Identification of Two Distinct Hybridization Groups in the Genus Hafnia by 16S rRNA Gene Sequencing and Phenotypic Methods

Abstract

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.The genus Hafnia presently consists of two or more distinct species (genomospecies) that are currently identified as a single taxon, H. alvei. DNA relatedness studies conducted by Steigerwalt et al. (12) in 1976 involving the genera Enterobacter and Serratia identified several strains of H. alvei ("Enterobacter hafniae") that exhibited widely divergent DNA binding ratios and degrees of mismatch (divergence) when hybridized with DNA from reference strain E. hafniae 4360-76. These results, although limited in scope by the number of strains studied, provided the first direct evidence for species diversity within the genus. Although definitive taxonomic studies regarding this species were not subsequently published, Brenner and colleagues at the Centers for Disease Control and Prevention (CDC) confirmed the existence of at least two distinct relatedness (hybridization) groups (HGs) within H. alvei (2, 3). Reference strains for these HGs include ATCC 13337 T and ATCC 29926 (CDC 5632-72) for HG 1 (H. alvei sensu stricto) and ATCC 29927 (CDC 4510-73) for HG 2 (unnamed) (4).Over the past several years, there has been renewed interest in the taxonomy of hafniae as causative agents of extraintestinal disease (6, 11) and their reputed association with bacterial gastroenteritis (7). One study found that 16S rRNA gene sequencing could be successfully used to distinguish Hafnia genomospecies (8), while a subsequent investigation achieved similar results using multilocus enzyme electrophoresis (10). This latter Australian survey also concluded that the two genomospecies differed in their genetic structure and host distribution based upon an analysis of strains recovered from a variety of mammals, reptiles, fish, amphibians, insects, and aquatic sources (10).HG 1 contains the type strain, ATCC 13337 T (Stuart's type 32011), and by definition constitutes H. alvei sensu stricto. HG 2, also currently identified as H. alvei, remains unnamed, since previous studies have failed to identify phenotypic characteristics useful in distinguishing these two species from each other (2, 3, 4). It is also unclear what the distribution and frequency of these two hybridization groups are in clinical specimens. Greipsson and Priest (5) published the last major numerical taxonomy study o...

The Genus Hafnia : from Soup to Nuts

“…16S rRNA sequencing of 22 H. alvei isolates revealed two clusters, one containing 5 strains (including ATCC 29926) that exhibited 0% to 0.19% sequence divergence from ATCC 13337 T (DNA group 1) and a second cluster (DNA group 2) of 17 strains that exhibited 0% to 0.09% sequence divergence from ATCC 29927 (0.95% to 1.04% divergence from ATCC 13337 T ) (71). A subsequent study of 52 Hafnia strains by the same group again found two DNA groups that were clearly distinguishable using 16S rRNA gene sequencing (70). An Australian study in 2003 investigated the genetic structures of 161 Hafnia isolates by multilocus enzyme electrophoresis at12 different loci (98).…”

Clinical and Laboratory Diagnostic Characteristics and Cytotoxigenic Potential of Hafnia alvei and Hafnia paralvei Strains

“…All strains were biochemically identified as Hafnia and were KCN, Voges-Proskauer, and glycerol positive and negative for glutamate decarboxylase activity, which distinguishes this group from Escherichia albertii (15). Each strain was additionally identified to the species level as H. alvei or H. paralvei using 16S rRNA gene sequencing as previously described (16). Some strains were also identified to the species level by DNA-DNA hybridization (13).…”

“…Hafnia strains were screened against a standard set of biochemical characteristics previously identified as useful in the separation of H. alvei from H. paralvei. These tests included malonate utilization (48 h), D-arabinose and salicin fermentation (96 h), and ␤-glucosidase activity (13,16,17). ␤-Glucosidase activity was measured by fluorescence after 5 min of incubation in the dark on bacterial impregnated disks from Key Scientific (Stamford, TX).…”

“…In our laboratory, tests such as motility at 24 h, inulin hydrolysis, and sodium acetate utilization did not discriminate between the two Hafnia species as previously noted by others (3). Still other potential discriminatory tests that were associated exclusively with only one species, such as raffinose fermentation, were found at such low frequencies to make their usefulness in laboratory identification meaningless (16). However, both malonate utilization and fermentation of D-arabinose, which were previously linked to specific Hafnia DNA groups or biotypes, appeared more useful in species differentiation.…”

“…These species could not easily be separated and identified biochemically, although both taxa were distinguishable using techniques such as 16S rRNA gene sequencing (16). The problem of identifying some differential biochemical characteristics has been recently overcome, and Huys and colleagues (13) have proposed a new Hafnia species, H. paralvei, which was previously referred to as H. alvei hybridization (DNA) group 2.…”