ABSTRACT
Objective:To investigate the dynamic changes of the luminal microbiota in the jejunum following administration of proton pump inhibitors (PPIs) in a rat model.Methods:Rats were randomized into six groups (n =6 each group).A group of rats were sacrificed just after anesthesia as normal control (0 d) and,other five groups were continuously administered with omeprazole (10 mg/kg twice daily,intraperitoneally) and were euthanized at 5,9,14,21,28 days following the treatment,respectively.Total DNA in the luminal contents of jejunum was extracted and was used for polymerase chain reaction (PCR) amplification with the primer set targeted the hypervariable V3 region of 16S ribosomal RNA genes.Subsequently,the amplicons were separated by denaturing gradient gel electrophoresis (DGGE).After the gels were stained and photographed,the bands were cut out and sequenced to determine the closest bacterial relatives with the BLAST.The DGGE profiles were analyzed to evaluate the shifts of the microbiota composition and diversity following treatments.Results:Changes of the jejunal microbiotas in rats were observed at 5 and 9 days post PPI administration,as characterized by outgrowth of Streptococcus pneumonia,Clostridium saccharolyticum and Lactococcus garvieae compared to those of the controls (0 d).With time extension of PPI treatment,the mictobiotas significantly shifted toward dysbiotic state,in which the opportunistic pathogens,including Ertterococcus faecalis and Clostridium difficile,were strikingly expanded,especially 21 days later.However,the commensals such as Lactobacillus reuteri and Weissella koreensis were markedly declined in PPI-treated animals compared with the controls.The similarity of the jejunal microbiotas between PPI-treated animals and controls was markedly reduced following PPI treatment,reaching (56.1 ± 16.7) ％ at 28 days.Conclusion:Our data demonstrate that the gastric acid suppression could induce shifts of the jejuna microbiota in a rat model.More importantly,long-term use (＞ 14 d) of PPI could lead to the dysbiosis of the jejunal microbiota,which might be related causally to increased susceptibility to enteric infection.
ABSTRACT
Lysozyme hydrolyses bacterial cell walls and acts as a nonspecific innate immunity molecule against the invasion of bacterial pathogens. We cloned the cDNA of lysozyme from Fenneropenaeus chinensis and named Fc-lysozyme (FcLyz in short). The full length of the gene was of 709 bp, and the open reading frame (477 bp) encoded 158 amino acids. The predicted protein had a signal peptide (-1--18 residue) and molecular weight of the mature protein (residue 1-140) was of 16.2 kD. A Lyz 1 domain (residue 1-130) in the lysozyme was found by SMART analysis. The results of semiquantity RT-PCR showed that FcLyz was constitutively expressed in tested tissues in a low level in normal shrimp, and up-regulated in hemocytes, heart, hepatopancreas and gill of bacterial challenged shrimp. The DNA fragment of mature Fc-Lys was subcloned to pET-30a (+) expression vector, the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and then induced by isopropylthio-beta-D-galactoside (IPTG). The antibacterial activity of the purified recombinant FcLys was analyzed and minimal inhibitory concentration (MIC) was assayed. The recombinant protein showed high antibacterial activity against some Gram-positive bacteria, and MIC reached 3.43 micromol/L, and relatively low activity against Gram-negative bacteria. All together, the Fc-Lys was regulated by pathogen infection and had antibacterial activity. This suggested that the FcLyz may be one of the important molecules against pathogens in innate immunity of the shrimp.