Objective:To explore the resistance of common clinical isolates to chlorhexidine gluconate (CHG) and the clinical characteristics of patients with the infections.Methods:A total of 1 000 isolates from the First Affiliated Hospital of Wenzhou Medical University in 2018 (from January to May) were collected, which included 200 strains each of Escherichia coli ( E. coli), Acinetobacter baumanii ( A. baumanii), Pseudomonas aeruginosa ( P. aeruginosa), Staphylococcus aureus ( S. aureus), and Enterococcus spp.. Minimum inhibitory concentration (MIC) of CHG against 1 000 isolates were determined by the agar dilution method. The correlation between the resistance of isolates and clinical characteristics of infected patients was analyzed. Chi-square test or Fisher exact probability test were used for statistical analysis. Results:A total of 57 CHG resistant strains were detected in 1 000 clinical isolates. These CHG-resistant strains were mainly isolated from sputum and intensive care unit ward, accounting for 49.1%(28/57)and 38.6%(22/57), respectively. The resistance rates of P. aeruginosa, A. baumanii, Enterococcus spp., S. aureus, and E. coli to CHG were 16.0%(32/200), 7.0%(14/200), 3.0%(6/200), 1.5%(3/200) and 1.0%(2/200), respectively. The CHG-resistant rates of P. aeruginosa to ceftazidime, ciprofloxacin, levofloxacin and gentamicin were 53.1%(17/32), 78.1%(25/32), 65.6%(21/32) and 50.0%(16/32), respectively, which were all higher than those of CHG-sensitive P. aeruginosa (25.0%(8/32), 25.0%(8/32), 21.9%(7/32) and 15.6%(5/32), respectively), with statistical significance ( χ2=5.317, 18.080, 12.444 and 8.576, respectively, all P<0.05). The hospital mortality was 22.8%(13/57) in patients infected with CHG-resistant bacteria, which was higher than that in patients infected with CHG-sensitive bacteria ((7.0%(4/57); Fisher exact probability test, P=0.018)). CHG-resistant group had a higher history of CHG exposure and antimicrobial treatment (61.4%(35/57) and 70.2%(40/57), respectively), which were both higher than those with CHG-susceptible isolates (17.5%(10/57) and 47.4%(27/57), respectively), the differences were both statistically significant ( χ2=22.947 and 6.118, respectively, both P<0.05). In addition, the multi-drug resistance rate of CHG-resistant strains was 54.4%(31/57), which was higher than that of CHG-susceptible strains (35.1%(20/57)), the difference was statistically significant ( χ2=4.293, P=0.039). Conclusions:CHG resistant strains have higher antimicrobial resistance. Hospital mortality in patients infected with CHG-resistant bacteria is higher than patients infected with CHG-sensitive bacteria. The important risk factors are CHG exposure and antimicrobial therapy.

Objective:To evaluate the effect of intra-articular injection of different concentrations of ozonated water on articular cartilage of rabbits with osteoarthritis (OA).Methods:Twenty-four clean-grade New Zealand white rabbits of both sexes, weighing 2.0-3.0 kg, aged 6 months, were divided into 4 groups ( n=6 each) using a random number table method: control group (group C), OA group, low concentration ozonated water group (L group) and high concentration ozonated water group (H group). The OA model was established by intra-articular injection of papain.At 2 weeks after the model was successfully established, 10.0 and 20.0 μg/ml ozonated water 1.0 ml was injected into the knee joint of rabbits in L and H groups, respectively, and 0.9% sodium chloride solution 1.0 ml was injected once a week, 3 times in total in OA group.At 1 week after the last injection, the cartilage tissue of the knee joint was removed and stained with toluidine blue for evaluation of Mankin score (under light microscope). The activity of caspase-3 in chondrocyte was detected by enzyme-linked immunosorbent assay. Results:Compared with group C, the Mankin score and caspase-3 activity were significantly increased in the other 3 groups ( P<0.05). Compared with group OA, the Mankin score and caspase-3 activity were significantly decreased in group L and group H ( P<0.05). Compared with group L, the Mankin score was significantly increased, and the activity of caspase-3 was decreased in group H ( P<0.05). Conclusion:Injecting ozonated water 10.0 μg/ml and 20.0 μg/ml into the knee joint cavity both can inhibit the apoptosis in chondrocytes and reduce the damage to articular cartilage, however, high concentration of ozonated water can cause the denaturation of the articular cartilage matrix in rabbits with OA.

