Objective To investigate the alternations in gene/amino acid sequence of penicillin-binding protein (PBP)2x from clinical isolates of Streptococcus pneumoniae, and to find the reasons for the rapid surge of penicillin and cefotaxime nonsusceptibility among pneumococcal isolates from Shenyang. Methods Thirty-four strains of Streptococcus pneumoniae were collected from January 2006 to February 2007. The antibiotics susceptibility of these strains was detected. PCR amplification and direct sequencing of pbp2x genes were performed. The sequence variations of PBP genes of the penicillin nonsusceptible Streptococcus pneumoniae(PNSP) in this region were studied by BLAST analysis. Results Two prominent substitutions were common to 12 PNSP isolates for which the MIC of penicillin resistance and cefotaxime were at least 0.5 mg/L, which included the replacement of Thr338→Ala in the first conservative motif STMK and Leu546→Val adjacent to third conservative motif KSG. The importance of the exchange of His394→Leu was identified in one PNSP isolate 15. The remarkable finding in this study was Met342→Ile following the first conservative motif STMK. pbp2x sequences of eight PRSP isolates shared Lys501-Glu505-Thr507 substitutions which might be served as a unique marker for PRSP in this region. Novel gene and amino acid sequence variants in 17 isolate were identified in this study, and these sequences have been deposited in the GenBank database and assigned accession No. EU044831, EU089706-EU089709, EU106881-EU106884 and EU124672. Conclusion It is likely that the emergence of penicillin and cefotaxime nonsusceptible Streptococcus pneumoniae in Shenyang might be associated with novel gene sequence variants.

Objective To detect the encoding gene of efflux pump and two-component system, and investigate the effect of efflux inhibition on the multiresistance of Acinetobacter baumarmii. Methods PCR was used to detect the adeB, adeR and aries gene. Agar dilution was used to determine the minimum inhibitory concentrations(MIC) of ciprofloxacin, cefotaxime, amikacin and imipenem of 50 multiresistance Acinetobacter baumannii, with or without 25 μg/ml reserpine. Results 94%, 96% and 92% of 50 muhiresistance of Acinetobacter baumannii were detected for adeB, adeR and aries gene,respectively. At least four fold decrease of MIC was observed in 49, 50, 50 and 46 isolates for ciprofioxacin, cefotaxime, amikacin and imipenem, respectively. Conclusion The multiresistance of Acinetobacter baumannii is related to the effect of the efflux system.

Objective To investigate the alternations in gene/amino acid sequence of penicillin-binding protein (PBP) 2b from clinical isolates of penicillin-nonsusceptible Streptococcus pneumonia(PNSP) in this region.Methods 24 strains of Streptococcus pneumonia were collected from January to December 2006.The antibiotics susceptibility of these strains was detected.PCR amplification and direct sequencing of pbp2b genes were performed.The sequence variations of PBP genes of the PNSP in this region were studied with sequence BLAST analysis.Results Three prominent substitutions were common tO 13 PNSP isolates with minimal inhibitory concentration(MIC) at least 0.1 mg/L.These included the replacement of Thr445→Ala following the conservative motif SSN,Glu475→Gly and Thr488→Ala/Ser.The exchange of Glu332→Gly was identified in 12 PNSP isolates of which the MIC was at least 0.25 mg/L.Seven penicillin resistant Streptococcus pneumonia (PRSP) isolates (MIC≥3 mg/L)shared the amino acid substitution Ala618→Gly adiacent to third conserved (KTG) motif and the PBP2b sequences of seven PRSP isolates were classified within Back's group Ⅱ and were very similar to those of the Korean J77 isolate.Novel gene and amino acid sequence variants in isolate 14,15,8,11 and 24 was identified in this study and these gene sequences have been deposited in the GenBank database and assigned accession no.EU035970,EU056919,EU056920,EU056921 and EU106886.Conclusion Analysis of pbp2b genes revealed highly similar patterns of nucleotide and amino acid sequence variation among most resistant isolates.while penicillin intermediate Streptococcus pneumonia might be associated with novel gene sequence variants.

