OBJECTIVETo study the drug resistance of Acinetobacter baumannii (AB) producing VIM-2-type metallo-β-lactamase (MBL) isolated from burn patients of our ward against carbapenem antibiotics and its homology.

METHODSA total of 400 strains of AB (identified) were isolated from sputum, urine, blood, pus, and wound drainage. of burn patients hospitalized in our ward from September 2011 to March 2014. Drug resistance of the 400 strains of AB to 15 antibiotics, including compound sulfamothoxazole, aztreonam, etc. , was tested using the automatic microorganism identifying and drug sensitivity analyzer. Among the carbapenems-resistant AB isolates, modified Hodge test was applied to screen carbapenemase-producing strains. The carbapenemase genes of the carbapenemase-producing strains, and the mobile genetic elements class I-integron (Intl1) gene and conserved sequence (CS) of carbapenemase-producing strains carrying blaVIM-2 gene were determined with PCR and DNA sequencing. For carbapenemase-producing strains carrying blaVIM-2 gene, synergism test with imipenem-ethylene diamine tetraacetic acid (EDTA) and enhancement test with imipenem-EDTA and ceftazidime-EDTA were used to verify the MBL-producing status. Drug resistance of the VIM-2-type MBL-producing AB strains was analyzed. For VIM-2-type MBL-producing AB strains, plasmid conjugation experiment was used to explore the transfer of plasmid; outer membrane protein (OMP) CarO gene was detected by PCR. For VIM-2-type MBL-producing AB strains carrying CarO gene, the protein content of CarO was analyzed with sodium dodecyl sulfate polyacrylamide gel electro- phoresis. The repetitive consensus sequence of Enterobacteriaceae genome PCR (ERIC-PCR) was carried out for gene typing of VIM-2-type MBL-producing AB strains to analyze their homology.

RESULTS(1) The resistant rates of the 400 strains of AB against levofloxacin and compound sulfamethoxazole were low. A total of 381 carbapenems-resistant AB strains were screened, including 240 carbepenemase-producing strains. (2) Out of the 240 carbepenemase-producing strains, 18 strains were found to harbor the blaVIM-2 gene, accounting for 7.5%; 133 strains carried the blaTEM-1 gene, accounting for 55.42%; 195 strains carried the blaOXA23 gene, accounting for 81.25%; 188 strains carried the bla(armA) gene, accounting for 78.33%. (3) Eighteen carbepenemase-producing strains which carried the bla(VIM-2) gene were found to carry the Intl1 gene, showing the Intl1-VIM linkage. Simultaneously, Intl1 variable area CS showed diversity. (4) Eighteen carbepenemase-producing strains which carried the blaVIM-2 gene were verified to produce MBL. The resistant rates of the 18 strains of AB against compound sulfamethoxazole were the lowest, followed by levofloxacin and cefoperazone/sulbactam, and those against the other antibiotics were above 60.00%. (5) Through multiple joint tests, plasmid conjugation experiment positive transfer strain was not found in 18 VIM-2-type MBL-producing AB strains. (6) Nine out of the 18 VIM-2-type MBL-producing AB strains were found to carry CarO gene. The OMP CarO of VIM-2-type MBL-producing AB strains carrying CarO gene was lost or lowered in the protein content. (7) The 18 VIM-2-type MBL-producing AB strains were classified into 6 genotypes by the ERIC-PCR. There were respectively 6, 4, 3, and 1 stain (s) in genotypes A, B, C, and F, and there were 2 strains in genotypes D and E respectively.

CONCLUSIONSThe resistance mechanism of AB against carbapenems is mainly mediated by blaTEM-1, blaOXA-23, and bla(arma); meanwhile, VIM-2-type MBL-producing and lack or change in OMP CarO are attributable to carbapenems resistance of clinically isolated AB from burn wards, and the Intl1 gene may take a part in blaVIM-2 gene transmission.

