OBJECTIVETo study the changes in expression of quorum sensing gene aba I in Acinetobacter baumannii (AB) strains isolated from burn patients during biofilm formation process, and its influences on the extracellular matrix of biofilm and drug resistance of AB.

METHODSSix drug-resistant and five drug-sensitive AB strains isolated from wound excretion, blood and venous catheter were collected from burn patients hospitalized in Ruijin hospital of Shanghai Jiao Tong University School of Medicine from January to October 2011. The AB standard strain ATCC 19606 was used as control. (1) Clinical strains and standard strain were normally cultured 10, 24, and 48 h respectively in vitro. The bacteria samples were stained with propidium iodide to measure biofilm thickness with confocal laser scanning microscope. (2) Clinical strains and standard strain were cultured in tubes 10, 24, and 48 h respectively in vitro under shaking condition. The bacteria floating in the medium were regarded as free bacteria, while those adhered to the tube wall as the bacteria within biofilm (biofilm bacteria). Relative expression value of genes aba I and pgaB was detected by real-time fluorescent quantitative PCR with the expression value of the standard strain set at 1. Data were processed with analysis of variance.

RESULTS(1) At post culture hour (PCH) 10, 24, 48, biofilm thickness of clinical strains was thicker than that of standard strain; biofilm thickness of drug-resistant strains [(28.8 ± 0.6), (31.7 ± 1.1), and (38.1 ± 3.1) µm] was respectively thicker than that of drug-sensitive strains [(17.1 ± 0.4), (20.1 ± 1.6), and (25.8 ± 1.7) µm, with F value respectively 1274.38, 206.60, and 61.73, P values all below 0.05]. (2) Biofilm bacteria: at PCH 10, 24, 48, expression values of aba I in drug-resistant strains (6.6 ± 1.7, 25.7 ± 3.5, 9.8 ± 3.6) were much higher than those of drug-sensitive strains (2.7 ± 1.0, 15.0 ± 3.5, 4.7 ± 3.2, with F value respectively 21.82, 25.24, and 6.22, P values all below 0.05); expression values of pgaB in drug-resistant strains (37.4 ± 1.1, 44.5 ± 3.6, 33.1 ± 11.5) were obviously higher than those of drug-sensitive strains (14.6 ± 0.8, 20.0 ± 6.9, 18.7 ± 6.8, with F value respectively 1488.44, 57.26, and 6.01, P values all below 0.05). (3) Free bacteria: at PCH 10, 24, 48, there was no significant statistical difference between drug-resistant strains and drug-sensitive strains in expression value of aba I (with F value respectively 0.24, 2.33, and 0.11, P values all above 0.05); expression values of pgaB in drug-resistant strains (13.8 ± 3.8, 12.5 ± 2.9, 23.7 ± 2.1) were obviously higher than those of drug-sensitive strains (7.0 ± 5.9, 5.0 ± 1.3, 15.6 ± 6.7, with F value respectively 5.44, 28.42, and 7.76, P values all below 0.05). (4) Comparison between biofilm bacteria and free bacteria in resistant strains: expression value of aba I in biofilm bacteria at each time point was respectively higher than that of free bacteria (with F value respectively 43.69, 286.61, and 9.98, P values all below 0.05); expression values of pgaB in biofilm bacteria at PCH 10, 24 were higher than those in free bacteria (with F value respectively 214.26 and 283.20, P values below 0.05). (5) Comparison between biofilm bacteria and free bacteria in sensitive strains: expression value of aba I in BF bacteria at PCH 24 was higher than that of free bacteria (F = 70.28, P < 0.05); expression values of pgaB in biofilm bacteria at PCH 10, 24 were higher than those of free bacteria (with F value respectively 8.03 and 22.62, P values below 0.05).

CONCLUSIONSDuring biofilm formation process, the increasing expression of quorum sensing gene aba I in drug-resistant AB strains isolated from burn patients may up-regulate the expression of gene pgaB, which leads to high production of extracellular matrix and biofilm formation, and enhances drug resistance of AB.

Acinetobacter Infections ; Acinetobacter baumannii ; genetics ; isolation & purification ; Biofilms ; Burns ; microbiology ; Drug Resistance, Bacterial ; Genes, Bacterial ; Humans

OBJECTIVETo observe expressions of pgaABC gene clusters and changes in biofilm (BF) phenotype in Acinetobacter baumannii (AB) isolated from burn patients.

