Safety Evaluation of Neo Transgenic Pigs by Studying Changes in Gut Microbiota Using High-Throughput Sequencing Technology.

The neo (neomycin phosphotransferase) gene is widely used as a selection marker in the production of genetically engineered animals and plants. Recent attention has been focused on safety concerns regarding neo transgene expression. In this study, neo transgenic and non-transgenic piglets were randomly assigned into Group A and Group B to evaluate effects of neo transgene by studying changes in gut microbiota using high-throughput sequencing. Group A pigs were fed a standard diet supplemented with antibiotic neomycin; Group B pigs were fed a standard diet. We examined horizontal transfer of exogenous neo gene using multiplex PCR; and investigated if the presence of secreted NPT II (neo expression product) in the intestine could lead to some protection against neomycin in transgenic pigs by monitoring different patterns of changes in gut microbiota in Group A animals. The unintended effects of neo transgene on gut microbiota were studied in Group B animals. Horizontal gene transfer was not detected in gut microbiota of any transgenic pigs. In Group A, a significant difference was observed between transgenic pigs and non-transgenic pigs in pattern of changes in Proteobacteria populations in fecal samples during and post neomycin feeding. In Group B, there were significant differences in the relative abundance of phyla Firmicutes, Bacteroidetes and Proteobacteria, and genera Lactobacillus and Escherichia-Shigella-Hafnia between transgenic pigs and non-transgenic pigs. We speculate that the secretion of NPT II from transgenic tissues/cells into gut microbiota results in the inhibition of neomycin activity and the different patterns of changes in bacterial populations. Furthermore, the neo gene also leads to unintended effects on gut microbiota in transgenic pigs that were fed with basic diet (not supplemented with neomycin). Thus, our data in this study caution that wide use of the neo transgene in genetically engineered animals should be carefully considered and fully assessed.

Hafnia psychrotolerans sp. nov., isolated from lake water.

A psychrotolerant, Gram-stain-negative, motile, aerobic, peritrichous bacterium, strain DJC1-1(T), was isolated from Lake Dajiaco, Tibetan Plateau, China. The strain was negative for citrate utilization, lipase activity and α-glucosidase, but positive for the Voges-Proskauer reaction and N-acetyl-ß-glucosaminidase. 16S rRNA gene sequence analysis indicated that Hafnia paralvei ATCC 29927(T), Hafnia alvei ATCC 13337(T), Serratia grimesii DSM 30063(T) and Serratia plymuthica DSM 4540(T) were the closest relatives of strain DJC1-1(T), with similarities of 97.76, 96.80, 97.71 and 97.58 %, respectively. The DNA G+C content of strain DJC1-1(T) was 53.9 mol%. The predominant fatty acids were C16 : 0 and C17 : 0 cyclo. Based on these characteristics, strain DJC1-1(T) can be assigned to the genus Hafnia. In DNA-DNA hybridization tests, strain DJC1-1(T) shared 50.6, 35.1, 36.5 and 18.1 % DNA-DNA relatedness with the type strains of H. paralvei, H. alvei, S. grimesii and S. plymuthica, respectively. The growth temperature ranged from 0 to 40 °C, with optimum growth at 15 °C. Physiological and biochemical tests differentiated strain DJC1-1(T) from the type strains of recognized species of the genus Hafnia. Therefore, strain DJC1-1(T) is identified as representing a novel species of the genus Hafnia, for which the name Hafnia psychrotolerans sp. nov. is proposed. The type strain is DJC1-1(T) ( = JCM 30077(T) = CGMCC1.12806(T)).

Conversion of glycerol to 1,3-propanediol by Citrobacter freundii and Hafnia alvei - newly isolated strains from the Enterobacteriaceae.