Acinetobacter baumannii ; drug effects ; enzymology ; genetics ; isolation & purification ; Anti-Bacterial Agents ; pharmacology ; therapeutic use ; Bacterial Proteins ; Burns ; drug therapy ; microbiology ; Carbapenems ; pharmacology ; Drug Resistance, Bacterial ; Genes, Bacterial ; Humans ; Imipenem ; pharmacology ; Microbial Sensitivity Tests ; Sulbactam ; pharmacology ; beta-Lactamases ; genetics

OBJECTIVETo investigate the effects of phosphorylated mitogen-activated protein kinases (MAPK), including the phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2), the phosphorylated protein p38 (p-p38), the phosphorylated c-Jun N-terminal kinase (p-JNK), on phosgene inhalation-induced lung injury and its relationship with matrix metalloproteinase 9 (MMP-9).

METHODSAccording to the random number table, 30 male Wistar rats were divided into air control group (C), phosgene inhalation group (P), PD98059 (specific inhibitor of ERK1/2) group, SB203580 (specific inhibitor of p38) group, and SP600125 (specific inhibitor of JNK) group, with 6 rats in each group. The number of neutrophils in the bronchoalveolar lavage fluid (BALF) was counted and the lung wet-dry ratio (W/D) was examined. The serum levels of inflammatory factors TNF-α, IL-1β, IL-6, and IL-8 were determined with ELISA. The protein expressions of p-ERK1/2, p-p38, p-JNK, and MMP-9 in lung tissue were detected with Western blotting. The mRNA level of MMP-9 in lung tissue was detected with real-time fluorescence quantitative PCR. Data were processed with one-way analysis of variance (among groups) and SNK method (paired comparison).

RESULTSCompared with those of group C [respectively (2.0 ± 0.7)×10(4) /mL and 3.7 ± 0.6], the number of neutrophils and W/D of group P [respectively (10.7 ± 1.4)×10(4) /mL and 7.6 ± 0.4] were increased. The number of neutrophils in group SB203580 and group SP600125 was respectively (8.3 ± 1.1)×10(4), (7.9 ± 1.3)×10(4)/mL, with W/D respectively 6.1 ± 1.4, 6.1 ± 0.9, all of which decreased as compared with those of group P (with P values all below 0.01). Compared with those of group C, the levels of TNF-a, IL-1β, IL-6, and IL-8 of group P were increased, but decreased in group SB203580 and group SP600125 compared with that of group P, though still higher than those of group C, and the differences were statistically significant (P < 0.05 or P<0.01). Protein quantities of p-p38 and p-JNK were higher in group P (respectively 1.19 ± 0.22 and 1.43 ± 0.14) than in group C (respectively 0.76 ± 0.06 and 0.74 ± 0.05). Compared with those of group P, the protein levels of p-ERK1/2 (0.47 ± 0.05) in group PD98059, p-p38 (0.88 ± 0.07) in group SB203580, and p-JNK (0.91 ± 0.07) in group SP600125 were significantly reduced (P < 0.05 or P < 0.01). The protein and mRNA levels of MMP-9 were higher in group P (respectively 2.23 ± 0.18 and 4.93 ± 0.12) than in group C (respectively 1.26 ± 0.14 and 1.80 ± 0.03). The protein and mRNA levels of MMP-9 in group SB203580 (respectively 1.58 ± 0.14 and 2.96 ± 0.09) and group SP600125 (respectively 1.55 ± 0.30 and 3.00 ± 0.13) were lower than those in group P (P < 0.05 or P < 0.01).

CONCLUSIONSThe phosgene inhalation can activate the MAPK signaling protein pathway by increasing expressions of p-p38 and p-JNK, which lead to an up-regulation of MMP-9, and this may contribute to the phosgene inhalation-induced lung injury.