METHODSFrom January 2009 to October 2010, 24 strains of AB isolated from burn patients hospitalized in our burn wards were collected for the study, while the standard strain ATCC 19606 was used as control. Expressions of pgaABC gene clusters were detected by real time fluorescence quantitative RT-PCR. All strains were cultured for 16 hours in vitro, BF with semi-quantitative detection was respectively evaluated by modified microtiter-plate test under stable condition and tube test under shaking condition for expression of absorbance value. All strains were cultured for 48 hours in vitro, then stained with fluorescent agent and collected for measurement of BF thickness with confocal laser scanning microscopy (CLSM). Data were processed with t test.

RESULTS(1) The expression of pgaB gene (27.91 ± 7.93) in clinical AB strains was much higher than that of standard strain ATCC 19606 (1.00, t = 5.77, P < 0.05). There was no statistical difference in expression of pgaA and pagC genes between standard strain ATCC 19606 (1.00) and clinical AB strains (1.01 ± 0.28, 1.15 ± 0.38, with t value respectively 0.04, 0.64, P values all above 0.05). (2) After being cultured for 16 hours, BF of clinical AB strains cultured under shaking condition formed distinct "purple circle", and its absorbance value (1.25 ± 0.31) was higher than that in standard strain ATCC 19606 (0.76 ± 0.03, t = 2.67, P < 0.05). There was no obvious difference in absorbance value between clinical AB strains and standard strain ATCC19606 cultured under stable condition. (3) After being culture for 48 hours, green fluorescence intensity and distribution in extracellular matrix of clinical AB strains were stronger as compared with those of standard strain ATCC 19606, and BF thickness in clinical AB strains [(27.3 ± 9.4)µm] was thicker than that in standard strain ATCC 19606 [(15.6 ± 1.7) µm, t = 2.09, P < 0.05].

CONCLUSIONSThe high expression of pgaB gene in AB strains isolated from burn patients can induce production of extracellular matrix, which may be related to increase in the ability and thickness of BF formation.

Acinetobacter Infections ; microbiology ; Acinetobacter baumannii ; genetics ; isolation & purification ; Biofilms ; Burns ; microbiology ; Genes, Bacterial ; Humans ; Multigene Family

Acinetobacter baumannii ; classification ; genetics ; isolation & purification ; Drug Resistance, Multiple, Bacterial ; genetics ; Humans ; Phylogeny

OBJECTIVETo investigate the prevalence of central venous catheter-related infection (CRI) in burn patients and its risk factors, so as to guide the clinical practice.

METHODSClinical data of 5 026 days of 480 cases of central venous catheterization altogether in 228 burn patients admitted to our ward from June 2011 to December 2014, conforming to the study criteria, were retrospectively analyzed. (1) The incidence of CRI and that of catheter-related bloodstream infection (CRBSI) in patients (the infection rates per thousand days were calculated) and mortality due to them, and detection of concerning bacteria were recorded after each case of catheterization. (2) The incidence of CRI after each case of catheterization in patients was recorded according to the classification of their gender, age, total burn area, full-thickness burn area, cause of injury, severity of inhalation injury, location of catheterization, whether catheterization through wound or not, duration of catheterization, and the data were processed with chi-square test. Indexes with statistically significant differences were selected, and they were processed with multivariate logistic stepwise regression analysis to screen the independent risk factors of CRI. (3) To all cases of catheterization and cases with catheterization through wound, incidence of CRI after each case of catheterization in patients at each time period was recorded according to the sorting of duration of catheterization. Data were processed with chi-square test and Fisher's exact test, and the values of P were adjusted by Bonferroni.

