Objective:To analyze the biological characteristics and genomic features of a Streptococcus pyogenes strain isolated from the pus of a child with an atypical brain abscess. Methods:The Streptococcus pyogenes strain, named 21SPY7071 ( emm22 type), was isolated from the brain abscess specimen of a child undergoing surgery for brain abscess. The biological characteristics of this strain were analyzed using blood agar culture, Gram staining, growth curve measurement, and hemolytic analysis. After whole-genome sequencing, core genome multilocus sequence typing (cgMLST) was performed to analyze the cgST type of the strain, and the distribution of virulence genes and drug resistance genes were analyzed. qRT-PCR was used to detect the differences in the expression of virulence genes at mRNA level between the strain 21SPY7071 and two Streptococcus pyogene strains of ATCC 19615 ( emm80 type) and GAS029 ( emm22 type, clinical isolate). Results:The strain 21SPY7071 was a Gram-positive Streptococcus with atypical weak β-hemolysis. Whole-genome sequencing and cgMLST revealed that this isolate belonged to the cgST type of 65937, containing 187 virulence genes and 97 drug resistance genes, with high sequence similarity (95.16%~100.00%) to the main virulence factor-encoding genes in the Virulence Factor Database. Compared with the strains of ATCC 19615 and GAS029, the strain 21SPY7071 showed reduced expression of genes encoding streptolysin S ( sagABCDEFGHI), streptolysin O ( slo), and hyaluronate lyase ( endA/ sdaB). Besides, compared with the strain ATCC 19615, the strain 21SPY7071 also showed decreased expression of virulence genes including speG, ideS/ mac, hylA, smeZ, lmb, and scpA/ scpB. Conclusion:This Streptococcus pyogenes strain isolated from the pus of a child with atypical brain abscess exhibits weak hemolysis and lower expression of virulence genes, which may be related to the mild clinical symptoms observed in the child.

Objective:To investigate the effects of cryopreservation on the results of nucleic acid detection and culture for Bordetella pertussis in residual culture-positive nasopharyngeal swab specimens, aiming to provide the basis for specimens preservation, transport and centralized detection. Methods:In this cross-sectional study, the residual nasopharyngeal swab specimens which were culture-positive for Bordetella pertussis were collected in the Children′s Hospital, Zhejiang University School of Medicine from January to August in 2022. The specimens were placed at ?20 ℃ and?70 ℃ by random number table method, respectively. Re-detection by culture and PCR for Bordetella pertussis were conducted after these specimens were frozen for 114.3±31.9 days. The specimens were grouped according to the cryopreservation temperature and the semi-quantitative results by bacteria culture. The positive rates of the results were compared with χ 2 test between groups. Results:A total of 244 nasopharyngeal swabs specimens were included and 166 were culture-positive after cryopreservation, the positive rate decreased by 32%. Among them, the positive rate of re-culture of specimens containing low bacterial loads decreased by 56% after cryopreservation. However, there was no significant difference in the positive rate of culture between the specimens freezing at ?70 ℃ and ?20 ℃ (χ2=1.65, P=0.20). The positive rate of DNA detection decreased by 10.6% (88.9% vs 78.3%) after cryopreservation. The positive rate of the ?70 ℃ storage group was significantly higher than that of the ?20 ℃ group (χ2=5.11, P=0.02). The positive rate of the re-detection of DNA of nasopharyngeal swabs with low bacteria loads in ?70 ℃ storage group was significantly higher than that of the ?20 ℃ group (χ2=4.86, P=0.03). While for the samples with a bacterial load of "+" or more, there was no significant difference in the positive rate of DNA detection after cryopreservation between the ?20 ℃ and -70 ℃ (χ2=1.25, P=0.26) groups. The positive rate of nasopharyngeal swab culture after cryopreservation was 68.0% (166/244), which was significantly lower than the DNA detection positive rate of 78.3% (191/244, χ2=6.52, P=0.01). Conclusions:Cryopreservation nasopharyngeal swabs specimens could be used for Bordetella pertussis culture and nucleic acid detection. The bacterial load in the original sample affects the positive detection rate after cryopreservation. Cryopreservation has less influence on the positive rate of the result of nucleic acid detection when compared with culture. Preservation at ?70 ℃ is superior to ?20 ℃.

