OBJECTIVE To construct two-hybrid genomic library of Rhodococcus equi,and study the pathogenic mechanism of R.equi.METHODS The genomic DNA of R.equi was partially digested and cloned into AD vector of two-hybrid system,the number of independent clone and the size of insert fragment were detected as the quality index of the library.RESULTS The independent clone of the library was 1?106 and the average size of insert fragment was 2.3 kb.CONCLUSIONS The pathogenic mechanism of R.equi is not clear and it is very important for this study to construct two-hybrid genomic library.

Hypervirulent Klebsiella pneumoniae ( hvKP) mainly infects healthy people and causes serious infections, such as liver abscess, meningitis, necrotizing fasciitis, endophthalmitis and severe pneu-monia. Studies have shown that hvKP is more virulent than classic Klebsiella pneumoniae characterized by ex-pressing more capsular polysaccharide and carrying the virulence factors including magA, rmpA and iron ac-quisition molecules. The greater survival and anti-phagocytosis abilities of hvKP strains contribute to the spread and metastasis of hvKP infection. This review describes the virulence factors, colonization and infec-tion of hvKP as well as the host immunity to hvKP.

Objective To study 8 isolates of multiple antibiotic-resistant Klebsiella oxytoca in 8 different patients from one surgical ward. Methods Susceptibility test, extended-spectrum ?-lactamases (ESBLs) screening methods and some molecular biological methods were performed. Results 8 isolates of Klebsiella oxytoca were verified as closely related homologous CTX-M-3-producing strains. Conclusions The little outbreak was caused by 8 closely related homologous strains in one ward.

Objective To study the function of aminophenylboronic acid(APB)and clavulanate(CA)for detecting ESBLs in Enterobacter cloacae.Methods The phenotype of ESBLs of 61 Enterobacter cloacae isolates was detected with adding single beta-lactamase inhibitor CA to ceftazidime(CAZ)and cefotaxime(CTX),and double beta-lactamase inhibitors CA/APB to ceftazidime(CAZ)and cefotaxime(CTX)respectively.PCR was used to detect ESBLs genes of 61 Enterobacter cloacae isolates.The results of the enzymatic inhibitor potentiation test and PCR were compared and analyzed.Results With adding single enzymatic inhibitor CA to CAZ,28 isolates of Enterobacter cloacae producing ESBLs were detected,while 14 isolates were detected with adding CA to CTX.With adding double enzymatic inhibitors CA/APB to CAZ,28 isolates of Enterobacter cloacae producing ESBLs were detected,while 44 isolates were detected with adding CA/APB to CTX.By PCR positive ESBLs genes were detected in 47 isolates of Enterobacter cloacaes.Conclusions The potentiation test with double beta-lactamase inhibtion can be used to detect ESBLs in Enterobacter cloacae.

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 detect the hypermucoviscosity phenotype , capsular serotype and virulence gene of Klebsiella pneumonia (K.pneumonia) from various kinds of clinical specimens and understand the characteristics of different K.pneumonia causing infections.Methods A retrospective study was conducted through collection of 178 K.pneumonia isolates from blood, sputum, bronchoalveolar lavage fluid , urine, normally sterilized fluid , puncture fluid from liver abscess and other abscesses between January 2010 and December 2012 in General Hospital of Chinese PLA.String test was carried out for detection of hypermucoviscosity phenotype.Capsular serotype and virulence gene ( rmpA) were checked by polymerase chain reaction.Analysis was made according to the hypermucoviscosity , capsular serotype , rmpA gene , as well as the sources of K.pneumoniae.Statistic data was analyzed by contingency table analysis and χ2 test.Results Eighty-three out of 178 ( 46.6%) strains of K.pneumonia were hypermucoviscous with positive string test, the positive rate of virulence gene rmpA was 92.8%(77/83).K1/K2/K57 capsular serotypes were the predominant serotypes in the group of puncture fluid from liver abscess and other abscesses (75.0%,27/36)than the group of blood (32.4%,12/37), urine(21.7%,5/23) and normally sterilized fluid(25.0%,5/20), and also more than in the group of sputum , bronchoalveolar lavage fluid (50.0%,22/44),χ2 =21.19,P<0.01.The positive rate of string test in the group of puncture fluid from liver abscess and other abscesses (77.8%, 28/36)was significantly higher than the group of blood (29.7%, 11/37), urine (30.4%, 7/23), or normally sterilized fluid (25.0%, 5/20),χ2 =27.90,P<0.01.The positive rate of rmpA gene in the isolates from puncture fluid of liver abscess and other abscesses was higher than other groups.Conclusions As the pathogens of various kinds of infections , mainly abscess and respiratory infection, hypermucoviscous strains of K.pneumonia were of great clinical significance.Capsular serotype K57, as well as K1 and K2, possessed hypermucoviscosity and hypervirulence in China.

ObjectiveTo establish the rapid molecular diagnosis of 16 common coagulase negative Staphylococcus(CNS).MethodsDNA sequencing of 16 CNS would be obtained with gap gene.After the alignment gap gene sequences which were available in the GenBank,the bacteria were identified with homological alignment and phylogenetic tree,and compared with the 16S rRNA gene.ResultsThe sequence similarity of the gap sequences ranged from 39% to 98% in 16 CNS.There were the highest similarity (98%) between S.hominis and S.hominis subsp,and the lowest(39% ) between S.saprophyticus and S.xylosus.The sequence similarity of the 16S rRNA sequences ranged from 96 to 98%,at least two species of bacteria similar rate of 99% and the most four species similar rate of 99%.Phylogenetic homology analysis showed that it was a high confidence(99% ) in the detection ofS.xylosus and S.lentus,S.chromogenes and S.intermedius,S.hominis and S.hominis subsp,but for 10 other species of bacteria,gap homology analysis has less unreliable confidence(49%,56% ) and 16S rRNA has more unreliable confidence(43%,43%,50%,56%,63%,65%,76% ).ConclusionAnalysis of gap sequence could identify 16 CNS timely and accurately,with higher confidence than 16S rRNA.

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 detect the main virulence genes and biofilm formation of Enterococci strains isolated from blood samples .Methods Twenty-eight strains of Enterococcus faecalis ( E.faecalis) and 54 strains of Enterococcus faecium ( E.faecium) were collected from blood samples .Five main virulence genes (asa1, esp, hyl, cylA and gelE) were detected by multiplex PCR.Biofilm formation was investigated by using microtiter dish biofilm formation assay .Results All E.faecalis strains were positive for at least one kind of virulence genes , of which 14 strains were concurrently positive for asa1, esp, cylA and gelE.asa1, cylA and gelE were only detected in E.faecalis strains, while hyl gene only existed in E.faecium strains. Twenty-seven strains of E.faecium were esp positive, of which 12 strains were both hyl and esp positive. None of the 5 virulence genes were identified in 10 strains of E.faecium.85.7% of E.faecalis strains and 63.0%of E.faecium strains could form biofilm.Conclusion Compared with E.faecium strains, more types of virulence genes were detected in E.faecalis strains with higher positive rates .Moreover , E.faecalis strains were more likely to form biofilms than E.faecium strains.

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.