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.

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.

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.

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.

Identification of two distinct hybridization groups in the genus Hafnia by 16S rRNA gene sequencing and phenotypic methods.

A collection of 52 strains belonging to the Hafnia alvei complex were subjected to molecular (16S rRNA gene sequencing) and biochemical analysis. Based upon 16S rRNA gene sequencing results, two genetic groups were identified which correspond with previously recognized DNA hybridization group 1 (ATCC 13337(T) and ATCC 29926; n = 23) and DNA hybridization group 2 (ATCC 29927; n = 29). Of 46 biochemical tests used to characterize hafniae, 19 reactions (41%) yielded variable results. Of these 19 tests, 6 were determined to have discriminatory value in the separation of DNA groups 1 and 2, with malonate utilization found to be the most differential test. Test results of malonate utilization alone correctly assigned 90% of Hafnia isolates to their correct DNA group.