Biofilm formation of Hafnia paralvei induced by c-di-GMP through facilitating bcsB gene expression promotes spoilage of Yellow River carp (Cyprinus carpio).

Hafnia paralvei, a Gram-negative foodborne pathogen, is found ubiquitously in various aquatic animals and seafoods, which can form biofilm as a dominant virulence factor that contributes to its pathogenesis. However, the biofilm formation mechanism of H. paralvei and its effect on food spoilage has not been fully characterized. Here we show that biofilm formation, is regulated by c-di-GMP which mediated by bcsB, can increase the spoilage ability of H. paralvei. We found that GTP was added exogenously to enhance the synthesis of c-di-GMP, which further promoted biofilm formation. The gene dgcC, one of 11 genes encoding GGDEF domain-containing proteins in H. paralvei, was significantly upregulated with GTP as substrate. The upregulation of dgcC contributes to a significant increase of c-di-GMP and the formation of biofilm. In addition, the overexpression of dgcC induced upregulation of bcsB, a reported effector protein encoding gene, which was further demonstrated that overexpression of bcsB can encourage the synthesis of bacterial cellulose and biofilm formation. The effect of biofilm formation induced by c-di-GMP on spoilage of Yellow River carp (Cyprinus carpio) was evaluated by sensory evaluation, the total viable count, and the total volatile basic nitrogen, which showed that biofilm formation can significantly increase the spoilage ability of H. paralvei on C. carpio. Our findings provide the regulation of c-di-GMP on expression of bcsB, that can contribute to biofilm formation and spoilage ability of H. paralvei, which is favor to understanding the pathogenesis of Hafnia paralvei and its role in food spoilage.

Intrinsic Resistance to Colistin in the Genus Hafnia.

A bacterial species is considered to be intrinsically resistant to an antimicrobial when nearly all of the wild-type isolates (i.e., those without acquired resistance) exhibit minimum inhibitory concentration (MIC) values that are sufficiently high such that susceptibility testing is unnecessary, and that the antimicrobial should not be considered for therapy. Accordingly, knowledge of intrinsic resistance influences both the selection of treatment regimens and the approach to susceptibility testing in the clinical laboratory, where unexpected results also facilitate the recognition of microbial identification or susceptibility testing errors. Previously, limited data have suggested that Hafnia spp. may be intrinsically resistant to colistin. We evaluated the in vitro activity of colistin against 119 Hafniaceae that were isolated from human samples: 75 (63%) from routine clinical cultures and 44 (37%) from stool samples of travelers undergoing screening for antimicrobial resistant organisms. Broth microdilution colistin MICs were ≥4 µg/mL for 117 of 119 (98%) isolates. Whole-genome sequencing of 96 of the isolates demonstrated that the colistin-resistant phenotype was not lineage-specific. 2 of the 96 (2%) isolates harbored mobile colistin resistance genes. Compared to whole-genome sequencing, VITEK MS matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and VITEK 2 GN ID failed to consistently distinguish between Hafnia alvei, Hafnia paralvei, and Obesumbacterium proteus. In conclusion, using a reference antimicrobial susceptibility testing method and a genetically diverse collection of isolates, we found Hafnia spp. to be intrinsically resistant to colistin. The recognition of this phenotype will help inform rational approaches by which to perform antimicrobial susceptibility testing and therapy for patients with infections that are caused by Hafnia spp.

Genomic analysis of a novel active prophage of Hafnia paralvei.

Little is known about the prophages in Hafniaceae bacteria. A novel Hafnia phage, yong2, was induced from Hafnia paralvei by treatment with mitomycin C. The phage has an elliptical head with dimensions of approximately 45 × 38 nm and a long noncontractile tail of approximately 157 × 4 nm. The complete genome of Hafnia phage yong2 is a 39,546-bp double-stranded DNA with a G+C content of 49.9%, containing 59 open reading frames (ORFs) and having at least one fixed terminus (GGGGCAGCGACA). In phylogenetic analysis, Hafnia phage yong2 clustered with four predicted Hafnia prophages and one predicted Enterobacteriaceae prophage. These prophages and members of the family Drexlerviridae together formed two distinct subclades nested within a clade, suggesting the existence of a novel class of prophages with conserved sequences and a unique evolutionary status not yet studied before in Hafniaceae and Enterobacteriaceae bacteria.