Animals ; Burns, Inhalation ; enzymology ; Cytokines ; metabolism ; Disease Models, Animal ; Flavonoids ; pharmacology ; Imidazoles ; pharmacology ; MAP Kinase Signaling System ; Male ; Matrix Metalloproteinase 9 ; metabolism ; Mitogen-Activated Protein Kinases ; metabolism ; Phosgene ; Phosphorylation ; Pyridines ; pharmacology ; Rats ; Rats, Wistar

OBJECTIVETo study the effect of cotransfection of genes of insulin-like growth factor I (IGF-I) and herpes simplex virus thymidine kinase (HSV-tk) on wound healing.

METHODSThirty male Wistar rats were inflicted with 30% TBSA full-thickness scald. They were then divided into A group (4.6 microg pcDNA3.1/IGF-I+Lipofectamine 2000+saline), B group (3.6 microg pcDNA3.1/HSV-tk+Lipofectamine 2000+saline), C1 group and C2 group (2.3 microg pcDNA3.1/IGF-I+1.8 microg pcDNA3.1/HSV-tk+Lipofectamine 2000+saline), and D group (3.0 microg pcDNA3.1+Lipofectamine 2000+saline) according to the random number table, with 6 rats in each group. The above-mentioned mixtures were subcutaneously injected into left back of each rat the moment after injury and on post scald day (PSD) 7, 14, 21, and 28. Gancyclovir (2.5 mg/100 g) was hypodermically injected into rats in C2 group on PSD 29, 30, 31, 32. Changes in body weight of rats were measured. Wound healing rates were calculated. On PSD 35, the expressions of IGF-I gene in local wound and liver tissue were determined with immunohistochemical staining. The serum expression of IGF-I was determined with radioimmunoassay. Expression of HSV-tk gene in local wound was determined with RT-PCR. Apoptosis of fibroblast in C1 and C2 groups was observed under transmission electron microscope. Data were processed with one-way analysis of variance and Turkey method.

RESULTSBody weight of rats in A, C1, and C2 groups increased from PSD 7 through 35, and the difference between former three groups and B, D groups was statistically significant (with F value respectively 2.764, 4.519, 5.009, 13.449, 5.877, P values all below 0.05). Wound healing rates of rats in A, C1, and C2 groups were higher than those in B, D groups (with F value respectively 5.286, 100.880, 152.380, 127.850, 147.750, P values all below 0.05). IGF-I gene was positively expressed in wound fibroblast in A, C1 and C2 groups, but negatively in liver tissues of all the rats. There was no significant statistical difference among groups in serum content of IGF-I [from (1185+/-170) to (1270+/-130) ng/mL, F=0.355, P=0.838]. HSV-tk gene was positively expressed in rat skin tissue in B, C1 and C2 groups. Fibroblast apoptosis was observed under transmission electron microscope in C2 group, but it was not observed in C1 group.

CONCLUSIONSCotransfection of pcDNA3.1/IGF-I and pcDNA3.1/HSV-tk mediated by liposome can promote wound healing, and inhibit the scar proliferation to some extent.

Animals ; Burns ; genetics ; metabolism ; therapy ; Insulin-Like Growth Factor I ; genetics ; metabolism ; Male ; Rats ; Rats, Wistar ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics ; metabolism ; Transfection ; Wound Healing

OBJECTIVETo investigate the drug-resistance of Acinetobacter baumannii (Ab) isolated from patients in burn ward, and study the incidence of 16S rRNA methylase genes mediated high-level aminoglycoside drug-resistance and its mechanism of transfer.

METHODSA total of 40 Ab clinical isolates were collected from burn ward in Gansu Province People's Hospital from May 2006 to Dec. 2007. The sensitivity of Ab for 20 antibiotics were determinated by K-B agar diffusion. The minimal inhibitory concentrations (MIC) of amikacin, gentamicin, tobramycin, netilmicin, isepamicin and kanamycin against Ab strains were determinated by agar dilution. Five kinds of 16S rRNA methylase genes including armA, rmtA, rmtB, rmtC, rmtD were amplified by PCR, the positive PCR-products were purified and sequenced, and the plasmid were extracted by alkaline lysis. The transferability of drug-resistance were determinated by conjugation and plasmid transformation tests.

