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Strain-resource engineering is often considered as an important infrastructure of microbiology related research and industry. The western developed countries took the lead in establishing the classical microbial resource utilization method, and continuously improved the preservation system, species annotation technology and global sharing mechanism, which realized the expansion and reserve of biological resources since end of the 19th century. The rich and diversified germplasm resources, standard strains and production strains not only have important economic values, but also maintain the advantages of scientific research, bioeconomy (such as antimicrobial agents, vaccines, detection reagent development and standard development, etc.) and national security. Although there has been a lot of progress in related research in recent years, compared with developed countries, there is still a big gap in related fields in China. The investment and top-level design in this area lag far behind the western developed countries, and it is not commensurate with the current level of economic and social development in my country. Drawing lessons from the practice of WFCC and WDCM (World Data Center for Microorganisms, Global microbial data Center, affiliated to WFCC), for the purpose of collecting new clinical species/strains, this paper puts forward some suggestions on the identification, preservation and upload system of isolates.
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Objective:To explore and evaluate a appropriate suitable method for detection of Campylobacter and antibiotic sensitivity test for foodborne diarrhea in clinical laboratories. Methods:Pre-experiment:a total number of 400 fecal samples of patients with foodborne diarrhea were prospectively collected from the intestinal disease clinic of Beijing Tongren Hospital from September 2017 to January 2018. Double-hole filtration culture method and modified cefoperazone charcoal deoxycholate (CCD) agar culture method were used for fecal culture in micro-aerobic environment for 48 hours, and then suspicious colonies were identified by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Meanwhile, C. jejuni and C. coli were detected by real-time quantitative polymerase chain reaction(qPCR). Large sample verification: 2 062 fecal samples of patients with foodborne diarrhea in three hospitals of different levels in different areas of Beijing were collected for qPCR detection and culture from April 2018 to March 2019. The antimicrobial sensitivity test (AST) of C. jejuni and C. coli was performed according to the disk diffusion method and agar dilution method recommended by Clinical and Laboratory Standards Institute and National Antimicrobial Resistance Monitoring System for Enteric Bacteria. The results of the three detection methods and the consistency of the two antibiotic sensitivity tests were compared. Results:In the pre-experiment, the positive rates of Campylobacter ( jejuni/coli) detected of qPCR, double-hole filtration culture and modified CCD agar culture were 9.0% (36/400), 5.0% (20/400)and 3.5% (14/400), and the difference was statistically significant ( P<0.01). The samples with negative result of qPCR were negative by both culture methods. The total positive rates of Campylobacter detected by qPCR was 8.1% (168/ 2 062)including 7.0% (144/2 062) for C. jejuni and 1.2% (24/2 062) for C. coli. The samples with positive qPCR results were cultured by double-hole filtration culture method and the positive rate was 61.9%(104/168), among which, the positive rate of C. jejuni and C. coli were 58.3%(84/144) and 83.3%(20/24) respectively, which was not significantly different from the detection rate and culture positive rate in the pre-test ( P>0.1). The resistance rates of C. jejuni and C. coli to ciprofloxacin were 94.0%(94/100) and 100.0%(24/24) and to erythromycin were 6.0%(6/100) and 33.3%(8/24). The results from two antibiotic sensitivity test methods were consistent (Kappa>0.75). Conclusions:qPCR is rapid, sensitive and easy to operate, so it is suitable for routine development in clinical laboratories. The double-hole filtration culture method is beneficial to the acquisition of strains and is essential for the further study of Campylobacter. There was no significant difference between agar dilution method and disk diffusion method in antibiotic sensitivity test. Campylobacter showed a very high resistance rate to quinolones, which was no longer suitable for the treatment of Campylobacter foodborne diarrhea in Beijing area. Macrocyclic lipid antibiotics should be preferred.