Objective:To investigate the role of type Ⅵ secretion system (T6SS) in the pathogenicity and antibiotic resistance of Acinetobacter baumanii. Methods:From January 1 to December 31, 2016, a total of 45 Acinetobacter baumanii isolates were collected from patients with bloodstream infection in the First Affiliated Hospital of Wenzhou Medical University. The susceptibilities to commonly used antimicrobial agents were determined by VITEK 2 Compact automatic microbiology analyzer. Detection of T6SS characteristic gene hemolysin coregulated protein ( hcp) was achieved by polymerase chain reaction. Biofilm formations, serum resistances and competition tests of T6SS-positive/negative Acinetobacter baumanii were performed in vitro. The clinical data of patients with bloodstream infection were collected and analyzed. Chi-square test, t test and Kruskal-Wallis test were conducted for statistical analysis. Results:The positive rate of T6SS in 45 Acinetobacter baumanii isolates was 53.3% (24/45). The resistance rates of T6SS-positive Acinetobacter baumanii to ceftazidime, ciprofloxdcin, gentamicin, imipenem, levofloxacin, piperacillin/tazobactam, tobramycin and cefepime (95.8%, 95.8%, 66.7%, 95.8%, 79.2%, 95.8%, 79.2%, 91.7%)were all higher than that of T6SS-negative Acinetobacter baumanii (28.6%, 28.6%, 28.6%, 28.6%, 9.5%, 23.8%, 23.8%, 28.6%), and the differences were all statistically significant ( χ2=22.12, 22.12, 6.51, 22.12, 21.83, 24.72, 13.79, 18.97, respectively, all P<0.05). The biofilm formation ability, serum resistance and competitive ability of T6SS-positive Acinetobacter baumanii were stronger than those of T6SS-negative Acinetobacter baumanii, and the differences were all statistically significant ( t=4.99, Z=-2.61 and -2.27, respectively, all P<0.05). The positive rate of T6SS isolated from intensive care unit (ICU) ward (80.0%, 16/20) was significantly higher than that from non-ICU ward (32.0%, 8/25; χ2=10.29, P<0.05). But T6SS had no effect on the prognosis of patients ( χ2=1.74, P=0.188). Conclusions:T6SS of Acinetobacter baumanii is associated with high pathogenicity, and the high drug resistance rate makes treatment extremely difficult. Physicians need to pay much attention, especially to the patients from ICU wards.

Objective:To evaluate the effect of orexin-A on programmed necrosis during cerebral ischemia-reperfusion (I/R) in rats.Methods:Thirty clean-grade healthy adult male Spraugue-Dawley rats, weighing 280-320 g, were divided into 3 groups ( n=10 each) using a random number table method: sham operation group (Sham group), cerebral I/R group (I/R group) and orexin-A group (OA group). In I/R and OA groups, a rat model of global cerebral I/R injury was established by transesophageal cardiac pacing-induced cardiac arrest and cardiopulmonary resuscitation in anesthetized animals.Orexin-A 30 μg/kg (diluted to 0.5 ml in phosphate buffer solution) was intravenously injected at 10 min before establishing the model in OA group.Phosphate buffer solution 0.5 ml was intravenously injected at 10 min before establishing the model in Sham and I/R groups.The neurological deficit score (NDS) was assessed at 24 h of reperfusion, then the rats were sacrificed, and bilateral hippocampal tissues were obtained.The morphological structure of pyramidal cells in the hippocampal CA1 region was examined after HE staining, and normal pyramidal cells were counted.Western blot was used to detect the expression of receptor-interacting protein 1 (RIP1), RIP3 and mixed-lineage kinase domain-like protein (MLKL). Immuno-histochemistry was used to count RIP1, RIP3 and MLKL positive cells in the hippocampal CA1 region.The activity of superoxide dismutase (SOD) in hippocampi was determined by xanthine oxidase method, and the content of malondialdehyde (MDA) in hippocampi was determined by thiobarbituric acid method. Results:Compared with Sham group, the normal pyramidal cell count in the hippocampal CA1 region was significantly decreased, NDS was increased, the expression of RIP1, RIP3 and MLKL protein was up-regulated, the positive cell count was increased, the content of MDA in hippocampi was increased, and the activity of SOD in hippocampi was decreased in I/R and OA groups ( P<0.05). Compared with I/R group, the count of normal pyramidal cells in the hippocampal CA1 region was significantly increased, NDS was decreased, the expression of RIP1, RIP3 and MLKL protein was down-regulated, the count of positive cells was decreased, the content of hippocampal MDA was decreased, and the activity of hippocampal SOD was increased in OA group ( P<0.05). Conclusion:The mechanism by which orexin-A reduces cerebral I/R injury may be related to inhibiting programmed necrosis in rats.