Objective To determine the possible genetic background and the source of our hospital's 43 clinical isolates of multidrug-resistant Acinetobacter baumannii, and the category of gene cassettes in type 1 integrons of all strains.Methods Restriction enzyme Apa I was chosed for all strains in pulsed-field gel electrophoresis (PFGE) methods.Multilocus sequence typing ( MLST) was used to compare the allelic profiles of all the strains. PCR method was used for amplify the integrons of all strains. Results PFGE results showed that 43 strains were divided into four types. A-type and B-type were divided into 4 and 2 subtypes, respectively. The MLST results showed the existing of three allelic profiles; 1-3-3-2-2-7-3, 1-3-3-2-2-11-3, and 1-3-3-2-2-14-3.B-type and D-type of PFGE have the same allelic profile(1-3-3-2-2-11-3).A-type strains were detected mainly in ICU, and in burn unit only found B- and D-type.The same integron was detected in 62.8% of the strains.The constituent ratio of A1,A2,A3,A4,B1,B2,C and D-type was 40.7% , 18.5% , 7.4% , 3.7% , 14.8% , 3.7% , 3.7% and 7.4% , respectively.Conclusions The coexistence of multiple cloning system in this region was proved by the PFGE and MLST, and the same clone can evolve to different subtypes when stimulated by different environmental conditions; and the different carrying-situationt of the same integron in strains prove the possibility of the change during the evolution of resistance mechanisms.

Objective To investigate the antibiotic resistance and resistance genes of carbapenem-resistant Enterobacteriaceaes (CRE) isolated from 5 hospitals in Northeast China.Methods This study collected 85 CRE isolates during January 2013 to June 2015 from five hospitals in Northeast China.Drug sensitivities of 14 antimicrobial agents were determined by the broth microdilution method.The phenotypes of carbapenemases were screened by modified Hodge test and EDTA test respectively.The genotypes of carbapenemases and other extended spectrum β-lactamases (ESBL) were detected by PCR gene amplification and DNA sequencing method.Using the PCR result as gold standard, the performances of other two carbapenemase detection methods were evaluated.Results Among the 85 CRE strains collected in this study, Klebsiella pneumoniae was the most frequently isolated species (61/85,71.8%).The results of antimicrobial agent sensitivity showed that the 85 CRE strains had resistance rate of cephalosporin and β-lactams/enzyme inhibitor (piperacillin-tazobactam) over 80.0%.The resistance rate of carbapenem was high, with ertapenem 100.0% (85/85), meropenem 65.9% (56/85), imipenem 71.8% (61/85).There were 36 isolates resistant to both meropenem and imipenem.For fluoroquinolones, the resistance rates of levofloxacin and ciprofloxacin were 72.9% (62/85) and 65.9% (56/85), respectively.The resistance rate to fosfomycin and amikacin were 65.0% (55/85) and 54.1% (46/85), respectively.The resistance rate of colistin (21.2%, 18/85) and tigecycline (20.5%, 17/85) were low.Forty-nine strains were modified Hodge test positive and 12 strains were EDTA test positive.By PCR gene amplification and DNA sequencing method, 64 strains carried carbapenemase-encoding genes, of which KPC-2 was the main type (53/85, 62.4%), followed by IMP-4 (10/85, 11.8%), NDM-5 (7/85, 8.2%) and NDM-6 (1/85, 1.2%).At the same time, 85 CRE isolates had the ESBL gene detection and 47 isolates were CTX-M type ESBLs (47/85, 55.3%), with no TEM or SHV type.Conclusions Klebsiella pneumoniae is the majority of CRE strains from 5 large hospitals in Northeastern China.The CRE strains are resistant to most of antimicrobials.Most carbapenemases-producing isolates have the KPC-2 type.Nearly half of the carbapenemase-producing strains also carry ESBL genes, which makes the resistance mechanisms more complicated.

