Objective To survey the bacterial species and drug resistance of bacteria isolated from blood, urine and other samples in 12 military hospitals located at different areas in China. Methods A total of 1099 non-repetitive bacterial isolates were collected from 12 military hospitals and sent to the General Hospital of PLA for re-identification and drug susceptibility test. The minimal inhibitory concentrations (MICs) of antimicrobial agents were determined by agar dilution method. The results were evaluated according to the standards of CLSI (2007) and analyzed by WHONET 5.4 software. ESBLs, AmpC ?-lactamases were detected using the confirmatory test and APB discs method, respectively. Results Gram positive cocci and gram negative bacilli constituted 39.7% and 60.3% of 1099 clinical isolates respectively. Methicillin-resistant Staphylococcus aureus (MRSA) accounted for 62%, and methicillin-resistant coagulase negative Staphylococcus (MRSCN) accounted for 92%. ESBLs-producing and AmpC-producing strains of Escherichia coli accounted for 51.1% and 11.3%, respectively, and of Klebsiella pneumoniae accounted for 45.1% and 16.2%, respectively. As to caftazidime, amikacin, cefotaxime, cefoxitin and levofloxacin, the resistance rate in Escherichia coli (E. coli) and Klebsiella pneumoniae isolated from blood was lower than that isolated from urine. However, as to meropenem, ceftazidime, polymyxin and minocyclin, the resistance rate in Pseudomonas aeruginosa and Acinetobacter spp isolated from blood was higher than that isolated from urine. Conclusion MRSA, MRSCN, and producers of ESBLs and AmpC ?-lactamases are common in military hospitals. Resistance pattern of bacteria from blood differs from that of bacteria from urine. It is necessary for military hospitals to take the bacterial distribution and resistance levels to antimicrobial agents under surveillance in order to guide the proper use of antibiotics for military doctors, and the results may serve as guidelines in the use of antimicrobial agents in war time.

OBJECTIVE To investigate the combined effect of cefoperazone/sulbactam with levofloxacin(group 1) and polymyxin B with rifampin(group 2) on 43 isolates of multi-drug-resistant Pseudomonas aeruginosa. METHODS The minimal inhibitory concentration(MIC) of all the antibiotics mentioned above was determined by agar dilution method.Fractional inhibitory concentration(FIC) index was calculated for all the selected isolates with all combinations,and the activities of antibiotics alone and in combination against the selected strains were evaluated. RESULTS The MIC of all the combined antimicrobials was reduced significantly(P

OBJECTIVE To investigate the phenotype and genotype of plasmid-encoded AmpC and extended spectrum beta-lactamases in Klebsiella pneumoniae.METHODS 3-Aminophenylboronic acid(APB) test and ESBLs confirmatory test were used for phenotypic detection of AmpC and ESBLs.Conjugation was conducted in order to understand the spread of plasmid in bacteria.The size and genotype of ampC and ESBL genes were studied by extraction and purification of plasmid,PCR and sequencing analysis.RESULTS A plasmid of about 15kb was extracted from K.pneumoniae.This plasmid carrying resistance genes to antibiotics could be spread from K.pneumoniae to recipient Escherichia coli NK5449 through conjugation.DHA-type ampC gene and SHV-type ESBLs gene could be amplified from plasmids extracted from both K.pneumoniae and its conjugant in E.coli,they were DHA-1 ampC gene and SHV-12 ESBLs gene confirmed by sequencing analysis.CONCLUSIONS DHA-1 ampC gene and SHV-12 ESBLs gene are detected from the plasmid of K.pneumoniae.