RESULTS(1) Infection rate of CRI per thousand days was 50.14‰ (252/5 026), resulting in the mortality rate of 3.51% (8/228). Infection rate of CRBSI per thousand days was 18.70‰ (94/5 026), resulting in the mortality rate of 2.19% (5/228). Respectively 319 and 105 strains of pathogens were detected in CRI and CRBSI, in which the top four bacteria detected were Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, and Klebsiella pneumoniae, and the most common fungus found was smooth Candida. (2) There were no statistically significant differences in the incidence of CRI after each case of catheterization among patients with different gender, age, cause of injury, severity of inhalation injury, and location of catheterization (with χ(2) values from 0.427 to 6.991, P values above 0.05). There were statistically significant differences in the incidence of CRI after each case of catheterization among patients with different total burn area, full-thickness burn area, whether catheterization through wound or not, duration of catheterization (with χ(2) values from 7.202 to 14.246, P<0.05 or P<0.01). (3) Total burn area, whether catheterization through wound or not, and duration of catheterization were the independent risk factors of CRI (with odd ratios respectively 1.495, 1.670, 1.924, 95% confidence intervals respectively 1.096-2.040, 1.077-2.590, 1.303-2.841, P<0.05 or P<0.01). (4) In all cases enduring catheterization, the incidence of CRI in patients after each episode of catheterization was close between cases enduring catheterization shorter than or equal to 3 days and those longer than 3 days and shorter than or equal to 5 days (χ(2) <0.001, P>0.05); the incidence of CRI in patients after each episode of catheterization was significantly higher in cases enduring catheterization longer than 5 days and shorter than or equal to 7 days, longer than 7 days and shorter than or equal to 14 days, and longer than 14 days than the former two periods (with χ(2) values from 3.625 to 13.495, P values below 0.05). In the cases with catheterization through wound, the incidence of CRI of patients after each episode of catheterization was close between cases enduring catheterization shorter than 5 days and those longer than or equal to 5 days and shorter than 7 days (P>0.05); the incidence of CRI of patients after each episode of catheterization was significantly higher in cases enduring catheterization longer than or equal to 7 days and shorter than 14 days and longer than or equal to 14 days than those with longer than or equal to 5 days and shorter than 7 days (with χ(2) values respectively 6.828 and 4.940, P values below 0.05).

CONCLUSIONSThe infection rate of CRI per thousand days in burn patients is relatively low, while that of CRBSI is relatively high, both resulting in relatively low mortality, and Acinetobacter baumannii is the main pathogen. Total burn area, whether catheterization through wound or not, and duration of catheterization are independent risk factors of CRI in burn patients, and with which its occurrence could be predicted. It is suggested that central venous catheterization should be removed within 5 days, and catheterization through wounds should be avoided as much as possible. If catheterization through wound is unavoidable, removal of the catheter within 7 days is recommended.

Acinetobacter baumannii ; isolation & purification ; Burns ; complications ; Catheter-Related Infections ; epidemiology ; Humans ; Incidence ; Prevalence ; Retrospective Studies ; Risk Factors

BACKGROUND: We evaluated the effectiveness of isolation measures using cohorting rooms and antimicrobial use in reducing the isolation rate of methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Acinetobacter baumannii (MDR-AB). METHODS: Four cohorting rooms (16 beds) for patients colonized or infected with multidrug-resistant organisms (MDRO) have been created in the general wards of our 894-bed hospital since October 2003. We prospectively evaluated the isolation rates of MRSA and MDR-AB, and amount of antimicrobial use during the 8-year study period. We also investigated the relationship between antimicrobial use density (AUD) and the isolation rates of MRSA and MDR-AB. RESULTS: After creating cohorting rooms, the isolation rates of MRSA decreased from 1.56 cases per 1,000 patient-days from 2004-2005 to 1.24 from 2006-2007 (P=0.57). The isolation rates of MDR-AB also decreased from 0.72 from 2004-2005 to 0.36 from 2010-2011 (P<0.01). The mean quarterly AUDs of glycopeptides and carbapenems were 30.17+/-6.80 and 19.5+/-7.10, respectively. There were no significant correlations between AUD values and the isolation rate of MRSA or MDR-AB. CONCLUSION: This study suggests that isolation measures using cohorting rooms to help limit the transmission of MDRO infection and colonization, especially MDR-AB, in resource-limited settings is feasible and efficacious.
Acinetobacter ; Acinetobacter baumannii ; Carbapenems ; Cohort Studies ; Colon ; Drug Resistance ; Glycopeptides ; Humans ; Methicillin Resistance ; Methicillin-Resistant Staphylococcus aureus ; Patient Isolation ; Patients' Rooms ; Prospective Studies

Acinetobacter Infections ; microbiology ; Acinetobacter baumannii ; drug effects ; isolation & purification ; Anthracosis ; microbiology ; Anti-Bacterial Agents ; pharmacology ; Drug Resistance, Bacterial ; Fosfomycin ; pharmacology ; Humans ; Imipenem ; pharmacology ; Pneumonia, Bacterial ; microbiology

Acinetobacter Infections ; etiology ; Acinetobacter baumannii ; drug effects ; isolation & purification ; Child, Preschool ; Drug Resistance, Bacterial ; Female ; Humans ; Infant ; Intensive Care Units, Pediatric ; Male ; Risk Factors

OBJECTIVETo study the transferrable character of Acinetobacter baumannii (AB) plasmids with 3 types of beta-lactamase gene.

