Listeria monocytogenes, an intracellular pathogen, is the causative agent of listeriosis, a serious epidemic and sporadic food-borne disease. The clinical manifestations of listeriosis include meningitis, meningoencephalitis, septicemia, spontaneous abortion, perinatal infections, and gastroenteritis. Although rare in comparison to other food-borne diseases, listeriosis has a high rate of lethality (about 30%), making L. monocytogenes an important pathogen. L. monocytogenes can survive in a broad range of ecological niches, including farm environments and food-processing plants and in a wide range of hosts, including humans and many species of mammals. Furthermore, the capacity to adapt and survive under extreme conditions allows this bacterium to exist ubiquitously in the environment and to survive and proliferate under conditions within the food supply. Although the study of L. monocytogenes has already been extensively reviewed, knowledge about this pathogen has been expanding rapidly. Against the background of the growing body of information on this bacterium, the present review mostly discusses advances made in the study of this pathogen over the last 5 years.
We investigated the effect of preserving samples with ethanol and acetone on molecular analyses of a complex microbial community in the gut of the termite Microcerotermes sp. Although the yield of DNA extracted from the guts decreased as the period of preservation increased (three days, one week, and one month), terminal-restriction fragment length polymorphism (T-RFLP) profiles of bacterial 16S rRNA genes amplified from the extracted DNA were conserved with respect to the occurrence of major taxa. Bacterial diversity detected in the analyses decreased only slightly after the preservation, and an analysis of clones demonstrated that the difference in terms of coverage was statistically insignificant before and one month after preservation with acetone, the treatment which resulted in the greatest dissimilarity in T-RFLP. However, the relative abundance of some taxonomic groups was remarkably changed after one month of preservation, especially in acetone. The results suggest that both ethanol and acetone well preserve bacterial DNA from a complex gut community, but that the DNA should be extracted as soon as possible. If the period of preservation exceeds one week, the molecular community profiles should be interpreted with caution, and it is better to avoid abundance-based comparisons.
The endophytic bacteria in the seeds of rice plants (Oryza sativa, cultivar Kinuhikari) cultivated on an experimental plot adjacent to a paddy field were studied as the seeds matured by comparing them with the bacteria on the surface of the seeds. Endophytic and surface bacteria were isolated using a nutrient broth and a diluted nutrient broth agar medium. The isolates were identified based on 16S rRNA gene sequences. Three genera (Paenibacillus, Acidovorax and Pantoea) and 2 genera (Stenotrophomonas and Rhizobium) were specific to the inside and to the surface of the seeds, respectively. Six genera (Bacillus, Curtobacterium, Methylobacterium, Sphingomonas, Xanthomonas and Micrococcus) were common to both the inside and the surface. As the seed matured, the flora of culturable endophytic bacteria changed in a different manner from that of culturable surface bacteria. More isolates tolerant of high osmotic pressure were found among the endophytes than among the surface bacteria, especially at the later stages of the maturation process. An increasing number of endophytic isolates exhibited amylase activity at the later stages.
Plasmid-mediated conjugation is an important mechanism through which bacteria establish new genetic traits in the environment. Here we used a combination of direct viable counting (DVC), fluorescence in situ hybridization (FISH), and green fluorescent protein (GFP) gene expression to estimate plasmid transfer frequencies (TFs) on nutrient-limited media at the single cell level. Conjugation experiments on nutrient-rich and -limited media were carried out using Pseudomonas fluorescens as a donor of a broad-host-range plasmid (RK2) tagged with the GFP gene and P. putida as the recipient. FISH-GFP and DVC-FISH-GFP allowed for the accurate detection of donor, recipient and transconjugants by fluorescence microscopy. The TFs obtained by these culture-independent approaches were 1 to more than 3 orders of magnitude higher than those determined by selective plate-counting. Lower and variable TFs obtained by selective plate-counting emphasize the importance of culture-independent approaches in plasmid transfer studies. The application of DVC before FISH-GFP on nutrient-limited medium produced elongated and/or fattened cells and resulted in a better fluorescence signal in target bacteria cells and better accumulation, maturation and fluorescence-emission of GFP within transconjugant cells, facilitating the detection and identification of cells compared with FISH-GFP alone. The DVC-FISH-GFP could be useful for other conjugation studies using bacteria with low metabolic activity under oligotrophic/stressful conditions or studies which require a precise determination of plasmid transfer events during short-term conjugation experiments.
Microbial diversity in forage crop seeds was examined using culture-independent methods. Environmental DNA samples were extracted from the surface of the seeds, and the intergenic spacer region between small subunit and large subunit RNA genes was then amplified by ribosomal intergenic spacer analysis (RISA) for the profiling of microbial community structures. The results suggested the presence of stable microbial communities in seeds and sequencing of the RISA amplicons identified a total of 33 unique microbial sequences in 1.6 g of seed material from Italian ryegrass (Lolium multiflorum) and timothy (Phleum pretense). These included several sequences showing high similarity to known plant-associated microbes, such as Pseudomonas fluorescens and Clavibacter michiganensis, the Cladosporium and Dioszegia group of fungi, and also several uncultured bacteria.
Three endophytic bacterial strains (Burkholderia sp. no. 25, Enterobacter sp. no. 35 and Klebsiella sp. no. 38) isolated from sweet potato and sugarcane were examined for their ability to incorporate the gene encoding green fluorescent protein (GFP) via conjugation and electroporation. Enterobacter sp. no. 35 and Klebsiella sp. no. 38 were successfully tagged with the gfp gene by conjugation using pTn5kmgfpmut1. Fluorescence microscopic observation of Brassica oleracea inoculated with gfp-tagged endophytes revealed that Enterobacter sp. no. 35 and Klebsiella sp. no. 38 colonized the junctions between the lateral roots and the main roots.
Genes expressed more intensively during dehydration were screened in the terrestrial cyanobacterium Nostoc sp. HK-01, which is phylogenetically very similar to the aquatic Anabaena sp. PCC 7120. A DNA microarray for Anabaena was used to determine the gene expression of Nostoc sp. HK-01 cells. The results showed that genes homologous with those of Anabaena were expressed increasingly more during dehydration. The expression was transient in Anabaena while in Nostoc it increased until the wet weight decreased to 10% of that before drying. It was concluded that the higher desiccation-tolerance of Nostoc was supported by the more intensive gene expression than in Anabaena.