Objective To investigate the mechanism of drug resistance mediated by micF gene and outer membrane porin F ( OmpF) in Shigella strains. Methods Shigella strains were isolated from stool samples of patients who presented to the Second Hospital of Tianjin Medical University with acute diar-rhea in 2015. Antibiotic susceptibility test was performed to screen out the multidrug-resistant and non-multi-drug-resistant strains. The ompF gene was amplified by PCR. The micF and ompF genes at transcriptional levels in the two groups of strains were detected by quantitative real-time RT-PCR. Intracellular concentra-tions of ciprofloxacin in the two groups of Shigella strains were measured by automatic microplate reader. Re-sults According to the result of antibiotic susceptibility test, 13 strains that were resistant to 3 or more than 3 antibiotics were classified into the multidrug-resistant group, while the other 8 strains that were sensitive to all antibiotics used in this study or only resistant to 1 or 2 antibiotic were classified into the non-multidrug-re-sistant group. All of the 21 Shigella strains carried the ompF gene. Compared with the non-multidrug-resist-ant strains, the multidrug-resistant strains showed higher expression of micF gene, but lower expression of ompF gene. The differences in micF and ompF genes between the two groups were statistically significant. The result of correlation analysis suggested that there was a negative correlation between micF and ompF genes (r=-0. 244). The intracellular concentrations of ciprofloxacin in multidrug-resistant strains were low-er than those in the non-multidrug-resistant strains (P<0. 001). Conclusion The decreased expression of OmpF was one of the possible mechanisms of multidrug-resistance in Shigella strains. The micF gene was negatively related to the expression of OmpF. Moreover, the decreased intracellular concentrations of cipro-floxacin in multidrug-resistant strains might be related to the decreased expression of OmpF.

Objective To understand genetic distribution, drug resistance, molecular typing and the epidemiological relativeness between strains of the Shigella boydii virulence. Methods Nine Shigella boydii strains were isolated form stool samples of patients with diarrhea from the Enteric Disease Clinic of the Second Hospital of Tianjin Medical University in June-October 2015. The strains were identified by biochemical test and serum agglutination test. Antibiotics susceptibility test was carried out using the Kirby-Bauer method. Polymerase chain reaction was used for detecting virulence genes. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) technique were used to determine the epidemiological relationship between nine Shigella boydii strains. Results There were three subtypes in nine isolated Shigella boydii samples, including one, three and five isolates inⅠ,Ⅱ,Ⅳsubtypes respectively. All of the 9 isolates were multi-drug resistant. The resistant rate of these strains for ampicillin was 100%(9/9), and then the resistant rates of these strains for ceftazidime, streptomycin, gentamicin, trimethoprim/sulfamethoxazole, cefotaxime, ceftriaxone, norfloxacin and levofloxacin were 1/9, 4/9, 4/9, 4/9, 5/9, 5/9, 6/9, 6/9 and 6/9, respectively. All of these strains were sensitive to amikacin, cefperazone-sulbactam and imipenem. The ipaH was carried by all the testing strains, and none of the strains carried the sen, set1A, set1B, ial, virA, icsA and SigA. The detective rates of pic, sepA and sat were 4/9, 5/9 and 7/9 strains, respectively. Nine shigella boydii strains were divided into 8 PFGE types. The similarity between the spectrums of PFGE was 63.21%-100%. Multilocus sequence typing showed that six isolates were belonged to ST648, two isolates were ST131 and one isolate was ST10. Conclusion Nine isolates of Shigella boydii (divided into three subtyping) isolated from our hospital are multi-drug resistant and they have distant relationships, belonging to the dissemination of case.

Objective:To establish a double antibody sandwich ELISA assay for detection of human IL-37 in serum.Methods: Mouse anti-human IL-37 monoclonal antibody was used as capturing antibody,rabbit anti-human IL-37 polyclonal antibodies served as detection antibody,HRP labeled goat anti-rabbit IgG employed as second antibody and recombinant human IL-37 protein used as reference standard for the establishment of a doubleantibody sandwich ELISA.The working conditions were optimized,such as sensitivity,linear range,reproducibility and evaluated the serum IL-37 in patients with Dengue fever.Results: The sensitivity of the established ELISA method was 1.465 μg/L approximately.Likewise,the linearity range of this method was about (1.465-46.875) μg/L.Further,the co efficient of variation (CV) of inter-batch and intra-batch in this study were 6.6％ and 11.7％,respectively.Notably,this method could be used in the detection of IL-37 in serum of the patients with Dengue fever,showing that the level of IL-37 in Dengue fever patients was much higher than that in healthy controls.Conclusion: The double antibody sandwich ELISA assay for the detection of human IL-37 was successfully established,which can be apply to detect of human IL-37 in clinical samples.