Objective To describe the clinical characteristics of hypervirulent Klebsiella pneumoniae (hvKP) infection.To analyze the antibiotic susceptibility of hvKP to provide the empiric antibiotic options.To investigate capsule serotype and sequence type (ST) of hvKP and their correlation with clinical profiles.Methods hvKP was defined as bacteria isolated from patients with community-acquired pyogenic liver abscess (CA-PLA) with co-infection sites outside liver or a bloodstream infection in a host without underlying biliary tract diseases.Patients with CA-PLA hospitalized in the First Hospital of China Medical University were retrospectively analyzed from January 2011 to December 2017.Antibiotic susceptibility was detected by automatic bacterial identification and antibiotic susceptibility analysis system in vitro.Polymerase chain reaction method and gene sequencing were used to detect the main capsule serotype and ST.Results A total of 140 cases with hvKP infection were enrolled.The co-infections outside liver abscess included 98 bloodstream infections,53 pneumonia,11 perianal abscess,10 urinary system infections,3 subphrenic abscess,3 endophthalmitis,2 spleen abscess,and other miscellaneous infections including 1 peritonitis,1 skin and soft tissue infection,1 myelitis,1 colitis,1 psoas major abscess and 1 myocardial abscess.Among the 140 cases,106 presented with single co-infection site,32 with 2 sites,and 2 with 3 sites.HvKP manifested high antibiotic susceptibility up to 80％ for most commonly used antibiotics.Capsule serotyping of 4,3 revived isolates indicated that K1 serotype accounted for 53.49％ (23/43),K2 34.88 (15/43),K54 2.33％ (1/43),K57 2.33％ (1/43),and other serotypes 6.98％(3/43).There was no significant distribution among K1,K2,K54,and K57 of hvKP capsule serotypes in patients with or without diabetes mellitus (P＞0.05).Multilocus sequence typing (MLST) suggested that ST23 and ST65 were predominant accounting for 39.53％ (17/4.3) and 25.58％ (11/4.3) respectively.No serotype or ST predominance was seen in any of the clinical infections.Conclusion HvKP is related to a wide spectrum of infectious diseases,including multiple extrahepatic sites and bloodstream infections besides CA-PLA with high antibiotic susceptibility.K1 and K2 are the predominant capsule serotypes,and ST 23 and ST65 are the predominant sequence types.

Objective To retrospectively analyze the clinical distribution and changes in antimicrobial resistance profiles of Staphylococcus aureus (S. aureus). Methods We collected clinical specimens of S. aureus from The First Hospital of China Medical University. The Vitek-2 and BD Phoenix 100 were performed for bacterial identification and drug sensitivity tests,and WHONET 5.6 was used to analyze the data. Results From 2007 to 2016,there were 3 377 unrepeatable strains of S. aureus,including 1 705 that were methicillin resistant S. aureus (MRSA). The isolation rate of S. aureus was 9.4 ％ and of these,50.5 ％ were MRSA. There were 776 S. aureus specimens from outpatients or the emergency department,including 16.8 ％ MRSA,and 2 011 S. aureus from inpatient departments,including 60.2 ％ MRSA. The main sources of specimens were sputum (41.8 ％),pus (17.9 ％),and body secretions (17.5 ％). The average resistance rates of MRSA for erythromycin,ofloxacin,ciprofloxacin,gentamycin,and tetracycline were higher than 75.0 ％. The average resistance rate of methicillin sensitive S. aureus (MSSA) for erythromycin was up to 76.8 ％,and for tetracycline,gentamycin,ciprofloxacin,and ofloxacin,were less than 25.0 ％. In 10 years,the average resistance rates of MRSA and MSSA for 11 kinds of common antibiotics had no obvious change. Conclusion The constituent rate of MRSA was high in The First Hospital of China Medical University,especially from the areas that were not sterile,suggesting that clinicians should pay attention to the identification of infection and sources for MRSA,which were from such areas. Hospital infection control should be focused on at the same time,in order to reduce the incidence of MRSA.