Objective To investigate the distribution and antimicrobial resistance of pathogens isolated from surgi-cal patients with infection.Methods Distribution and antimicrobial resistance of 1 208 pathogens isolated from sur-gical patients with infection from January 2013 to January 2014 were analyzed retrospectively.Results Of 1 208 pathogenic isolates,gram-negative bacteria,gram-positive bacteria and fungi accounted for 64.57% (n = 780 ), 24.92%(n = 301 )and 10.51 % (n = 127 )respectively.The main specimens were sputum (44.78%),urine (21 .11 %),blood(11 .51 %),and pus(10.26%).Antimicrobial susceptibility testing results showed that the produ-cing rate of extended-spectrumβ-lactamases (ESBLs)of Escherichia coli and Klebsiella pneumoniae was 62.60%and 33.61 % respectively,resistant rate to imipenem was 0.76% and 15.57%,respectively.The resistant rate of Pseudomonas aeruginosa and Acinetobacter baumannii to imipenem was 38.93% and 75.80% respectively.Methi-cillin-resistant Staphylococcus aureus and methicillin-resistant coagulase negative Staphylococcus was 71 .68% and 87.93% respectively.Conclusion The major pathogens isolated from surgical patients with infection are gram-neg-ative bacteria,the main infection sites are respiratory tract and urinary tract in this hospital;multidrug resistance is serious,especially carbapenem resistance,which should be paid attention.

Objective To understand pathogen spectrum of bacterial and fungal infection of central nervous system (CNS),and evaluate the etiological diagnostic value of universal primer polymerase chain reaction (PCR).Methods Data about patients with suspected or confirmed bacterial and fungal infection of CNS from January 2009 to March 2015 were collected,species of pathogens from cerebrospinal fluid (CSF)were analyzed,DNA from patients’CSF were performed PCR amplification and sequencing with universal primers of bacterial 16S rRNA and fungal 28S rRNA, PCR detection results were compared with CSF culture during the same period.Results A total of 400 patients were with confirmed or suspected bacterial or fungal infection of CNS,132 of whom were with positive CSF culture.150 pathogenic isolates were detected,including 48 isolates of gram-positive bacteria,90 gram-negative bacteria,and 12 fungi;the top three isolated bacteria were Acinetobacter baumannii (n =32 ),coagulase negative staphylococcus (n=16)and Klebsiella pneumoniae (n=13);the most common fungus was Cryptococcus neoformans (n=8).CSF from 88 infected patients and 20 non-infected patients were selected for PCR amplification,the sensitive of PCR am-plification assay was higher than the culture method (35.23% [31/88]vs 28.41 %[25/88],χ2 =4.17,P <0.05).

OBJECTIVE To investigate the genes of carbapenemases and integrases in multi-drug resistant Acinetobacter baumannii(MDRAba).METHODS PCR was used for detection of genes of carbapenemases: OXA-23-like,OXA-24-like,OXA-51-like,and OXA-58-like,and integrases Ⅰand Ⅱ from 70 clinical strains of carbapenem-resistant A.baumannii(CRAba).PCR products of OXA-23-like and OXA-58-like were analyzed by sequencing.Agar dilution method was carried out for antimicrobial susceptibility test.RESULTS Genes for OXA-23-like and OXA-51-like were positive from 56 and 69 isolates,accounted for 80.0% and 98.6%, respectively;gene of OXA-58-like was detected from only one strain.Sixty one strains showed positive for integrase Ⅰ gene.OXA-23 or OXA-58 was the exact gene type by sequencing.All 70 strains were highly resistant to ciprofloxacin,ceftazidime,and gentamicin,but susceptible to polymyxin B.CONCLUSIONS CRAba strains distributed in General Hospital of PLA mainly possess OXA-23 type carbapenemase and integraseⅠ.

Objective To develop a novel multiplex polymerase chain reaction (PCR) to detect multidrug-resistant Acinetobacter baumannii.Methods One hundred and five strains of multidrug-resistance A. baumannii were isolated from January 2006 to April 2007. The bacterial DNA was obtained by boiling the pure growth of A. baumannii. All isolates were subjected to the multiplex PCR to detect genes of blaOXA-23-like,blaOXA-24-like,blaOXA-51-like,blaOXA-58-like,intI 1 and intI 2.Results Among 105 isolates,76 were positive for blaOXA-51-like,blaOXA-23-like,and intI 1,18 were positive for blaOXA-51-like and intI 1,10 were positive for blaOXA-51-like and blaOXA-23-like,1 was positive for blaOXA-51-like and blaOXA-23-like,1 was positive for blaOXA-51-like,blaOXA-23-like,and blaOXA-58-like,and all were negative for blaOXA-24-like and intI 2.Conclusion The presence of OXA carbapenemase and integrase genes was correlated with multidrug resistance in A.baumannii.