Characterization of autochthonal Hafnia spp. strains isolated from Spanish soft raw ewe's milk PDO cheeses to be used as adjunct culture.

The present work was performed to study the enterobacteria involved in the ripening of the artisanal raw ewe's milk PDO cheeses 'Torta del Casar' and 'Queso de la Serena' produced in Extremadura (Spain). These isolates were strain-typed, safety tested and characterized for some important technological properties. A total of 485 enterobacterial isolates were clustered by RAPD-PCR and subsequently identified by partial sequencing of the 16S rRNA gene. Among the 17 different species identified, Hafnia paralvei was the predominant species; H. alvei and Lelliottia amnigena were present to a lesser extent. Therefore, 55 Hafnia spp. strains, selected according to their genetic profile and dairy origin, were tested for the safe application. Overall, they were able to produce the biogenic amines putrescine and cadaverine under favourable conditions, presented α-haemolytic activity and did not produce cytolytic toxin active against HeLa cells or contain virulence genes. In addition, antibiotic susceptibility profiles showed that 17 Hafnia spp. strains were less resistant to the 33 antibiotics tested; subsequently, they were further technologically characterized. Although they showed differences, in general, they were well adapted to the stress conditions of cheese ripening. Among them, two strains, H. alvei 544 and 1142, are highlighted mainly due to their proteolytic activity at refrigeration temperatures and their low or null gas production. Although further studies are necessary before industrial application, these two strains are proposed for potential use as adjunct cultures to favour the homogeneity of these PDO cheeses, preserving their unique sensory characteristics.

Induction and Genomic Analysis of a Lysogenic Phage of Hafnia paralvei.

Hafnia paralvei is a bacterium that can cause zoonoses. No research has been reported on H. paralvei prophage. In this study, a Hafnia phage yong1 was induced from pathogenic H. paralvei LY-23 by mitomycin C. The phage showed a Myoviridae-like morphology having a hexagonal head of approximately 65 nm in diameter and a contractile tail of approximately 95 nm in length and 17 nm in width. Its genome was sequenced by using the Illumina Miseq platform. The complete genome of Hafnia phage yong1 is 43,329 bp with a G + C content of 47.65%. BLASTn analysis revealed that Hafnia phage yong1 had the highest sequence similarity with the predicted prophages of Enterobacter chengduensis strain WCHECl-C4 = WCHECh050004 recovered from a human blood sample and Escherichia coli strain L103-2 recovered from a goose farm in China. Hafnia phage yong1 contains a tRNA gene and 76 predicted open reading frames, 33 of which were annotated. Gene strings similar to the bacteriophage λ cro-cI-rexA-rexB operon conferring Imm and Rex to lysogenic cells were found in Hafnia phage yong1 genome. Hafnia phage yong1 is the first Myoviridae-like phage found to contain such contiguous genes. Hafnia phage yong1 formed an independent branch between two families, Chaseviridae and Drexlerviridae, in the Proteomic tree.

Single-Cell Biodetection by Upconverting Microspinners.

Near-infrared-light-mediated optical tweezing of individual upconverting particles has enabled all-optical single-cell studies, such as intracellular thermal sensing and minimally invasive cytoplasm investigations. Furthermore, the intrinsic optical birefringence of upconverting particles renders them light-driven luminescent spinners with a yet unexplored potential in biomedicine. In this work, the use of upconverting spinners is showcased for the accurate and specific detection of single-cell and single-bacteria attachment events, through real-time monitoring of the spinners rotation velocity of the spinner. The physical mechanisms linking single-attachment to the angular deceleration of upconverting spinners are discussed in detail. Concomitantly, the upconversion emission generated by the spinner is harnessed for simultaneous thermal sensing and thermal control during the attachment event. Results here included demonstrate the potential of upconverting particles for the development of fast, high-sensitivity, and cost-effective systems for single-cell biodetection.