Objective To investigate the antimicrobial resistance ,molecular phenotypes ,virulence gene profiles of Salmonella A gona (S .A gona) isolated from patients with acute diarrhea ,and to better understand its epidemic trend ,prevention and treatment .Methods Clinical data and stool samples of patients with acute diarrhea during April to October in 2013 and 2014 from the Second Hospital of Tianjin Medical University were collected .Enrichment culture ,biochemical identification and serotyping analysis were used to isolate and identify S .A gona strains .The isolated strains were further analyzed with antibiotics susceptibility test ,pulsed field gel electrophoresis (PFGE) ,multiple locus sequence typing (MLST ) , Quinolone resistance determining region (QRDR) .Plasmid-mediated quinolone resistance (PMQR) and β-lactamases genes (TEM ,SHV ,OXA ,and CTX-M) were amplified by polymerase chain reaction (PCR) and sequencing .The representative genes carried by Salmonella pathogenicity islands (SPI) 1 — 6 ,9 — 12 and virulence plasmids were amplified by PCR .And the clinical characteristics of S .Agona infection were analyzed .Results Among 119 non-repetitive (non-typhoidal salmonella ,NTS) isolates during the two years ,eight isolates (6 .7％ ) of S .A gona were identified . The resistance rate of S .A gona strains to streptomycin was 100 .0％ , those to ampicillin and gentamicin were 62 .5％ ,to levofloxacin ,ciprofloxacin and nalicixic acid were 25 .0％ ,to chloramphenicol ,amoxillin/clavulanic acid and piperacillin tazobactam were 12 .5％ .The strains were susceptible to other drugs .All 8 isolates had the identical ST13 genotype .PFGE showed 5 clones ,and 4 out of 5 isolates had the exact same patterns of PFGE and drug susceptibility .Two (fluoroquinolones ,FQ) resistant strains carried gyrA mutation leading to amino acid substitutions at position 87 in GyrA ,and no PMQR genes was detected ,while one of which was sensitive to ciprofloxacin by K-B method .All five ampicillin-resistant isolates were positive for TEM-1b gene and one isolate of them was resistant to β-lactam/β-lactamase inhibitor complex .The representative genes carried by SPI 1 — 6 , 9 ,11 ,12 (hilA ,sseL ,mgtC ,siiE ,sopB ,pagN ,bapA ,pagC and sspH2) were 100 .0％ positive ,while the genes carried by SPI10 (sef A ) virulence plasmids (spvB , prot6E) were negative . Two patients with FQ resistant strains infection were clinically diagnosed with bacillary dysentery ,and the remaining six cases with FQ susceptible strains infection were clinically diagnosed with acute gastroenteritis .Conclusions FQs-resistant and multi-drug resistant S .A gonaisolates have emerged in clinical settings .These isolates carry a variety of virulence genes .Resistance to FQ of S .Agonamay cause more severe illness .ST13 might be the dominant genotype of S . A gona in China ,and we should try to prevent the infection outbreak of S .A gona .

Objective To investigate the mechanism of efflux pump AcrAB-TolC involved in the cross-resistance between quaternary ammonium compounds ( QAC) and fluoroquinolones ( FQ) in Escherich-ia coli. Methods Seventy-eight Escherichia coli strains were isolated from clinical samples and then tested for the sensitivity to benzalkonium bromide by ager dilution method. Six strains were randomly selected and induced with benzalkonium bromide. Changes in the sensitivity of these strains to benzalkonium bromide and ciprofloxacin were analyzed after induction. Expression of the efflux pump genes acrA, acrB and tolC at mRNA level in the induced strains and their parent strains were detected by quantitative real-time PCR. Re-sults Among the 78 strains, the minimum inhibitory concentrations ( MIC) ranged from 8 μg/ml to 64μg/ml and 47. 4％ of strains have higher MIC than the standard strain. Compared with the parent strains, the induced strains showed higher expression of acrA and tolC genes, and the differences between the two groups were statistically significant. The MIC values of ciprofloxacin to the six induced strains were 4-8 times higher than those to their parent strains. Conclusions It was speculated that the increased expression of acrA and tolC genes at transcription level in the induced strains were related to the cross-resistance between quaternary ammonium compounds and fluoroquinolone antibiotics.

BACKGROUND:Although the clinical application of Masquelet technology has achieved extensive success,the research on optimizing all aspects of Masquelet technology is still being carried out.The focus of doctors is to speed up bone healing and shorten bone healing time after bone grafting. OBJECTIVE:To observe the effect of calcium phosphate combined with recombinant human bone morphogenetic protein-2 in repairing tibial infectious bone defects. METHODS:Thirty-one patients with tibial infectious bone defects were selected from The People's Hospital of Jianyang City from June 2017 to June 2022.They were treated with the Masquelet membrane induction technique.During the second stage of operation,they were divided into a control group(n=15)and a study group(n=16)according to different bone graft materials.Patients in the control group were implanted with autologous bone/allogeneic bone particles,and those in the study group were implanted with calcium phosphate combined with recombinant human bone morphogenetic protein-2/autologous bone particles.Six months after the second stage operation,peripheral blood inflammatory indexes such as white blood cell count,C-reactive protein,and erythrocyte sedimentation rate were detected.Imaging bone healing time,bone healing X-ray score,bone defect healing classification,and adjacent joint function were recorded.The presence of nail track infection,implant absorption,pain,and infection in the bone extraction area were observed. RESULTS AND CONCLUSION:(1)White blood cell count,erythrocyte sedimentation rate,and C-reactive protein levels of the two groups 6 months after the second stage operation were significantly lower than those before the first stage operation(P<0.05).There was no significant difference in each index between the two groups(P>0.05).(2)Bone healing time in the study group was shorter than that in the control group(P<0.05).(3)The Samantha X-ray score of the study group 6 months after the second stage operation was higher than that of the control group(P<0.05).The excellent and good rate of bone defect healing and adjacent joint function of the study group was higher than that of the control group(P<0.05).There was no significant difference in the recurrence rate and complication rate between the two groups(P>0.05).(4)These findings indicate that the effect of calcium phosphate combined with recombinant human bone morphogenetic protein-2 during the second stage operation of the Masquelet membrane induction technique in the treatment of tibial infectious bone defect is good and safe.