OBJECTIVE To investigate the prevalence of DHA AmpC ?-lactamases mediated by plasmid in Klebsiella pneumoniae in China.METHODS Antimicrobial susceptibility test was conducted by the methods of double agar dilution and ESBLs confirmatory in K-B method according to the criteria of guidelines of CLSI.AmpC ?-lactamases were detected on the basis that AmpC ?-lactamases could be inhibited by 3-aminophenylboronic acid(APB).Gene chip technology and PCR were used to detect ESBLs and AmpC gene.RESULTS Among total 34 isolates of K.pneumoniae 32(94.1%) produced AmpC ?-lactamases and ESBLs.The most common(38.3%) were types DHA and TEM and SHV.MIC50 and MIC90 of all strains to all tested antimicrobial agents were lower than 34 strains tested 0.25?g/ml and 0.5?g/ml.Fourteen strains AmpC and ESBLs were conjugated successfully.CONCLUSIONS DHA AmpC ?-lactamases mediated by plasmid are the most common in K.pneumoniae in General Hospital of PLA of China.The most common(38.3%) are types DHA and TEM and SHV.Fourteen(41.2%) strains can be spreaded by plasmid.

Objective To evaluate the value of amplification and sequencing of 16S rRNA gene in the identification of clinical rare pathogenic bacteria,and guide the diagnosis and treatment for related clinical infection.Methods 12 bacterial isolates that were difficult,or unable to be identified with conventional laboratory methods,or with special phenotypes were collected. The 16S rRNA gene was amplified by polymerase chain reaction (PCR),then sequenced for identifying bacterial species through BLAST comparison,clinical characteristics of related infection were ana-lyzed.Results 12 clinical isolates were all positive for PCR amplification (about 1500 bp),species were all identi-fied (similarity≥99% ),the identified strains were Listeriamonocytogenes(n= 2),Brucellamelitensis(n= 2),Fu-sobacteriummortiferum(n= 1),Rothiaaeria(n= 1),Nocardiafarcinica(n= 1),Staphylococcussaccharolyticus (n= 1 ),Rhizobiumradiobacter(n= 1 ),Prevotellabivia(n= 1 ),Ralstoniamannitolilytica(n= 1 ),and Atopobium vaginae(n= 1 ). The sensitivity of 16S rRNA gene amplification was high,and the minimum detection limit of Escherichiacoli ATCC 25922 was 1.5×101 CFU/mL. Clinical data of 12 patients revealed that these strains can cause multi-sites and multi-types of infection,after patients received targeted antimicrobial therapy,11 improved, and 1 died.Conclusion Sequencing for 16S rRNA gene can rapidly and accurately identify rare,anaerobic,and difficult cultured bacteria,provide laboratory evidence for etiological diagnosis and treatment of different types of infection.

OBJECTIVE To study the phenotypic existence,genetic type and gene transfer of extended spectrum beta-lactamases(ESBLs) and AmpC beta-lactamase from Klebsiella pneumoniae and K.oxytoca. METHODS Disk confirmation test and 3-aminophenylboronic acid(APB) disk potentiation test were used to detect ESBLs and AmpC beta-lactamase.The genetic types of these two kinds of beta-lactamases were examined by gene chip technology and sequence analysis.The transfer of resistance genes was conducted by conjugation. RESULTS From 72 strains of K.pneumoniae and 20 strains of K.oxytoca which were not susceptible to cefoxitin,coexistence of AmpC(beta-lactamase) with ESBLs together was very common,accounted for 54.2% and 75.0%,single ESBLs accounted for 22.2% and 25.0%,respectively.There were 12.5% single AmpC in(K.pneumoniae).DHA type ampC gene and SHV type ESBLs gene were the main molecular types.These genes could be transferred from clinical isolates to recipient E.coli J53. CONCLUSIONS ESBLs as well as AmpC(beta-lactamase) are the most important resistance mechanism in K.pneumoniae and K.oxytoca.The resistance could be transferred through the bacterial conjugation.