RESULTSThe drug-resistance rates of Ab against six aminoglycosides antibiotics was 72.5%, 72.5%, 70.0%, 67.5%, 70.0%, 70.0%, respectively. Twenty five strains were resistant to six aminoglycosides antibiotics (62.5%), among which 10 isolates were armA-positive (40.0%); rmtA, rmtB, rmtC and rmtD-positive isolates were not found. Ten transformants and 10 conjugates showed high-level resistance against aminoglycosides antibiotics, all of which the value of MIC > or = 256 microg/mL carried armA gene.

CONCLUSIONSThe drug-resistance of Ab clinical isolates have high drug-resistance. 16S rRNA methylases gene exists in Ab and locates in plasmid chromosome.

Acinetobacter baumannii ; enzymology ; genetics ; isolation & purification ; Burn Units ; Burns ; microbiology ; Drug Resistance, Bacterial ; genetics ; Genes, Bacterial ; Genes, rRNA ; Humans ; Methyltransferases ; genetics ; Plasmids

OBJECTIVETo investigate the effects of taurine on myocardial mitochondria and their enzyme activities in rats with severe burn.

METHODSOne hundred and twenty healthy adult Wistar rats were subjected to 30% TBSA full-thickness burn. They were randomly divided into burn group (B, with intraperitoneal injection of isotonic saline), treatment group (T, with intraperitoneal injection of taurine, 200 mg/kg),with 60 rats in each group . Ten rats with sham scald were used as control (S group). The myocardial tissue samples in B and T groups were harvested at 1, 3, 6, 12, 24 and 48 postburn hours (PBH) for determination of activity respectively of succinate dehydrogenase (SDH), cytochrome oxidase (CCO), the superoxide dismutase (SOD), Ca2+ -ATPase in mitochondria, and contents of cytochrome c (Cyt c), cytochrome aa3 (Cyt aa3), malondialdehyde (MDA), and Ca2+ in mitochondria and cytoplasm . The myocardial tissue samples of controls were harvested at 1 PBH for determination of above indices.

RESULTSThe activity of CCO in B group was decreased at 1 PBH , especially at 6 ,12 PBH. The activity of SDH in B group was decreased to lowest level at 6 PBH, and its value was lower than that of S group at each time point. The activity of CCO or SDH in T group was not obviously decreased, and the activity of CCO at 3, 6, 12 PBH showed significant difference compared with B group (P < 0.05). The contents of Cyt aa3 and Cyt c in B group at 3, 6, 12, 24 PBH were obviously decreased, which were significantly lower than those in T group (P < 0.05). The activity of SOD in B group at 3, 6, 12 PBH was obviously decreased, the activity of Ca2+ -ATPase at 3, 6, 12 and 24 PBH was decreased to different extent, which was significantly lower than those in T group (P < 0.05). The MDA contents in B and T groups were higher than that in S group at 3-48 PBH ,and it was highest in B group (P < 0.05). The Ca2+ content of mitochondria in B group at 1 PBH was increased (13.7 +/- 1.5), and it was (24.8 +/- 2.6), (29.7 +/- 3.1), (16.3 +/- 1.9) and (13.5 +/- 1.7) at 3, 6, 12, 24 PBH respectively,and they were all higher than that of S group (10.7 +/- 1.6, P < 0.05). The Ca2 contents of cytoplasm in group B at 3 - 24 PBH were also higher than that in S group (P < 0.05). The Ca2+ content of mitochondria in T group at 3, 6, 12, 24 PBH was (16.8 +/- 2.8), (18.7 +/- 1.9), (10.5 +/- 1.8) and (13.3 +/- 1.7)respectively, which were lower than that in B group at every time point.

CONCLUSIONTaurine have protective effect on mitochondria and their enzyme activities in myocardium in rats with severe burn, and it may be attributable to improving the ability of eradicating oxygen free radicals and alleviating Ca2+ overload in the mitochondria.

