25Pyrosequencing followed by conventional PCR and sequencing was used to determine 26 the complete nucleotide sequence of three plasmids (pRCEID2.9, pRCEID3.2 and 27 pRCEID13.9) from the Lactobacillus casei strain TISTR1341. The plasmid sequences were 28 found to be almost identical, respectively, to those of pLA106, pLA105 and pLA103 from
29Lactobacillus acidophilus strain TK8912, suggesting these strains may be related. Sequence
Many microbial species have been recognized as enteropathogens for humans. Here, we predicted the causative agents of acute diarrhea using data from multiplex quantitative PCR (qPCR) assays targeting 19 enteropathogens. For this, a case-control study was conducted at eight hospitals in Thailand. Stool samples and clinical data were collected from 370 hospitalized patients with acute diarrhea and 370 non-diarrheal controls. Multiple enteropathogens were detected in 75.7% and 13.0% of diarrheal stool samples using multiplex qPCR and bacterial culture methods, respectively. Asymptomatic carriers of enteropathogens were found among 87.8% and 45.7% of individuals by qPCR and culture methods, respectively. These results suggested the complexity of identifying causative agents of diarrhea. An analysis using the quantification cutoff values for clinical relevance drastically reduced pathogenpositive stool samples in control subjects from 87.8% to 0.5%, whereas 48.9% of the diarrheal stool samples were positive for any of the 11 pathogens. Among others, rotavirus, norovirus GII, Shigella/ EIEC, and Campylobacter were strongly associated with acute diarrhea (P-value < 0.001). Characteristic clinical symptoms, epidemic periods, and age-related susceptibility to infection were observed for some enteropathogens. Investigations based on qPCR approaches covering a broad array of enteropathogens might thus improve our understanding of diarrheal disease etiology and epidemiological trends. Diarrheal diseases are one of the major causes of mortality and morbidity worldwide, especially during the first 5 years of life for individuals subjected to malnutrition 1-3. Diarrhea can be defined by increased stool frequency, liquidity, or volume 4. A wide range of enteropathogens including bacteria, viruses, and protozoa have been
There is an increasing interest to develop various lactic acid bacteria (LAB) species as mucosal delivery vehicles, for which the development of a variety of cloning and expression systems for these bacteria is of primary importance. This study reports the complete nucleotide sequence of the cryptic plasmid pRCEID7.6 derived from the chicken probiotic LAB strain Lactobacillus casei TISTR1341. Sequence analysis and comparison showed that pRCEID7.6 is composed of nine putative open reading frames. The replicon origin of pRCEID7.6 consisted of untranslated origin of replication and translated replication protein B sequences. This region was used to construct Escherichia coli/L. casei shuttle vectors carrying erythromycin and chloramphenicol resistance genes as selective markers. Segregation and structural stability of the vectors in L. casei was sufficient for most genetic applications. The feasibility of this vector for heterologous protein expression in L. casei was determined by cloning in pRCEID-LC7.6, the gene encoding the nucleocapsid protein (NP), from the influenza A virus under the control of the homologous promoter from the lactate dehydrogenase gene. L. casei carrying this recombinant plasmid was shown to successfully express the NP protein. Therefore, this shuttle vector can be used for further study in the development of mucosal delivery vehicles.
Aims: In this study, lactic acid bacteria (LAB) were isolated from 42 healthy infants and determined for probiotic properties. Twelve LAB isolates with potential probiotic properties were selected and screened for their feasibility of heterologous protein expression by selection of erythromycin sensitive isolates. Methodology and results: One of eleven erythromycin-sensitive LAB isolates identified and designated as Lactobacillus fermentum 47-7 was able to acquire and stable maintain the Escherichia coli-Lactobacillus shuttle vector, pRCEID-LC13.9. Further electrotransformation of L. fermentum 47-7 with the recombinant pLC13.9:LDH-PRO1:GFPuv containing green fluorescent protein (GFP) gene found that recombinant L. fermentum can express GFP. Conclusion, significance and impact of study: The probiotic L. fermentum isolate can be used as host for expression of heterologous proteins and could possibly be further developed as the alternate oral delivery system for various biomolecules for biotechnological application.
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