Objective To investigate the chlorhexidine acetate-resistance in Klebsiella pneumoniae ( K. pneumoniae) clinical isolates and to analyze the possible mechanisms and molecular epidemiology of re-sistant isolates. Methods A total of 332 K. pneumoniae clinical isolates were collected in the First Affilia-ted Hospital of Wenzhou Medical University in 2015. Standard agar dilution was used to screen chlorhexidine acetate-resistant isolates. The minimum inhibition concentrations ( MIC) of chlorhexidine acetate to resistant isolates with and without the presence of carbonyl cyanide m-chlorophenyl hydrazone ( CCCP) , which was an efflux pump inhibitor, were analyzed. Efflux pump genes of cepA, qacE and qacΔE1 that carried by and ex-pressed in those isolates were detected by polymerase chain reaction ( PCR) and quantitative real-time PCR ( RT-qPCR) , respectively. The biofilm formation ability was measured by crystal violet staining. The homol-ogy among the chlorhexidine acetate-resistant isolates was investigated with multilocus sequence typing ( MLST) and pulsed-field gel electrophoresis ( PFGE) . Results Twenty-five K. pneumoniae strains were re-sistant to chlorhexidine acetate. The MIC values of chlorhexidine acetate for them were reduced by at least four-fold in the presence of CCCP. Strains carrying the genes of cepA, qacE and qacΔE1 accounted for 100％, 40％ and 40％, respectively. The expression of the efflux pump genes in the chlorhexidine acetate-resistant isolates was higher than that in the susceptible isolates. The biofilm formation ability of the chlo-rhexidine acetate-resistant isolates was better than that of the susceptible isolates. Furthermore, negative, weak-positive and positive biofilm formation ability was observed in four ( 16％) , 20 ( 80％) and one (4％) strains, respectively. The results of MLST and PFGE showed that the 25 chlorhexidine acetate-resist-ant isolates belonged to 19 different sequence types ( ST) with diverse PFGE patterns. Conclusions This study suggested that active efflux was the main mechanism of chlorhexidine acetate resistance in K. pneumoni-ae. The 25 chlorhexidine acetate-resistant K. pneumoniae strains possessed different biofilm formation ability and shared low homology.

Objective To evaluate the development of cerebral anoxia during controlled hypoten-sion with nicardipine or urapidil after carotid endarterectomy in patients. Methods Forty-four patients of either sex, aged 48-64 yr, scheduled for elective carotid endarterectomy under general anesthesia, requi-ring controlled hypotension after operation, were divided into nicardipine group ( group N ) and urapidil group ( group U) using a random number table method, with 22 patients in each group. Nicardipine at 2. 5μg·kg-1 ·min-1 was intravenously infused in group N, and urapidil 2μg·kg-1 ·min-1 was intravenously infused in group U. After systolic blood pressure was decreased to 130-140 mmHg, the consumption of nicardipine was adjusted to 0. 2 - 0. 5 μg·kg-1 ·min-1 and the consumption of urapidil to 1-2μg·kg-1 ·min-1 in group N and group U, respectively, to maintain systolic pressure at 130-140 mmHg. Heart rate ( HR) , cardiac index ( CI) , bispectral index ( BIS) value, regional cerebral oxygen saturation (rSO2) and end-tidal pressure of carbon dioxide (PETCO2) were recorded after entering the operating room ( baseline) , at the beginning of controlled hypotension ( T1 ) , and at 5, 10, 20, 30, 60 and 120 min af-ter systolic blood pressure was decreased to the target hypotension ( T2-7 ) . Development of cerebral anoxia( the relative decrease in rSO2>12％ of the baseline value) was recorded in controlled hypotension period. Results Compared with the value at T1 , the HR at T2,3 and CI at T3-7 were significantly increased ( P<0. 05), and no significant change was found in rSO2, PETCO2 or BIS value at the other time points in group N (P>0. 05), and rSO2 was significantly decreased at T3-7 (P<0. 05), and no significant change was found in HR, CI, PETCO2 or BIS value at the other time points in group U (P>0. 05). Compared with group N, the HR at T2,3, CI at T3-7 and rSO2 at T3-7 were significantly decreased in group U (P<0. 05). The incidence of cerebral anoxia was significantly higher in group U than in group N ( P<0. 05) . Conclu-sion Controlled hypotension with nicardipine is recommended after carotid endarterectomy in order to avoid the development of cerebral anoxia in the patients.

