Effects of ppk1 deletion on the drug susceptibility of uropathogenic Escherichia coli producing ESBLs / 中华预防医学杂志

To investigate the effect and the mechanism of ppk1 gene deletion on the drug susceptibility of uropathogenic Escherichia coli producing extended-spectrum beta-lactamases (ESBLs-UPEC). The study was an experimental study. From March to April 2021, a strain of ESBLs-UPEC (genotype was TEM combined with CTX-M-14) named as UE210113, was isolated from urine sample of the patient with urinary tract infection in the Laboratory Department of Guangzhou Eighth People's Hospital, meanwhile its ppk1 gene knock-out strain Δpk1 and complemented strain Δpk1-C were constructed by suicide plasmid homologous recombination technique, which was used to study the effect of ppk1 gene on ESBLs-UPEC drug sensitivity and its mechanism. The drug susceptibility of UE210113, Δpk1, and Δpk1-C were measured by Vitek2 Compact System and broth microdilution method. The quantitative expression of ESBLs, outer membrane protein and multidrug efflux systems encoding genes of UE210113, Δpk1 and Δpk1-C were performed by using qRT-PCR analysis. By using two independent sample Mann-Whitney U test, the drug susceptibility results showed that, compared with UE210113 strain, the sensitivities of Δpk1 to ceftazidime, cefepime, tobramycin, minocycline and cotrimoxazole were enhanced (Z=-2.121,P<0.05;Z=-2.236,P<0.05;Z=-2.236,P<0.05;Z=-2.121,P<0.05), and the drug susceptibility of Δpk1-C restored to the same as which of UE210113 (Z=0,P>0.05). The expression levels of ESBLs-enconding genes blaTEM and blaCTX-M-14 in Δpk1 were significantly down-regulated compared with UE210113, but the expression was not restored in Δpk1-C. The expression of outer membrane protein gene omp F in Δpk1 was significantly up-regulated, while the expression of omp A and omp C were down-regulated. The results showed that the expression of multidrug efflux systems encoding genes tol C, mdt A and mdtG were down-regulated in Δpk1 compared with UE210113. The expression of all of the outer membrane protein genes and the multidrug efflux systems genes were restored in Δpk1-C. In conclusion,the lost of ppk1 gene can affect the expression of the outer membrane protein and multidrug efflux systems encoding genes of ESBLs-UPEC, which increase the sensitivity of ESBLs-UPEC to various drugs.

Effects of ppk1 deletion on the drug susceptibility of uropathogenic Escherichia coli producing ESBLs / 中华预防医学杂志

To investigate the effect and the mechanism of ppk1 gene deletion on the drug susceptibility of uropathogenic Escherichia coli producing extended-spectrum beta-lactamases (ESBLs-UPEC). The study was an experimental study. From March to April 2021, a strain of ESBLs-UPEC (genotype was TEM combined with CTX-M-14) named as UE210113, was isolated from urine sample of the patient with urinary tract infection in the Laboratory Department of Guangzhou Eighth People's Hospital, meanwhile its ppk1 gene knock-out strain Δpk1 and complemented strain Δpk1-C were constructed by suicide plasmid homologous recombination technique, which was used to study the effect of ppk1 gene on ESBLs-UPEC drug sensitivity and its mechanism. The drug susceptibility of UE210113, Δpk1, and Δpk1-C were measured by Vitek2 Compact System and broth microdilution method. The quantitative expression of ESBLs, outer membrane protein and multidrug efflux systems encoding genes of UE210113, Δpk1 and Δpk1-C were performed by using qRT-PCR analysis. By using two independent sample Mann-Whitney U test, the drug susceptibility results showed that, compared with UE210113 strain, the sensitivities of Δpk1 to ceftazidime, cefepime, tobramycin, minocycline and cotrimoxazole were enhanced (Z=-2.121,P<0.05;Z=-2.236,P<0.05;Z=-2.236,P<0.05;Z=-2.121,P<0.05), and the drug susceptibility of Δpk1-C restored to the same as which of UE210113 (Z=0,P>0.05). The expression levels of ESBLs-enconding genes blaTEM and blaCTX-M-14 in Δpk1 were significantly down-regulated compared with UE210113, but the expression was not restored in Δpk1-C. The expression of outer membrane protein gene omp F in Δpk1 was significantly up-regulated, while the expression of omp A and omp C were down-regulated. The results showed that the expression of multidrug efflux systems encoding genes tol C, mdt A and mdtG were down-regulated in Δpk1 compared with UE210113. The expression of all of the outer membrane protein genes and the multidrug efflux systems genes were restored in Δpk1-C. In conclusion,the lost of ppk1 gene can affect the expression of the outer membrane protein and multidrug efflux systems encoding genes of ESBLs-UPEC, which increase the sensitivity of ESBLs-UPEC to various drugs.

Dynamic Changes and Differences of B-cell Responses between Severe Dengue Disease and Mild Dengue Fever / 中山大学学报(医学科学版)

@#【Objective】 To find out the possible marker for early warning or therapeutic target of severe dengue disease ，we studied B cell responses during natural dengue virus（DENV）infection and their association with disease severity.【Methods】Sixty-two cases and their blood samples were collected from the patients hospitalized during September to December in 2014 and three groups were designated as mild dengue（DF，n=33），severe dengue（SD，n=29）and control group（Control ，n=6）. Multicolor flow cytometrywas used to analyze the dynamic changes of B cells and plasmablasts in the peripheral blood of patients during the acute phase and the critical phase. The plasmablasts in a succession of samplesfrom the same infected patients （including 3 mild and 3 severe cases） were further analyzed. We also observed the changes of B cells and their subsets ，including naive B cells ，memory B cells and plasmablasts，during primary and secondary infections. 【Results】 The expansion of B cells in the severe group was significantly higher than that in the mild group（P=0.013），especially on day 5 and day 6 after the symptom onset（P = 0.017 and 0.002，respectively）. Compared with those in the control group，the plasmablasts in the mild and severe groups showed significant proliferation（P=0.011 and 0.032，respectively），but no statistical difference was observed between these two groups. The analysis of the serial blood samples showed that the plasmablasts，in mild cases，peaked on day 7 and 8 and returned to baseline on day 10，whereas in the severe group，peaked on day 7 to 9，and still existed at a certain rate after day 10. In mild dengue group，more proliferative B cells，less naive B cells and memory B cells were found in secondary infection than in primary infection（P = 0.028，0.010 and 0.037，respectively），but plasmablasts revealed no difference. In severe dengue group， only naïve B cells significantly decreased in secondary infection （P = 0.018）. 【Conclusion】 B cell responses between mild and severe dengue after DENV- 1 infection present different trends. The significant proliferation of B cells in the early stage of the disease and the persistent existence of plasmablasts may be related to the severity of the dengue disease.