Objective To investigate the homology of carbapenem-resistant Klebsiella pneumoniae (CR-KPN) isolates by multilocus sequence typing (MLST) method,and profile CR-KPN infections in terms of MLST sequence types (STs).Methods A total of 61 CR-KPN isolates were collected in five hospitals from Liaoning Province from January 2013 to June 2015.The MICs of 14 antimicrobial agents against these isolates were determined by broth microdilution method.The genotypes of carbapenemases were analyzed by PCR and DNA sequencing techniques.The homology of CR-KPN isolates were analyzed by MLST method.The clinical data of patients with CR-KPN infection were reviewed to characterize CR-KPN infections.Results A total of 18 STs were identified among the 61 CR-KPN strains according to MLST data.More than 50 ％ of the isolates belonged to ST11 (53.3 ％).ST11 strains showed higher resistance rate to carbapenems and higher prevalence of KPC-2 type carbapenemase.Univariate analysis indicated that more ST 11-infected patients were treated in ICU and with mechanical ventilation than non-ST11 CR-KPN-infected patients (P＜0.05).ST2033,ST2135,ST2193,ST2194,ST2195 and ST2196 were the STs firstly registered in the world.The eBURST analysis showed that ST2193,ST2194,ST2195 and ST 11 were closely related.Clinical data indicated that the prevalent CR-KPN strains during the same period in the same hospital usually belonged to the same ST clone.Conclusions MLST of CR-KPN showed 18 sequence types,of which ST11 was the predominant type.Clinical data indicated that the prevalent CR-KPN strains during the same period in the same hospital usually belonged to the same ST clone.This suggests the potential of local CR-KPN outbreak.The ICU patients and those receiving mechanical ventilation may be prone to CRKPN (especially ST11) infection.Such patients should be managed appropriately.

Objective:To investigate the epidemiological characteristics of community acquired pyogenic liver abscess (CA-PLA) as a reference for its early identification, early diagnosis and rational antibacterial treatment.Methods:A single center retrospective study was carried out in patients with CA-PLA hospitalized in First Hospital of China Medical University from January 2011 to December 2017.The symptoms, signs and treatment results were concluded. The underlying diseases and onset symptoms of the cases were grouped by year, and the change trend of the disease characteristics was analyzed. The etiology results were grouped according to whether the patients had underlying diseases of biliary tract, and the etiology characteristics were analyzed. Statistical analysis was performed by using chi-square test.Results:A total of 1 063 CA-PLA cases were included in this study. The analysis on underlying diseases grouped by year showed that the number of patients admitted to the hospital increased annually, and the percentage of patients with underlying hepatobiliary diseases decreased from 17.3% (19/110) in 2011 to 7.3% (14/191) in 2017, the difference was statistically significant ( χ2=13.648, P=0.034), while that of patients with diabetes mellitus kept high at 31.6% to 46.5% in the past seven years without increasing trend. There were 274 patients (25.8%) with extrahepatic manifestations. Totally 445 cases were microbiologically diagnosed, among which single Klebsiella pneumoniae infection was found in 371 cases (83.4%). Klebsiella pneumoniae was the leading pathogen in patients without underlying hepatobiliary diseases (91.6%, 362/395), in contrast to 18.0%(9/50) in patients with underlying hepatobiliary diseases. The other pathogens were Escherichia Coli (32.0%, 16/50) and mixed infection (18.0%, 9/50). The susceptibility rate to second generation and above cephalosporins of clinically defined hypervirulent Klebsiella pneumoniae was ≥97.5%, and that to carbapenems was 100.0%. Most patients had good prognosis, and 1 049 cases were cure or improvement discharged, six cases left hospital voluntarily, and eight cases died. Conclusions:Most of the CA-PLA patients have no underlying hepatobiliary diseases, and more than half of patients have no history of diabetes mellitus. Most of the pathogens are Klebsiella pneumoniae, which are relatively sensitive to antimicrobial agents.

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 .