Objective: To investigate the in vitro antibacterial activity of triclosan combined with different antibacterial agents against triclosan-resistant multidrug-resistant Acinetobacter baumannii (A.baumannii). Methods: A total of 626 A. baumannii strains were collected from the First Affiliated Hospital of Wenzhou Medical University from 2016 to 2017. The sensitivity of these A. baumannii strains to common antibiotics was detected by VITEK 2-compact automatical microbiological analyzer and the minimum inhibitory concentrations (MIC) of triclosan were detected by agar dilution method. Checkerboard method was used to detect the changes in MIC values of triclosan against 16 triclosan-resistant multidrug-resistant A. baumannii strains after it was used in combination with four external ointments, including gentamicin, erythromycin, chloramphenicol and kanamycin, and three common antibiotics of imipenem, meropenem and ciprofloxacin, respectively. Fractional inhibitory concentration index (FICI) was used to evaluate the joint bacteriostatic effects. Results: Among the 626 A. baumannii strains, 17 were resistant to triclosan with a drug resistance rate of 2.7% (17/626). These triclosan-resistant strains had high MIC values for ciprofloxacin, imipenem, ceftazidime and other commonly used clinical antibiotic and most of them were multidrug-resistant. After triclosan was used in combination with seven different antibacterial drugs, the MIC values of all drugs decreased to various degrees compared with those when they were used alone. Triclosan in combination with gentamicin, chloramphenicol and ciprofloxacin showed synergistic effects on 62.5%, 56.25% and 62.5% of the 16 strains and additive effects on 37.5%, 43.75% and 37.5%, respectively. When it was used in combination with erythromycin, kanamycin, imipenem and meropenem, synergistic effects on 37.5%, 25%, 12.5% and 12.5%, additive effects on 37.5%, 56.25%, 62.5% and 62.5%, and indifferent effects on 25%, 18.75%, 25% and 25% of the strains were detected, respectively. No antagonistic effect was found between triclosan and any of the above antibiotics. Conclusions Triclosan combined with gentamicin, chloramphenicol and ciprofloxacin had better in vitro antibacterial effects against the triclosan-resistant multidrug-resistant A. baumannii strains in this study with synergistic and additive effects. Some indifferent effects were found between triclosan and kanamycin, erythromycin, imipenem and meropenem, but no antagonistic effects were detected.

Objective: To investigate the distribution and expression of T3SS virulence genes in clinically isolated Pseudomonas aeruginosa (P. aeruginosa) strains and its correlation with drug resistance. Methods: A total of 68 P. aeruginosa isolates were collected from the First Affiliated Hospital of Wenzhou Medical University in 2015. The antimicrobial susceptibility was detected by the agar dilution method. The distribution of virulence genes such as exoU, exoS, exoT and exoY from different isolates was detected by PCR. The expression levels of transcriptional regulator genes (ptrA and exsA) and effector-related genes (exoT and exoS) in some isolates were determined by real-time fluorescence quantitative PCR, and the Pearson correlation analysis was used to analyze the results. Results: The detection rates of exoT and exoY in 68 P. aeruginosa isolates were higher, accounting for 79.4% and 75.0%, respectively. The detection rate of exoT in wound-sourced isolates was significantly higher than that in sputum (97.0% vs 61.8%, P＜0.01). In addition, the genotype of exoU - /exoS + was the most common, accounting for 51.5% (35/68). The resistance rates of sputum-sourced isolates to imipenem and meropenem were significantly higher than that of wound-sourced isolates (47.1% vs 8.8%, 47.1% vs 14.7%, P＜0.01). The resistance rates of isolates carrying exoU gene to carbapenems, aminoglycosides and fluoroquinolones were higher than those of isolates carrying exoS, exoT or exoY genes. Pearson correlation analysis showed that the expression level of ptrA gene was negatively correlated with those of exoT, exoS and exsA genes (P＜0.05). Conclusion The P. aeruginosa isolates from our hospital carrying T3SS virulence genes exoT and exoY are common, and the virulence genes are related to the drug resistance of P. aeruginosa. In addition, ptrA may be a potential negative regulatory gene for the expression of T3SS virulence genes.

