH2-P2,PcH1P2 and PcW-P2 ) of variable regions in class 1 integron.MethodsaadA2 gene was amplified by polymerase chain reaction(PCR) and cloned into the expression plasmid pET19b.After inducing,the recombined aminoacy1-3″-adenylyltransferase[ AAD(3″)] with His-tag was expressed,purified and immunized rabbits to get anti- AAD(3″) specific serum.The prepared antiserum was used to detect the translation levels of aadA2 gene that downstream of different promoters of variable regions in class 1 integron by Western blotting (WB).Broth microdilution method was used to detect the minimal inhibitory concentrations (MIC) to streptomycin in Escherichia coli JM109 with aadA2 gene downstream of different promoters of variable regions.ResultsRecombined AAD (3″) expression plasmid pET19b-aadA2 was constructed successfully and was verified by sequence analysis.After transformed into E.coli BL21 ( DE3 ),a resoluble recombined AAD(3″) high expression strain was obtained.After fermentation,recombined AAD(3″) was purified and immunized rabbits.The anti- AAD(3″) specific serum was obtained with titer ＞ 1∶100 000.WB was used to detect the expression levels of AAD (3″),the translation product of aadA2 gene,that downstream of 8 different promoters of variable regions.The relative expression level of AAD (3″) that downstream of PcW was assumed to be 1,then the relative expression levels of AAD(3″),which all were detected 3 times independently,that downstream of PcS,PcH2,PcH1,PcS-P2,PcH2-P2,PcH1-P2 and PcW-P2 were 12.9±2.3,9.1±1.0,2.0±0.4,16.0±1.3,14.1 ±1.3,10.5±0.7 and 8.9 ±1.7 respective.Very different expression levels of AAD (3″) that downstream of different promoters of variable regions were obtained( F =32.421,P ＜ 0.01 ).The mean values of MIC,which all were detected 3 times independently,to streptomycin in E.coli JM109 with aadA2 gene downstream of PcS,PcH2,PcH1,PcW,PcS-P2,PcH2P2,PcH1-P2 and PcW-P2 were 256,256,64,128,32,128,4 and 64 mg/L respective.These results indicated the different expression levels of aadA2 gene that downstream of different promoters of variable regions can confer their host bacteria different resistance levels to streptomycin.Conclusions Resoluble recombined AAD(3″) is purified successfully and high titer anti- AAD(3″) specific antiserum is obtained from the immunized rabbits.This laid foundation for further investigation on the correlationship between the expressions of intI1 gene and the gene cassettes within variable regions.The expression levels of antibiotic gene cassettes that downstream of different promoters of variable regions are very different,so are the very different antibiotic resistance levels of the host bacteria.Therefore more attentions should be paid to the researches on the classification of promoters of variable regions when molecular epidemiology studies on the class 1 integrons in clinical isolates were conducted.

Determining the start codons of aadA2 gene in class 1 integron / 中华微生物学和免疫学杂志

ObjectiveTo determine whether aadA2 gene can be translated from the ATG triplet,which there was no plausible ribosome binding site preceding it,and synthetized a functional protein in class 1 integron.MethodsSite-specific mutagenesis was used to construct aadA2 gene cassette with different start codons,together with their upstreamed promoters of variable regions were cloned into plasmid pACYC184 respective.The constructed plasmids were then transfored into Escherichia coli JM109,Western blot was used to detect the translation products of aadA2 gene with different start codons.Broth microdilution method was used to detect the minimal inhibitory concentrations to streptomycin in Escherichia coli JM109 containing aadA2 gene with different start codons.ResultsaadA2 gene can initiate translation from both ATG and GTG triplets in aminoacyl -3-adenylyltransferase protein synthesis,though there was no plausible ribosome binding site preceding the ATG triplet.Besides GTG and ATG triplets,there was other start codon downstream of the GTG triplet in aadA2 gene.The translated products that initiated from the start codons that described above were all functional AAD(3) proteins that can be detected by anti- aminoacyl -3-adenylyltransferase polyclonal antisera in Western blot and conferred different resistance levels to streptomycin in E.coli.ConclusionWhen inserted as the first gene cassette in class 1 integron,aadA2 gene can initiate translation from ATG triplet and synthetized a functional protein,though there was no plausible ribosome binding site preceding it.This structural characterization of class 1 integron can initiate translation of the open reading frame harbored in gene cassette that integrated into class 1 integron,though there was no plausible RBS preceding the start codon.This make class 1 integron be more convenience to express the genes that capture from environment.