Comparative genomic analysis of the Hafnia genus reveals an explicit evolutionary relationship between the species alvei and paralvei and provides insights into pathogenicity.

BACKGROUND: The Hafnia genus is an opportunistic pathogen that has been implicated in both nosocomial and community-acquired infections. Although Hafnia is fairly often isolated from clinical material, its taxonomy has remained an unsolved riddle, and the involvement and importance of Hafnia in human disease is also uncertain. Here, we used comparative genomic analysis to define the taxonomy of Hafnia, identify species-specific genes that may be the result of ecological and pathogenic specialization, and reveal virulence-related genetic profiles that may contribute to pathogenesis. RESULTS: One complete genome sequence and 19 draft genome sequences for Hafnia strains were generated and combined with 27 publicly available genomes. We provided high-resolution typing methods by constructing phylogeny and population structure based on single-copy core genes in combination with whole genome average nucleotide identity to identify two distant Hafnia species (alvei and paralvei) and one mislabeled strain. The open pan-genome and the presence of numerous mobile genetic elements reveal that Hafnia has undergone massive gene rearrangements. Presence of species-specific core genomes associated with metabolism and transport suggests the putative niche differentiation between alvei and paralvei. We also identified possession of diverse virulence-related profiles in both Hafnia species., including the macromolecular secretion system, virulence, and antimicrobial resistance. In the macromolecular system, T1SS, Flagellum 1, Tad pilus and T6SS-1 were conserved in Hafnia, whereas T4SS, T5SS, and other T6SSs exhibited the evolution of diversity. The virulence factors in Hafnia are related to adherence, toxin, iron uptake, stress adaptation, and efflux pump. The identified resistance genes are associated with aminoglycoside, beta-lactam, bacitracin, cationic antimicrobial peptide, fluoroquinolone, and rifampin. These virulence-related profiles identified at the genomic level provide insights into Hafnia pathogenesis and the differentiation between alvei and paralvei. CONCLUSIONS: Our research using core genome phylogeny and comparative genomics analysis of a larger collection of strains provides a comprehensive view of the taxonomy and species-specific traits between Hafnia species. Deciphering the genome of Hafnia strains possessing a reservoir of macromolecular secretion systems, virulence factors, and resistance genes related to pathogenicity may provide insights into addressing its numerous infections and devising strategies to combat the pathogen.

Occurrence of Selected Zoonotic Fecal Pathogens and First Molecular Identification of Hafnia paralvei in Wild Taihangshan Macaques (Macaca mulatta tcheliensis) in China.

Rhesus macaques (Macaca mulatta) are hosts to a range of zoonotic and potentially zoonotic pathogens. The present study firstly provides a broader investigation of the presence and prevalence of zoonotic fecal pathogens in wild Taihangshan macaques, a subspecies of rhesus macaque in China. A total of 458 fecal samples were collected between September 2015 and November 2016. Fourteen genera of intestinal parasites (four genera of protozoans and ten genera of helminths) and twelve genera of bacteria were tested for using PCR amplification. The overall samples prevalence of parasitic infection was 98.25%. Entamoeba spp. (89.96%), Balantidium coli (70.09%), and Isospora spp. (28.38%) were the most prevalent protozoa, whereas the predominant prevalent helminths were Trichuris sp. (93.23%), Strongyloides spp. (73.36%), and Oesophagostomum sp. (31.66%). Ten genera of intestinal bacteria were detected in samples of rhesus macaques, including Shigella (31.66%), Escherichia coli (29.91%), Klebsiella pneumoniae (28.38%), Leptospira (26.64%), Campylobacter jejuni (18.34%), Salmonella (13.32%), etc. Eight samples (1.75%) were tested Hafnia-positive based on sequences analysis of 16S rRNA and ampC gene. This is the first molecular characterization of Hafnia infection in NHPs. Our cross-sectional prevalence study provides important information for monitoring the potential transmission of zoonotic infections from wild rhesus macaques.