Animals ; Burns ; drug therapy ; enzymology ; metabolism ; Calcium ; metabolism ; Calcium-Transporting ATPases ; metabolism ; Cytochromes c ; metabolism ; Electron Transport Complex IV ; metabolism ; Female ; Male ; Mitochondria, Heart ; enzymology ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Succinate Dehydrogenase ; metabolism ; Superoxide Dismutase ; metabolism ; Taurine ; therapeutic use

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

OBJECTIVETo investigate the relationship among antibiotic resistance, integron, homology of multi-drug resistant Acinetobacter Baumannii isolated from burn ward.

METHODSThirty-one strains of multi-drug resistant Acinetobacter baumannii were isolated from samples of burn wound exudate in hospitalized patients of Gansu Province People's Hospital. The minimum inhibitory concentrations (MIC) of these strains against 11 antibiotics was examined by agar dilution method. Homology of these strains was analyzed by pulse-field gel electrophoresis (PFGE). Class 1, 2 and 3 integrase, integron genes and genotype of carbapenemases were amplified by PCR and verified by DNA sequencing.

RESULTSAcinetobacter baumannii were highly resistant to all antibiotics except imipenem, meropenem, cefoperazone-sulb-actam, piperacillin-tazobactam (antibiotic resistance rate was 45.2%, 48.4%, 48.4%, 41.0%, respectively). All strains were classified into 3 types of clones (A, B, C clone included 18, 7, 6 strains respectively) based on PFGE pattern. Integrons of 20 strains of Acinetobacter Baumannii harbored aadA1, aadA5, aacA4, aac3, catB8, aacC1, aac (6')-Ib, drfA17 and drf8 gene.

CONCLUSIONMulti-drug resistance Acinetobacter baumannii (major in clone A) spread widely in burn ward of Gansu Province People's Hospital. Integrons of Acinetobacter baumannii mediated drug resistance against aminoglycoside antibiotics, chloramp-phenicol. All carbapenem-resistant Acinetobacter Baumannii can produce OXA-23 carbapenemase.

Acinetobacter Infections ; Acinetobacter baumannii ; enzymology ; genetics ; isolation & purification ; Bacterial Proteins ; genetics ; Burns ; microbiology ; Drug Resistance, Multiple, Bacterial ; genetics ; Genes, Bacterial ; Genotype ; Humans ; Integrons ; Molecular Sequence Data ; beta-Lactamases ; genetics

OBJECTIVE: To observe the changes of expression of phosphorylation c-Jun NH2-terminal kinase (p-JNK) during the skin burned wound healing in patients and discuss the molecular mechanism of burned wound healing. METHODS: The staining intensity and distribution of p-JNK were detected by immunohistochemistry and routine histology in burned skin samples of 12 patients and normal skin samples of 12 control subjects. RESULTS: In normal skin, the positive signals of p-JNK were mostly localized in basal layer cells of the epidermis, with a positive rate of (8.8+/-1.3)%. In the burned group, the positive signals of p-JNK were mainly localized in the epidermal cells and some inflammatory cells, with a significantly higher positive rate of (31.2+/-3.3)% than the normal group(P<0.01). CONCLUSION The changes of p-JNK expression after skin burned might correlate with wound healing.
Adult ; Burns/enzymology* ; Female ; Forensic Medicine ; Humans ; JNK Mitogen-Activated Protein Kinases/metabolism* ; Male ; Middle Aged ; Phosphorylation ; Random Allocation ; Skin/injuries* ; Wound Healing

OBJECTIVETo investigate the role of c-Jun NH (2)-terminal kinase (JNk) in insulin resistance after burn and its mechanism.

