Comparison of Class 2 Integron Integrase Activities.

Integrons play important roles in the dissemination of antimicrobial resistant genes among bacteria. Class 2 integrons usually has an internal stop codon, TAA, in integrase genes (intI2), leading to a truncated integrase, IntI2*. However, a few class 2 integrons with a natural full-length integrase have been reported. In this study, the sequences of natural full-length intI2 were extracted from INTEGRALL database and analyzed. A total of 236 sequences of intI2 were retrieved from INTEGRALL database, only seven of which were natural full-length intI2 genes and could be divided into five types according to their coding amino acid sequence. Quantitative real-time PCR was used to detect gene cassette sat2 integration and excision efficiency catalyzed by different natural full-length IntI2s. The results showed that all five IntI2s could catalyze attI2 × attCsat2 integration and attCdfrA1/sat2 × attCsat2/aadA1 excision in Escherichia coli. Integration and excision frequency catalyzed by IntI2A176 was highest and was about twofold as high as those catalyzed by IntI2S175_A176. The secondary structure of the IntI2 was predicted by online software. Polymorphisms of these five IntI2s were limited within residues 172, 174, 175, 176 and 256, and these residues were all far away from the predicted DNA binding regions or catalyzed sites. Influence of amino acid sequence polymorphisms of these natural full-length IntI2s on their catalyzed activities is limited.

Characterization of Beta-Lactamases in Bloodstream-Infection Escherichia coli: Dissemination of blaADC - 162 and blaCMY- 2 Among Bacteria via an IncF Plasmid.

OBJECTIVES: To describe the molecular characteristics of beta-lactamases in bloodstream-infection Escherichia coli isolated from elderly patients, and to determine the genotypic patterns of bla CMY - 2 and bla ADC - 162. METHODS: A total of 50 bloodstream-infection E. coli isolates were obtained from patients aged > 50 years at Shanghai Sixth People's Hospital South Campus during 2015-2018. The isolates were subjected to beta-lactamase detection using phenotypic and molecular methods. Beta-lactamase genes were verified by sequencing and the phylogenetic relationships of the isolates were analyzed by multilocus sequence typing (MLST). The transferability of plasmids carrying bla CMY- 2 and bla ADC- 162 genes was verified by conjugation experiments and plasmid replicon typing. RESULTS: Eight beta-lactamase subtypes were detected in 50 isolates of bloodstream-infection E. coli. bla TEM- 1 (21/50) was the most common beta-lactamase gene, followed by bla CTX-M- 14 (8/50), bla OXA- 27 (5/50), bla CTX-M- 27 (3/50), bla CTX-M- 65 (1/50), bla ADC- 162 (1/50), and bla CMY- 2 (1/50). Of these, bla ADC- 162 (ST95-A), and bla CMY- 2 (ST95-B2) have not previously been reported in bloodstream-infection E. coli. In 21 isolates, beta-lactamase genes were located on conjugative plasmids belonging to incompatibility groups FrepB (n = 7), FIA (n = 1), FIC (n = 2), K (n = 8), N (n = 1), and I (n = 1), and bla CTX-M was associated with the common elements ISEcp1, IS903, and IS26, but with special sequences (region V, region Y, and region W) for ISEcp1 in 14 isolates. CONCLUSION: To the best of our knowledge, this study provides the first molecular characterization of beta-lactamase genes in E. coli isolated from the bloodstream in elderly patients. Beta-lactamase genes were detected at a relatively high frequency in elderly patients with bloodstream E. coli infections. Plasmid replicon analysis showed that horizontal dissemination of beta-lactamase genes was mainly mediated by IncK and IncF plasmids, which could encode multidrug resistance genes. The study also provides the first report of ISAba1-bla ADC - 162-tnpA and ISEcp1-bla CTX-M- 14-IS903-bla CMY- 2-blc-sugE in E. coli, and demonstrates IncF plasmid-mediated bla ADC - 162 and bla CMY- 2 gene dissemination among bacteria.