The study on epidemic characteristics of an outbreak of panresistance Klebsiella pneumoniae / 中华微生物学和免疫学杂志

Objective To understand the epidemic characteristics of an outbreak of panresistance Klebsiella pneumoniae occurred between 2006 and 2009 in a university hospital of Shanghai, China. Methods A total of 57 panresistance K. pneumoniae isolates were collected from August 2006 to December 2009.Antibiotic susceptibility of the isolates were determined by Kirby-Bauer disc diffusion method and microbroth dilution (MBD). ESBLs-producing initial screen test and phenotypic confirmatory test and carbapenemase-producing modified Hodge test ( MHT) were performed to detect the resistance phenotype of the isolates. Be-ta-lactamases were studied by IEF, PCR and the product sequencing. While conjugation assay were conducted to understand the transferability of these genes. The genetic relationship between isolates was established by ERIC-PCR and multilocus sequence typing (MLST). Except for the antibiotics recommended by CLSI guideline in the routine test, the other antibiotics were added to find out the effective drugs to treat the infection. Results All 57 isolates were highly resistant to all examined antibiotics. All isolates produced ESBLs and carbapenemase. IEF revealed that each isolate produced four beta-lactamases. All isolates carried blaKPC-2,blaCTX-M-14,blaSHV12,blaTEM-1,qnrB and aac(6') - I b-cr. Forty-four of the 57 (77.2% ) isolates were successful to transfer their resistance genes to E. coli recipient J53 by conjugation assay. By RAPD, all 57 isolates were grouped into two genotypes that were further identified as members of MUST types 423 and 11.Sequence types 423(ST423) only occurred before May 2008 and ST11 occurred (52 isolates) after May 2008. Most of isolates of the outbreak were ST11 (91. 2% ). A part of isolates were susceptive to added antibiotics. Conclusion The outbreak of panresistance K.pneumoniae was caused by those isolates which carried multiple resistant genes. There is a different ability of dissemination between different ST types K. pneumoniae isolate. It was necessary to add the antibiotics to find out the effective drugs to treat the infection.

Rapid identification and susceptibility testing of positive blood culture caused by gram negative bacteria / 中华微生物学和免疫学杂志

Objective To reduce the turnaround time for laboratory diagnosis of bacteremia, the feasibility of rapid identification and susceptibility testing using samples taken directly from positive blood culture bottles was evaluated. Methods The growth of microorganisms in blood culture bottles was screened by the BACTEC 9000 blood culture system. 65 positive blood culture bottles containing gram-negative bacteria were adopted to test. Culture fluid was injected into BD SST vacutainer and centrifuged to pellet blood cells. After collecting required McFarland units, they were cultured on Phoenix 100 NMIC/ID-4(identification-gram-negative bacteria and susceptibility testing) cards using 0.25 McF and 0.5 McF methods respectively. They were also evaluated by the standard method, involving subculture tests from positive blood culture bottles. Results 63 of 65 gram-negative bacteria (96. 9% ) were correctly identified with 0. 25 McF method. 59 of 65 gram-negative bacteria(90.8% ) were correctly identified with 0.5 McF method. For antimicrobial susceptibility testing, the 0.25 McF direct method had an agreement rate more than 94% , the 0.5 McF method was more than 85.7% and direct blood sample KB method was more than 93.8% compared to the standard method. But the overall minor error rate in susceptibility testing of direct blood sample KB method is higher than other methods. Conclusion Applying 0. 25 McF and 0. 5 McF rapid identification and susceptibility test was practical. During to possessing more prominent advantages, laboratory put the 0. 25 McF direct method into practice had a timely, remarkable significance.

Gene detection of vancomycin-resistant Enterococcus isolated from a teaching hospital in Shanghai / 中华检验医学杂志