METHODSTwenty-four Sprague-Dawley rats were randomized to control, burn and burn + anisomycin groups. The rats in control group received sham burn trauma, and burn and burn + anisomycin groups received 30% total body surface area (TBSA) full thickness burn injury. Anisomycin (5 mg/kg) together with 250 microl dimethyl sulfoxide (DMSO) was injected to the rats in anisomycin group intravenously, and only 250 microl DMSO in the other two groups. Euglycemic-hyperinsulinemic glucose clamps was performed 2 hours after the injection. The changes of phospho-serine 307, phospho-tyrosine of insulin receptor substrate (IRS)-1 and phospho-JNK in muscle tissues were determined and compared using immunoprecipitation and Western blot analysis or immunohistochemistry in the three groups.

RESULTSThe infusing rates of total 10% glucose (mg x kg(-1) x min(-1)) in control, burn and burn + anisomycin group were 12.3 +/- 0.4, 6.6 +/- 0.3, 6.5 +/- 0.4, respectively. The level of IRS-1 Serine 307 phosphorylation and phospho-JNK in muscle increased significantly, while insulin-induced tyrosine phosphorylation of IRS-1 decreased markedly after burn.

CONCLUSIONSThe activation of JNK elevates the level of IRS-1 phospho-serine 307 and might play a role in insulin resistance after burn in rats.

Adaptor Proteins, Signal Transducing ; metabolism ; Animals ; Anisomycin ; administration & dosage ; Anti-Bacterial Agents ; administration & dosage ; Blotting, Western ; Burns ; enzymology ; metabolism ; physiopathology ; Dimethyl Sulfoxide ; administration & dosage ; Disease Models, Animal ; Female ; Glucose Clamp Technique ; Immunohistochemistry ; Injections, Intravenous ; Insulin ; administration & dosage ; Insulin Receptor Substrate Proteins ; Insulin Resistance ; physiology ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Male ; Muscles ; metabolism ; Phosphorylation ; drug effects ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Serine ; metabolism ; Tyrosine ; metabolism

OBJECTIVETo investigate the role of p38 mitogen-activated protein kinase (MAPK) signal transduction pathway in the Kupffer cells production of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta in severely burns rats.

METHODSMale health adult Sprague-Dawley rats were randomized into four groups: sham burn rats given vehicle, sham burn rats given the p38 MAP kinase inhibitor SB203580, rats given a 30% total body surface area (TBSA) full-thickness burn and fluid resuscitation plus vehicle, and burn rats given injury and fluid resuscitation plus SB203580. Rats from each group were killed at 24 h after burn or sham burn and Kupffer cells (KCs) were isolated. After 18 h incubation, KCs next were stimulated with 50 ng/ml of LPS for 18 h. After stimulation, supernatants were removed for analysis of TNF-alpha and IL-1beta levels by ELISA. The TNF-alpha and IL-1beta mRNA expressions (by quantitative real-time RT-PCR) and the activities of p38 MAPK and JNK (by Western blot analysis) in KCs were examined.

RESULTSEighteen hours after 50 ng/ml LPS stimulation, KCs from burn rats released significantly higher levels of TNF-alpha and IL-1beta than did shams. The mRNA levels of TNF-alpha and IL-1beta in KCs increased significantly postburn. Western blot analysis suggested that expression of phosphorylated p38 MAPK and JNK were increased in KCs harvested from burn group after stimulation with LPS compared with those from sham group. In vivo administration of SB203580 markedly suppressed both the release of TNF-alpha and IL-1beta and the mRNA expressions of TNF-alpha and IL-1beta in KCs from both sham and burn rats. p38 MAPK activity in KCs was abolished by administration with SB203580, whereas JNK was not.

CONCLUSIONSp38 MAPK signal transduction pathway mediates KCs production of proinflammatory cytokines TNF-alpha and IL-1beta in severely burned rats.

Animals ; Blotting, Western ; Burns ; enzymology ; physiopathology ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; Interleukin-1 ; genetics ; metabolism ; Kupffer Cells ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Tumor Necrosis Factor-alpha ; genetics ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism ; physiology