Hafnia, an enterobacterial genus naturally resistant to colistin revealed by three susceptibility testing methods.

Objectives: To determine the susceptibility to colistin of Hafnia alvei and Hafnia paralvei, and to compare methods for colistin resistance detection in the Hafnia genus. Methods: A collection of 25 Hafnia isolates was studied. Species were identified by using 16S rRNA gene sequencing with subsequent phylogeny analysis. Susceptibility to colistin was determined using the broth microdilution (BMD) reference method, the Phoenix automated system, the Rapid Polymyxin NP test, the Etest system and the disc diffusion method. Results: The collection consisted of 15 H. alvei and 10 H. paralvei isolates. Based on the 16S rRNA analysis, a close relationship of the Hafnia genus with naturally colistin-resistant enterobacterial genera (Proteus, Morganella, Providencia and Serratia) was identified. Susceptibility testing performed using the BMD method, the Phoenix automated system and the Rapid Polymyxin NP test revealed a high rate of colistin resistance (96%). Underestimation of colistin resistance using Etest strips (72%) and the disc diffusion method (0%) was observed. Conclusions: The high rate of colistin resistance observed within the Hafnia genus and its close phylogenetic relationship with naturally colistin-resistant genera suggest that Hafnia is a naturally colistin-resistant enterobacterial genus.

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.

Binary combination of epsilon-poly-L-lysine and isoeugenol affect progression of spoilage microbiota in fresh turkey meat, and delay onset of spoilage in Pseudomonas putida challenged meat.

Proliferation of microbial population on fresh poultry meat over time elicits spoilage when reaching unacceptable levels, during which process slime production, microorganism colony formation, negative organoleptic impact and meat structure change are observed. Spoilage organisms in raw meat, especially Gram-negative bacteria can be difficult to combat due to their cell wall composition. In this study, the natural antimicrobial agents ε-poly-L-lysine (ε-PL) and isoeugenol were tested individually and in combinations for their activities against a selection of Gram-negative strains in vitro. All combinations resulted in additive interactions between ε-PL and isoeugenol towards the bacteria tested. The killing efficiency of different ratios of the two antimicrobial agents was further evaluated in vitro against Pseudomonas putida. Subsequently, the most efficient ratio was applied to a raw turkey meat model system which was incubated for 96 h at spoilage temperature. Half of the samples were challenged with P. putida, and the bacterial load and microbial community composition was followed over time. CFU counts revealed that the antimicrobial blend was able to lower the amount of viable Pseudomonas spp. by one log compared to untreated samples of challenged turkey meat, while the single compounds had no effect on the population. However, the compounds had no effect on Pseudomonas spp. CFU in unchallenged meat. Next-generation sequencing offered culture-independent insight into population diversity and changes in microbial composition of the meat during spoilage and in response to antimicrobial treatment. Spoilage of unchallenged turkey meat resulted in decreasing species diversity over time, regardless of whether the samples received antimicrobial treatment. The microbiota composition of untreated unchallenged meat progressed from a Pseudomonas spp. to a Pseudomonas spp., Photobacterium spp., and Brochothrix thermosphacta dominated food matrix on the expense of low abundance species. We observed a similar shift among the dominant species in meat treated with ε-PL or the antimicrobial blend, but the samples differed markedly in the composition of less abundant species. In contrast, the overall species diversity was constant during incubation of turkey meat challenged with P. putida although the microbiota composition did change over time. Untreated or ε-PL treated samples progressed from a Pseudomonas spp. to a Pseudomonas spp. and Enterobacteriaceae dominated food matrix, while treatment with the antimicrobial blend resulted in increased relative abundance of Hafnia spp., Enterococcaceae, and Photobacterium spp. We conclude that the blend delayed the onset of spoilage of challenged meat, and that all antimicrobial treatments of unchallenged or challenged meat affect the progression of the microbial community composition. Our study confirms that the antimicrobial effects observed in vitro can be extrapolated to a food matrix such as turkey meat. However, it also underlines the consequence of species-to-species variation in susceptibility to antimicrobials, namely that the microbial community change while the CFU remains the same. Addition of antimicrobials may thus prevent the growth of some microorganisms, allowing others to proliferate in their place.