Objective: To evaluate the in vitro antibiotic effects of colistin combined with meropenem, levofloxacin or fosfomycin against colistin-heteroresistant Acinetobacter baumannii (A.baumannii). Methods: A total of 576 A. baumannii clinical isolates were collected from the First Affiliated Hospital of Wenzhou Medical University from 2014 to 2015. Minimal inhibitory concentrations (MICs) of colistin against A. baumannii were detected by broth dilution method. Colistin-heteroresistant A. baumannii isolates were screened using population analysis profiles (PAPs). MICs of colistin combined with meropenem, levofloxacin or fosfomycin, and the four drugs used alone against colistin-heteroresistant A. baumannii were detected by checkerboard method and broth dilution method. Fractional inhibitory concentration index (FICI) was calculated to evaluate antibiotic effects. Results: None of the 576 A. baumannii isolates was resistant to colistin as indicated by the broth dilution method. Nine colistin-heteroresistant A. baumannii isolates were identified using PAPs. Compared with the MICs of colistin used alone, the MICs of colistin used in combination with meropenem, levofloxacin or fosfomycin against colistin-heteroresistant isolates were all decreased. Colistin-meropenem combination showed synergistic (55.6%), additive (33.3%) and indifferent effects (11.1%), but no antagonistic effect. Colistin-levofloxacin combination showed synergistic (55.6%), additive (22.2%) and indifferent effects (22.2%), but no antagonistic effect. Colistin-fosfomycin combination showed synergistic (77.8%) and additive (22.2%) effects, but no indifferent or antagonistic effect. Conclusion In vitro use of colistin in combination with meropenem, levofloxacin or fosfomycin has synergistic and additive antibacterial effects against colistin-heteroresistant A. baumannii. Combinations of colistin-meropenem and colistin-levofloxacin have fewer indifferent effects and no antagonistic effect.

Objective To investigate the effect of Kinesio Taping on interleukin-1β(IL-1β),tumor necrosis factor α(TNF-α)and matrix metalloproteinase-3 (MMP-3)in synovial fluid of patients with knee osteoarthritis in early and middle stage.Methods A total of 84 patients with knee osteoarthritis who met the inclusion/exclusion criteria were randomly selected and divided into the control group treated with placebo(kinesiology tape without elasticity)and the experimental group treated with Kinesio Tape.Both two tapes were changed every two days,with 15 times of changes as a course of treatment.Synovial fluid sample was drawn before and one week after treatment,and was used for measuring Levels of 1L-1β,TNF-α and MMP-3 by enzyme-linked immunosorbent assay (ELISA).and knee function was evaluated before and 1 week after treatment.Results Compared with pre-treatment,the levels of IL-1β,TNF-α and MMP-3 in synovial fluid were significantly decreased in the experimental group after treatment [(20.07 ± 6.94) ng/Lvs.(38.12 ± 5.93) ng/L,(42.42±8.76)ng/Lvs.(58.23±9.54)ng/L,(11.28±1.99)μg/L vs.(15.67±2.21)μg/L,t =12.81,7.91 and 9.57,all P＜0.05].The levels of IL-1β,TNF-α and MMP-3 in synovial fluid were lower in the experimental group after treatment than before treatment[(20.07±6.94)ng/L vs.(38.12±5.93)ng/L,(42.42±8.76)ng/L vs.(58.23±9.54)ng/L,(11.28 ±1.99)μg/L vs.(15.67±2.21)ng/L,t =12.81,7.91 and 9.57,all P＜0.05],and were lower in the experimental group after treatment than in control after treatment[(20.07±6.94)ng/L vs.(37.97±6.21)ng/L,(42.42±8.76)ng/L vs.(57.04 ±8.73)ng/L,(11.28± 1.99)μg/Lvs.(15.01± 2.56)μg/L,t =12.46,7.66 and 7.46,all P＜0.05]Lysholm score was significantly higher in the experimental group after treatment than before treatment[(74.5 ± 2.6) vs.(44.7 ± 2.8),t =50.54,P ＜0.05],and was also higher in the experimental group after treatment than in the control group after treatment[(74.5±2.6) vs.(50.2± 2.3),t =45.37,P＜0.05].Conclusions Kinesio Taping can significantly reduce the levels of IL-1β,TNF-α and MMP-3 in synovial fluid of patients with knee osteoarthritis through inhibiting the inflammatory response in articular cavity.