In this study, nearly 4000 bacterial strains from the family of Enterobacteriaceae isolated from different environments were screened for ability to convert glycerol to 1,3-propanediol (1,3-PD). The aim of the research was to isolate 1,3-PD producers from the natural environment, identify and characterize the best isolates. Three selective media were tested to usefulness in the isolation of bacteria from the family Enterobacteriaceae. Only, 28% of examined isolates could synthesize 1,3-PD from glycerol. 1,3-PD producing bacteria were identified by API 20E tests and 16S rRNA sequences to be Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter freundii and Hafnia alvei. It is the first time, when the fermentation glycerol to 1,3-PD by H. alvei was investigated. The selected strains (C. freundii AD119 and H. alvei AD27) were analyzed on a bioreactor scale under constant pH value 7.0 at temperature of 30°C and 37°C. After 40h in batch fermentation, H. alvei AD27 produced 11.3g/L of 1,3-PD at 37°C. For C. freundii AD119, the best results were obtained at temperature of 30°C. After 24h of fermentation, the 1,3-PD concentration reached above 23 g/L of 1,3-PD.

A case of bacteremia caused by Hafnia paralvei.

The genus Hafnia, a member of the Enterobacteriaceae, is widespread in nature and rarely causes human infection. We describe a case of an 85-year-old Japanese man hospitalized consequent to suspected cholecystitis, in which Hafnia sp. was recovered from the blood culture concomitantly with Enterococcus faecalis. Sequencing of the 16S ribosomal RNA gene sequence and phenotyping with ID 32 E revealed that the recovered Hafnia sp. was considered to be Hafnia alvei genomosp. 2 (ATCC 29927), recently reclassified as Hafnia paralvei. The patient recovered uneventfully with antimicrobial therapies.

Hafnia paralvei sp. nov., formerly known as Hafnia alvei hybridization group 2.

It has been shown previously, based largely on DNA-DNA hybridizations and partial 16S rRNA gene sequencing, that Hafnia alvei is genotypically heterogeneous and consists of at least two DNA hybridization groups (HGs). In the present study, the taxonomic status of H. alvei HGs 1 and 2 was reassessed. A panel of 24 reference strains and isolates previously assigned to one of the two HGs in H. alvei was subjected to (GTG)5-PCR fingerprinting; this resulted in the delineation of two (GTG)5-PCR clusters in perfect accordance with the respective HG designations. Based on full 16S rRNA gene sequencing of a selection of reference strains, H. alvei HGs 1 and 2 showed internal sequence similarities of 99.8 and 99.5%, respectively. Between the two groups, sequence similarities ranged from 98.8 to 99.1%. Mean DNA-DNA hybridization values of 74.7-99.9% were obtained within each of the two HGs, whereas cross-hybridizations between members of H. alvei HG 1 (including ATCC 13337T) and HG 2 revealed only 32.7-48.7 % DNA-DNA hybridization. Previously published and new phenotypic data revealed that a combination of malonate assimilation and beta-glucosidase activity enabled correct assignment of Hafnia isolates to one of the two HGs. Collectively, taxonomic data from this study confirm that H. alvei comprises at least two taxa at the species level, of which HG 1 corresponds to H. alvei sensu stricto because it includes the type strain ATCC 13337T. Strains formerly classified as members of H. alvei HG 2 represent a novel species, for which the name Hafnia paralvei sp. nov. is proposed; ATCC 29927T (=CDC 4510-73T =LMG 24706T), the former reference strain of H. alvei HG 2, is designated the type strain.

The genus Hafnia: from soup to nuts.

The genus Hafnia, a member of the family Enterobacteriaceae, consists of gram-negative bacteria that are occasionally implicated in both intestinal and extraintestinal infections in humans. Despite the fact that the genus currently contains only a single species (H. alvei), more extensive phylogenetic depth (two or more species) is apparent based upon DNA relatedness and 16S rRNA gene sequencing studies. Hafnia causes a variety of systemic infections, including septicemia and pneumonia; however, its role as a gastrointestinal pathogen is controversial. Many of the data supporting a role for hafniae as enteric pathogens were incorrectly attributed to this genus rather than to the actual pathogen, Escherichia albertii. There are numerous gaps in our understanding of this genus, including ecologic habitats and population genetics, disease-producing role in animals, phenetic and genetic methods useful in distinguishing genomospecies within the H. alvei complex, and bona fide pathogenicity factors.