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Objective:To quantitatively analyze the clinical and drug resistance feature of diarrhea of adults patients in 2016 and 2019 induced by the Escherichia coli (diarrheagenic Escherichia coli, DEC), and to reveal the difference of DEC′s epidemiological features of before and after measuring to strengthen food hygiene and safety in Beijing. Methods:A total number of 3 408 patients with food-borne adult diarrhea were received diagnosis and treatment in the intestinal clinic department of Beijing Tongren Hospital in 2016 and 2019.There were 1 926 patients in 2016 and 1 482 in 2019, respectively. The clinical information of patient were entered into the intestinal early warning system and were carefully preserved. The clinical specimens (the stool samples) were isolated and the DECs were identified by culturing. The colony of DECs was identified by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Five pathogenic types of Escherichia coli were classified by multiplex PCR methods. The drug-susceptibility test was performed according to the standards of the American Society for Clinical and Laboratory Standardization in 2019. The categorical data were analyzed by χ 2 test or Fisher′s exact test to verify the statistical difference. Results:A total number of 581 DECs strains were detected in 3 408 specimens. Among the subtypes of E Coli, the Enterotoxigenic Escherichia coli (ETEC) accounted for 53.36% (310/581), and Enterohemorrhagic Escherichia coli (EHEC) was detected. In 2016, the total detection rate of DEC was 14.54% (280/1 926), enteroaggregative Escherichia coli (EAEC) accounted for 18.21% (51/280), and ETEC accounted for 71.79% (173/280). In 2019, the total detection rate of DEC was 20.31% (301/1 482), EAEC accounted for 41.23% (116/301), and ETEC accounted for 48.93% (137/301). Compared with 2016, the detection rate of EAEC in 2019 increased significantly (χ2=29.26, P<0.001), followed by EPEC (χ2=9.37, P=0.002), and ETEC decreased (χ2=15.43, P<0.001). Compared with other pathogenic types, EAEC can easily cause nausea(χ2=32.72, P<0.001).The red blood cells(χ2=16.44, P=0.001) or the white blood cells (χ2=26.82, P<0.001) could be easily observed in stool specimens of patients infected with enteroinvasive Escherichia coli (EIEC). The resistance rates of EIEC to ampicillin, ampicillin/sulbactam and gentamicin were 80.95% (17/21), 66.67% (14/21) and 57.14% (12/21), respectively. Three strains of EAEC resistant to carbapenem antimicrobials were discovered in 2019 and of which two strains were resistant to ertapenem and imipenem, and the other one strain was only resistant to ertapenem. The whole genomic sequencing showed that there are multiple resistance mechanisms: including the mainly drug-resistant nodular cell differentiation family efflux pump, penicillin binding site mutation, and New Delhi metal-β-lactamase 5 production. Conclusions:The detection rate of DECs in adult patients with food-borne diarrhea is high, and the foremost subtype of DECs is ETEC. Compared with 2016, the detection rates of ETEC in clinical specimens decreased in 2019, and the detection rate of EAEC increased significantly, respectively. In 2019, a carbapenem-resistant antibacterial drug-resistant Escherichia coli strain was isolated. It is of great significance to focus on the biological characteristics and epidemiological changes of DEC.