Objective To investigate the prevalence of VRE in Huashan hospital of Shanghai from 2007 to 2009, and to examine the molecular characteristics of the VRE isolates.Methods A total of 890 non-repetive clinical isolates of Enterococcus were screened by the agar screening method ( ADSP method).Broth dilution susceptibility test was performed to determine the antimicrobial susceptibility of Enterococcus isolates to vancomycin and teicoplanin.The resistant genes and virulent genes of VRE isolates were investigated by PCR and sequencing methods.VRE isolates were classified by MLST and six isolates of VRE from 2007 to 2008 were analyzed by PFGE.Results Thirteen VRE isolates were identified by ADSP method and broth dilution susceptibility test. Six of them were resistant to vancomycin but sensitive to teicoplanin ( vancomycin MICs were from 64 to 256 μg/ml).The sequencing data of PCR products indicated these isolates might harbor a potential novel vancomycin resistant gene, which was different from the one reported in previous studies. The rest 7 isolates harbored vanA gene. The MICs of these isolates to vancomycin and teicoplanin were 32 - 64 μg/ml and 16 - 32 μg/ml, respectively.MLST results revealed 4 STs were identified in 13 VRE isolates.Eleven isolates belonged to clonal complexes(CC) 17.The positive rates of esp gene and hyl gene were 69.2% and 30.8%, respectively.Conclusions This study suggests that the most common VRE clone in Huashan Hospital was CC17.A potentially novel vancomycin resistance gene was identified, and further work needs to be done to investgate the function and the location of this novel gene.

Analysis of cefoxitin-sensitive, penicillin-resistant Staphylococcus tested by disk diffusion method / 中华检验医学杂志

Objective To evalue the ability of detecting the resistance of cefoxitin-sensitive,penicillin-resistant Staphylococcus by different methods and analyze the antibiotic susceptibility spectrum of coagulase-negative Staphylococcus which are non-mecA-mediated oxacillin resistance. Methods All the isolates were collected from Huashan hospital between 2007 and 2009. The isolates were recovered from various clinical sources, including respiratory tract, urine, secretion and sterile fluids samples. The oxacillin susceptibility of Staphylococcus aureus was determined by cefoxitin disk diffusion test, cefoxitin MIC test,oxacillin disk diffusion test and oxacillin MIC test Likewise, the oxacillin susceptibility of coagulasenegative Staphylococcus was determined by cefoxitin disk diffusion test and oxacillin MIC test. All the isolates with sensitive to cefoxitin were screened for the mec A gene by PCR Finally, the MIC of non-mecA-mediated oxacillin-resistant Staphylococcus were determined. Results Among 255 cefoxitin disk diffusion test sensitive and penicillin-resistant Staphylococcus aureus, 6 isolates were intermediated to oxacillin and 4 were resistant by oxacillin disk diffusion test, but all the isolates were sensitive by the cefoxitin disk diffusion test,cefoxitin MIC test and oxacillin MIC test. Among 75 cefoxitin disk diffusion test sensitive and penicillin-resistant coagulase-negative Staphylococcus, 16 isolates were resistant to oxacillin by oxacillin MIC method and 4 carried mecA gene. Among 12 non-mecA-mediated oxacillin-resistant Staphylococcus, the susceptible isolates of gentamicin is 10, clindamycin is 8, ciprofloxacin is 11, erythrornycin is 6, trimethoprim/sulfamethoxazo]e is 11 ,and cephalosporins, teicoplaninl, vancomycin, piperacillin/tazobactam, tetracycline are all 12. Conclusions The cefoxitin disk diffusion test can reliably predict mecA-mediated oxacillin resistant Staphylococcus aureus. It would be best to combine cefoxitin disk diffusion test and oxacillin MIC test to improve accuracy of detection of mecA-mediated oxacillin resistant coagulase-negative Staphylococcus.Furthermore, infections due to the non-mecA-mediated oxacillin resistant coagulase-negative Staphylococcus can be treated by penicillinase-stable penicillins, β-lactam/β-lactam inhibitor combinations, relevant cephems and carbapenems.

Applying high resolution melt analysis to discriminate VEB-3 hypotype of the clinical gram negative isolates / 中华微生物学和免疫学杂志

Objective To establish a new method, applying high resolution melt, to discriminate the VEB-3 hypotype from the clinical gram negative isolates. Methods From January to December 2003,292 consecutive and non-repetitive gram-negative bacteria producing VEB extended spectrum β-lactamase (ESBL) were collected. Extract the DNA of clinical gram negative isolates with phenol-chloroform. PCR was performed to amplify the VEB gene with the DNA being template. After that, we amplify the fragment of VEB gene containing the position 168. Then we detect the high resolution melt curve and analyze them. At last, we analyze the results of sequence and high resolution melt( HRM ). Results VEB-1 and VEB-3 gene are markedly different through HRM analysis. Conclusion It is accurately and quickly for us to identify the VEB-3 from other hypotype through the technology of HRM.