Tetramethylpyrazine attenuates atherosclerosis development and protects endothelial cells from ox-LDL.

PURPOSE: We assessed whether tetramethylpyrazine (TMP), an active ingredient of Ligusticum wallichii Franchat, attenuates atherosclerosis (AS) development in rabbits and protects endothelial cells injured by ox-LDL. METHODS: In vivo, rabbits subjected to atherosclerosis were treated with TMP (75 and 150 mg/kg) by oral gavage for 12 weeks. In vitro, rat aortic endothelial cells (RAECs) were stimulated by ox-LDL. RESULTS: TMP treatment with 75 and 150 mg/kg significantly reduced the relative atherosclerosis area ratio in the aorta (0.41 ± 0.042, 0.27 ± 0.047 vs. 0.66 ± 0.058 in AS), the ratio of intimal/medial thickness (0.54 ± 0.09, 0.39 ± 0.07 vs. 1.1 ± 0.3 in AS) and the number of monocytes in intimal (10.1 ± 2.8, 8.2 ± 2.0 vs. 14.1 ± 4.9 counts/mm(2) in AS). TMP also decreased levels of TC (15 ± 4.2 to 6.1 ± 1.2 mmol/L), TG (1.8 ± 0.3 to 1.08 ± 0.24 mmol/L), LDL-C (20.1 ± 4.3 to 10.2 ± 1.6 mmol/L) and increased HDL-C levels (0.40 ± 0.08 to 0.85 ± 0.17 mmol/L) in atherosclerosis rabbit plasma. TMP decreased the MCP-1 (187.3 ± 38.4 to 86.1 ± 17.2 pg/ml) and ICAM-1 (350.6 ± 43.7 to 260.6 ± 46.1 pg/ml) levels in plasma and inhibited LOX-1 expression in the rabbit aortas. Moreover, our in vitro study revealed that TMP suppressed monocyte adhesion to RAECs, inhibited RAEC migration, and down-regulated MCP-1 and ICAM-1 expression in ox-LDL-injured RAECs. Likewise, TMP inhibited LOX-1 and 5-LOX expression, and prevented nuclear accumulation of RelA/p65 and IκB degradation in ox-LDL-injured RAECs. Furthermore, TMP suppressed ox-LDL-induced activations of p-ERK, p-p38, and p-JNK MAPK. CONCLUSION: TMP produces a tangible protection in atherosclerosis and endothelial cells. TMP might be a potential protective agent for atherosclerosis.

Preparation and characterization of insulin-loaded bioadhesive PLGA nanoparticles for oral administration.

Poly(D,L-lactide-co-glycolide) nanoparticles (PLGA-NP) have been extensively used as a drug delivery system for proteins and peptides. However, their negative surface charge decreases bioavailability under oral administration. Recently, cationically modified PLGA-NP has been introduced as novel carriers for oral delivery. The characteristics of the nanoparticles, such as particle size, surface charge, and bioadhesion are considered the most significant determinants of the effect of these nanoparticles both in vitro and in vivo. Our aim was to introduce and evaluate the physiochemical characteristics, bioadhesion, and biological activity of positively charged chitosan-coated PLGA-NP (CS-PLGA-NP), using insulin as a model drug. Results were compared to those of common negatively charged PLGA-NP and the in vitro cytotoxicity of the two types of nanoparticles was examined. These results indicate that both CS-PLGA-NP and PLGA-NP had a narrow size distribution, averaging less than 150 nm. CS-PLGA-NP was positively charged (+43.1 ± 0.3 mV), exhibiting the cationic nature of chitosan, whereas PLGA-NP showed a negative surface charge (-1.72 ± 0.2 mV). CS-PLGA-NP exhibited stronger bioadhesive potency than PLGA-NP and much greater relative pharmacological availability with regard to orally delivered insulin. In addition, an evaluation of cytotoxicity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed no increase in toxicity in either kind of nanoparticle during the formulation process. The study proves that CS-PLGA-NP can be used as a vector in oral drug delivery systems for proteins and peptides due to its positive surface charge and bioadhesive properties.