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Objective:To investigate the effects of early applying of basic fibroblast growth factor (bFGF) on corneal haze formation after surface ablation surgery in rabbits.Methods:The right eyes of 60 healthy New Zealand white rabbits received photorefractive keratectomy (PRK) and were randomized into PRK+ normal saline group, PRK+ bFGF group and simple PRK group, with 20 rabbits in each group.Normal saline solution and bFGF were topically administered according to grouping, respectively, 3 times per day, 1 drop for each time until the sacrifice of the animals, and no drug was used in the PRK group.Another 8 normal rabbits were served as blank control group.The corneal healing response and haze formation were evaluated by anterior segment photography and anterior segment optical coherence tomography (AS-OCT) and graded based on Fantes criteria.Corneal histopathology was examined by hematoxylin-eosin staining.Immunohistochemistry was used to detect the expression of transforming growth factor-β 1(TGF-β 1), α-smooth muscle actin (α-SMA) and matrix metalloproteinase-2 (MMP-2) in cornea.This study protocol was approved by the Experimental Animal Ethic Committee of Affiliated Hospital of Binzhou Medical University (20180209-03). The use and care of the animals complied with the Statement of ARVO. Results:The corneal epithelium was completely healed in 3-4 days following surgery and there was not significantly different in healing time among the three groups.( F=0.57, P=0.57). The haze grading was significantly different among different groups at different time points ( Fgroup=41.736, P<0.01; Ftime=129.445, P<0.01) and showed the highest score in the PRK+ bFGF group on the 28th day after operation.On the 7th day after surgery, AS-OCT image showed that the surface reflection of corneal epithelium was continuous and smooth and corneal epithelium was not tightly attached to the superficial stromal layer; the reflection of the superficial stromal layer was enhanced in all the operation groups.The proliferation of corneal epithelial cells and superficial stromal layer in the operation area were seen under the optical microscope, and the arrangement of collagen fibers in the stromal layer was disordered with the most obvious changes in the PRK+ bFGF group in comparison with the PRK+ normal saline group and the simple PRK group, and these findings became worse on postoperative 28 days.The corneal epithelial surface reflection in the blank control group was continuous and smooth.Immunohistochemistry showed that a few MMP-2 positive cells were seen in the blank control group.TGF-β 1, α-SMA and MMP-2 proteins were positively expressed in the corneas 7 days after surgery in the three groups, and their expressions were the most obvious in the PRK+ bFGF in comparison with the PRK+ normal saline group and the PRK group and were enhanced 28 days after operation, showing statistically differences (all at P<0.05). Conclusions:Early application of bFGF following surface ablation surgery promotes the proliferation of corneal epithelial cells and irregular arrangement of collagen in the superficial stromal layer, which is associated with the expressions of haze-related factors TGF-β 1, α-SMA and MMP-2 in corneas.
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Objective To investigate the effect of conbercept on rabbit's haze after photorefractive keratectomy (PRK).Methods Sixty-four pedigree New Zealand white rabbits were randomly divided into PRK group,normal saline solution group,conbercept 0.5 mg group,conbercept 1.0 mg group,with 16 rabbits in each group.PRK was performed on right eyes,PRK group only received PRK,the other three surgery groups were given postoperative subconjunctival injection of 0.05 ml normal saline solution,0.5 mg (0.0:5 ml) conbercept and 1.0 mg(0.10 ml) conbercept,respectively.In addition,another 8 rabbits were randomly chosen as normal control group.The healing of postoperative corneal epithelial was observed by slit lamp biomicroscope,and the degrees of haze were graded based on Fantes.Eight rabbits in the surgery groups and 4 rabbits in the normal control group were killed in the first week and the fourth week.The corneal tissue was stained by hematoxylin-eosin,and the expressions of transforming growth factor-β1 (TGF-β1),α-smooth muscle actin (α-SMA) and matrix metalloproteinase-2 (MMP-2)were detected by immunochemistry.The use and feeding of experimental animals followed the Relevant Regulations of the Animal Management Committee of Binzhou Medical University.This study protocol was approved by Ethic Committee of the Affiliated Hospital of Binzhou Medical University (No.201701-08).Results Corneal epithelium of all operative rabbits healed completely at 3-5 days and no significant difference in healing time between the groups after operation (F=0.37,P =0.77).The degree of haze in each surgery group reached the highest value at about 4 weeks after operation,and haze in the conbercept 1.0 mg group was the most serious,followed by PRK group and normal saline solution group,haze in conbercept 0.5 mg group was significantly alleviated (Fgroup =20.114,P =0.000;Ftime =8.084,P =0.006).Hematoxylin-eosin staining showed that corneal epithelial cells and fibroblasts in superficial stroma proliferated in one week after PRK,which lead to the disorder of cells and collagen fibers,and the extent of hyperplasia was the same as that of haze.Immunohistochemistry showed that at one week after operation,the expressions of factors in PRK group and normal saline solution group were apparently lower than that of conbercept 1.0 mg group,but were apparently higher than that of conbercept 0.5 mg group,and the expressions of the factors were the weakest in normal control group,with significant differences between them (all at P ＜ 0.05).Conclusions Subconjunctival injection of appropriate conbercept can inhibit the formation of haze and the proliferation of corneal epithelium and superficial stroma,but overdose of conbercept leads to opposite effects.