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.

Role of 30 kDa antigen of enteric bacterial pathogens as a possible arthritogenic factor in post-dysenteric reactive arthritis.

BACKGROUND: Reactive arthritis (ReA)/Reiter's syndrome (RS) may be caused as a sequel of infections caused by enteric bacterial pathogens, although the mechanisms through, which different pathogens cause similar disease are not clear. AIM: This study was done to look for the presence and role of any common bacterial antigen among the pathogens isolated from such patients. MATERIALS AND METHODS: A total of 51 patients of ReA and 75 controls (three groups of 25 subjects each: Group 1: Patients who did not develop arthritic complications within 3 months after bacillary dysentery/diarrhea; Group 2: Patients with other arthritic diseases and Group 3: Normal healthy subjects) were included. The isolated enteric pathogens were tested to detect the immunodominant antigens. RESULTS AND CONCLUSIONS: A common 30 kDa antigen was found to be specifically present among seven arthritogenic enteric bacterial strains belonging to three genera, Salmonella, Shigella and Hafnia. Post-dysenteric ReA patients' sera show higher levels of immunoglobulin G, immunoglobulin M and immunoglobulin A antibodies against this antigen as compared to the controls. Lymphocytes of ReA patients recognize this antigen, proliferate and produce interleukin-2 in response to this antigen more than the lymphocytes of controls. 30 kDa antigen may be a common arthritogenic factor associated with post-dysenteric ReA/RS. The association of Hafnia alvei with post-dysenteric ReA is described for the first time. Four cases of mycobacterial ReA had an association with this antigen, suggesting that the arthritogenic antigen of mycobacteria and enteric bacteria may be of a similar nature.

Clinical and laboratory diagnostic characteristics and cytotoxigenic potential of Hafnia alvei and Hafnia paralvei strains.

A collection of 68 Hafnia strains previously identified to the species level by 16S rRNA gene sequencing were investigated for simple phenotypic properties that could aid in their recognition in the clinical laboratory. Four tests, including malonate utilization, fermentation of salicin and d-arabinose, and expression of ß-glucosidase activity, correctly assigned each strain to either Hafnia alvei or H. paralvei. Antibiotic susceptibility profiles were generated for 35 H. alvei and H. paralvei isolates using Etest strips for 24 antibiotics. All strains were susceptible to aminoglycosides, quinolones, carbapenems, and monobactams. Most of the Hafnia isolates had a colistin MIC of ≥2 µg/ml. Sequencing of an internal ampC gene fragment allowed genotypic differentiation of the two Hafnia species. Approximately 70% of the hafniae tested additionally produced a cytolytic toxin active on Vero cells which may play a role in gastroenteritis.

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.

[Gene cloning, expression and characterization of a novel phytase from Hafnia alvei].

A gene appA encoding a novel phytase was firstly cloned from Hafnia alvei by PCR and sequenced. The gene was consisted of 1335 bp, encoding 444 amino acids. The calculated molecular weight of the mature APPA was about 45.2 kD. The gene appA was expressed in E. coli BL21 (DE3). Recombinant APPA was purified and its enzymatic properties were determined. The optimum pH for the enzyme was 4.5 and the optimum temperature was 60 degrees C. The pH stability of r-APPA is good, the relative phytase activity was above 80% after treated in buffers of pH 2.0-10.0. The specific activity of r-APPA is 356.7 U/mg, and the Km value was 0.49 mmol/L and Vmax of 238 U/mg. The enzyme showed resistance to pepsin and trypsin treatment.