Objective:To analyze the biological characteristics and genomic features of a Streptococcus pyogenes strain isolated from the pus of a child with an atypical brain abscess. Methods:The Streptococcus pyogenes strain, named 21SPY7071 ( emm22 type), was isolated from the brain abscess specimen of a child undergoing surgery for brain abscess. The biological characteristics of this strain were analyzed using blood agar culture, Gram staining, growth curve measurement, and hemolytic analysis. After whole-genome sequencing, core genome multilocus sequence typing (cgMLST) was performed to analyze the cgST type of the strain, and the distribution of virulence genes and drug resistance genes were analyzed. qRT-PCR was used to detect the differences in the expression of virulence genes at mRNA level between the strain 21SPY7071 and two Streptococcus pyogene strains of ATCC 19615 ( emm80 type) and GAS029 ( emm22 type, clinical isolate). Results:The strain 21SPY7071 was a Gram-positive Streptococcus with atypical weak β-hemolysis. Whole-genome sequencing and cgMLST revealed that this isolate belonged to the cgST type of 65937, containing 187 virulence genes and 97 drug resistance genes, with high sequence similarity (95.16%~100.00%) to the main virulence factor-encoding genes in the Virulence Factor Database. Compared with the strains of ATCC 19615 and GAS029, the strain 21SPY7071 showed reduced expression of genes encoding streptolysin S ( sagABCDEFGHI), streptolysin O ( slo), and hyaluronate lyase ( endA/ sdaB). Besides, compared with the strain ATCC 19615, the strain 21SPY7071 also showed decreased expression of virulence genes including speG, ideS/ mac, hylA, smeZ, lmb, and scpA/ scpB. Conclusion:This Streptococcus pyogenes strain isolated from the pus of a child with atypical brain abscess exhibits weak hemolysis and lower expression of virulence genes, which may be related to the mild clinical symptoms observed in the child.

Objective:To investigate the effects of cryopreservation on the results of nucleic acid detection and culture for Bordetella pertussis in residual culture-positive nasopharyngeal swab specimens, aiming to provide the basis for specimens preservation, transport and centralized detection. Methods:In this cross-sectional study, the residual nasopharyngeal swab specimens which were culture-positive for Bordetella pertussis were collected in the Children′s Hospital, Zhejiang University School of Medicine from January to August in 2022. The specimens were placed at ?20 ℃ and?70 ℃ by random number table method, respectively. Re-detection by culture and PCR for Bordetella pertussis were conducted after these specimens were frozen for 114.3±31.9 days. The specimens were grouped according to the cryopreservation temperature and the semi-quantitative results by bacteria culture. The positive rates of the results were compared with χ 2 test between groups. Results:A total of 244 nasopharyngeal swabs specimens were included and 166 were culture-positive after cryopreservation, the positive rate decreased by 32%. Among them, the positive rate of re-culture of specimens containing low bacterial loads decreased by 56% after cryopreservation. However, there was no significant difference in the positive rate of culture between the specimens freezing at ?70 ℃ and ?20 ℃ (χ2=1.65, P=0.20). The positive rate of DNA detection decreased by 10.6% (88.9% vs 78.3%) after cryopreservation. The positive rate of the ?70 ℃ storage group was significantly higher than that of the ?20 ℃ group (χ2=5.11, P=0.02). The positive rate of the re-detection of DNA of nasopharyngeal swabs with low bacteria loads in ?70 ℃ storage group was significantly higher than that of the ?20 ℃ group (χ2=4.86, P=0.03). While for the samples with a bacterial load of "+" or more, there was no significant difference in the positive rate of DNA detection after cryopreservation between the ?20 ℃ and -70 ℃ (χ2=1.25, P=0.26) groups. The positive rate of nasopharyngeal swab culture after cryopreservation was 68.0% (166/244), which was significantly lower than the DNA detection positive rate of 78.3% (191/244, χ2=6.52, P=0.01). Conclusions:Cryopreservation nasopharyngeal swabs specimens could be used for Bordetella pertussis culture and nucleic acid detection. The bacterial load in the original sample affects the positive detection rate after cryopreservation. Cryopreservation has less influence on the positive rate of the result of nucleic acid detection when compared with culture. Preservation at ?70 ℃ is superior to ?20 ℃.

Objective To investigate the effects of Dermatophagoides pteronyssinus ( Der p) on the expression of TLR4 and IL-4 in P815 mast cells and to further analyze the immunoregulatory effects of 1,25-(OH)2D3 on Der p treated P815 mast cells.Methods Different concentrations of Der p and 1,25-( OH) 2 D3 were used alone or in combination to stimulate P815 mast cells.The supernatants of the stimulated cell culture were analyzed by enzyme-linked immunosorbent assay ( ELISA) for the detection of IL-4.The stimulated cells were collected and analyzed by real-time PCR and Western blot assays for the detection of TLR4atmRNAandproteinlevels,respectively.Results (1)TLR4expressionwasdetectedinP815 cells.The expression of TLR4 was enhanced in P815 cells treated with various concentrations of Der p.A significant dose-dependent up-regulation of TLR4 was observed in P815 cells after incubation with Der p for 36 h.(2) Der p promoted the release of IL-4 in P815 cells (P<0.05).(3) No significant differences with the expression of TLR4 and IL-4 were observed among 1,25(OH)2D3 treatment groups as compared with the control group (P>0.05).(4) 10-8 mol/L of 1,25(OH)2D3 promoted the Der p-induced expression of TLR4 in P815 cells (P<0.01).However, 1,25(OH)2D3 inhibited the release of IL-4 in a dose-dependent manner(P<0.05orP<0.01).Conclusion (1)Derpcouldpromotetheinflammationandallergicreac-tion through up-regulating TLR4 and IL-4 in mast cells.(2) The possible mechanism for the inhibitory of 1, 25(OH)2D3 on Der p-induced immune responses was due to the suppression of Th2-type immune responses through inhibiting the synthesis and secretion of IL-4 in mast cells.