METHODSThe plasmid of multi-drug resistant AB (donor) isolated from burn wound were transferred to E. coil ATCC25922 (receptor) through conjugation, and drug sensitivity was also observed. Drug-resistant gene and stability of filial generation and zygote were analyzed by PCR.

RESULTSThe drug-resistance of donor plasmids to Sulfamethoxazole, Ampicillin, Cefalotin, Cefpodoxime, Cefuroxime, Imipenem/Cilastatin and Ampicillin/Sulbactam, and three types of beta-lactamase gene were transferred to the receptor, and were also stably transmitted for passages. The minimum inhibitor concentration of receptor to Sulfamethoxazole was > 2 mg/L after conjugation with donor, and inhibitory character could be transferred to next generation.

CONCLUSIONbla(TEM-1), bla(PER-1) and bla(OXA-23) genes carried in the plasmid of AB can be transferred through conjugation and stably transmitted for passages, and it is one of the molecular mechanisms for AB with multi-drug resistance after burn infections.

Acinetobacter Infections ; Acinetobacter baumannii ; enzymology ; genetics ; isolation & purification ; Burns ; microbiology ; Drug Resistance, Multiple, Bacterial ; genetics ; Genes, Bacterial ; Humans ; Microbial Sensitivity Tests ; Plasmids ; beta-Lactamases ; genetics

OBJECTIVE: To explore the expression level of class I integrase (intI 1) mRNA in Acinetobacter baumannii from biofilm cells and planktonic cultured cells ,and to analyze the drug-resistance of Class I integron positive strains. METHODS: Acinetobacter baumannii were collected from hospitals,and Class I integron strains were screened by gene amplification. Total RNA of Class I integron positive strains was extracted, and the intI1 mRNA expression in the bioflim cells and planktonic cultured cells was measured by RT-PCR. Susceptibilities to antibiotics of Class I integron positive strains were also examined. RESULTS: The intI1 gene mRNA was expressed under 2 conditions, and the mRNA expressed in the biofilm cells was about 4 times higher than that in the planktonic cultured cells. Among the 64 strains of Acinetobacter baumannii, 46 strains were Class I integron positive strains. The antibiotic resistance of intI1 gene cassette-positive strains was higher than that of gene cassette-negative strains. CONCLUSION The intI1 gene mRNA can be up-regulated in Acinetobacter baumannii biofilm cells.Class I integron plays an important role in drug resistance. It is much easier to capture gene cassettes for bacteria under biofilm condition.
Acinetobacter Infections ; microbiology ; Acinetobacter baumannii ; genetics ; isolation & purification ; Base Sequence ; Biofilms ; Drug Resistance, Multiple ; genetics ; Humans ; Integrases ; biosynthesis ; genetics ; Molecular Sequence Data ; RNA, Messenger ; biosynthesis ; genetics

BACKGROUND: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) and Acinetobacter baumannii (CRAB) are the leading causes of nosocomial infections. A rapid and sensitive test to detect CRPA and CRAB is required for appropriate antibiotic treatment. We optimized a loop-mediated isothermal amplification (LAMP) assay to detect the presence of bla(VIM-2), bla(IMP-1), and bla OXA-23, which are critical components for carbapenem resistance. METHODS: Two sets of primers, inner and outer primers, were manually designed as previously described. The LAMP buffer was optimized (at 2mM MgSO4) by testing different concentrations of MgSO4. The optimal reaction temperature and incubation time were determined by using a gradient thermocycler. Then, the optimized bla(VIM-2), bla(IMP-1), and bla(OXA-23) LAMP reactions were evaluated by using 120 P. aeruginosa and 99 A. baumannii clinical isolates. RESULTS: Only one strain of the 100 CRPA isolates harbored bla(IMP-1), whereas none of them harbored bla(VIM-2). These results indicate that the acquisition of bla(VIM-2) or bla(IMP-1) may not play a major role in carbapenem resistance in Korea. Fifty two strains of the 75 CRAB isolates contained bla(OXA-23), but none contained bla(VIM-2) and bla(IMP-1) alleles. CONCLUSIONS: Our results demonstrate the usefulness of LAMP for the diagnosis of CRPA and CRAB.
Acinetobacter baumannii/genetics/*isolation & purification ; Anti-Bacterial Agents/*pharmacology ; Carbapenems/*pharmacology ; Drug Resistance, Bacterial/*genetics ; *Genes, Bacterial ; Nucleic Acid Amplification Techniques ; Pseudomonas aeruginosa/genetics/*isolation & purification ; Sensitivity and Specificity