Revisiting host identification of bacteriophage Enc34: from biochemical to molecular.

In our 2012 genome announcement (J Virol 86:11403-11404, 2012, https://doi.org/10.1128/JVI.01954-12), we initially identified the host bacterium of bacteriophage Enc34 as Enterobacter cancerogenus using biochemical tests. However, later in-house DNA sequencing revealed that the true host is a strain of Hafnia alvei. Capitalizing on our new DNA-sequencing capabilities, we also refined the genomic termini of Enc34, confirming a 60,496-bp genome with 12-nucleotide 5' cohesive ends. IMPORTANCE: Our correction reflects the evolving landscape of bacterial identification, where molecular methods have supplanted traditional biochemical tests. This case underscores the significance of revisiting past identifications, as seemingly known bacterial strains may yield unexpected discoveries, necessitating essential updates to the scientific record. Despite the host identity correction, our genome announcement retains importance as the first complete genome sequence of a Hafnia alvei bacteriophage.

Effect of Hafnia alvei on Morphophysiologic Parameters and Gut Microbiota of Mice with Inherited Type 2 Diabetes Mellitus.

Inbred mouse strains KK.Cg-a/a and KK.Cg-Ay/a known as genetic models of type 2 diabetes mellitus significantly surpassed the control strain C57BL/6J in the body weight, relative weight of extractable fat, and basal blood glucose levels. Real-timePCR of fecal samples from KK.Cg-a/a and KK.Cg-Ay/a mice revealed dysbiosis typical of type 2 diabetes mellitus in humans and animals. Long-term intragastric administration of a suspension of Hafnia alvei bacteria had no effect on the above morphometric and biochemical parameters. At the same time, recovery of the Bacteroides spp. population in KK.Cg-Ay/a mice and a decrease in the number of Bifidobacterium spp. in KK.Cg-a/a mice were observed. The possibility of therapeutic use of the probiotic based on H. alvei is discussed.

Recovery of Hafnia alvei from diseased brown trout, Salmo trutta L., and healthy noble crayfish, Astacus astacus (L.), in Bulgaria.

Hafnia alvei was isolated in Bulgaria from healthy noble crayfish, Astacus astacus (L.), and then from farmed diseased brown trout, Salmo trutta L., with signs of haemorrhagic septicaemia. The isolates were identified initially with conventional phenotyping and commercial Merlin Micronaut and API 20E rapid identification systems, followed by sequencing of the 16S rRNA gene. Hafnia alvei Bt1, Bt2 and Aa4 were of low virulence to rainbow trout and brown trout, although cytotoxicity was demonstrated by Bt1 and Bt2, but not by Aa4.

The complete genome sequence of Hafnia alvei A23BA; a potential antibiotic-producing rhizobacterium.

OBJECTIVES: The urgent need for novel antibiotics cannot be overemphasized. Hafnia alvei A23BA was isolated from plant rhizosphere as part of an effort to recover novel antibiotic-producing bacterial strains from soil samples. The genome of the isolate was sequenced to facilitate mining for potential antibiotic-encoding biosynthetic gene clusters and to gain insights into how these gene clusters could be activated. DATA DESCRIPTION: Here, we report the complete genome sequence of H. alvei A23BA obtained from the hybrid assembly of Illumina HiSeq and GridION reads. The genome, consisting of a circular chromosome and a circular plasmid, is 4.77 Mb in size with a GC content of 48.77%. The assembly is 99.5% complete with genomic features including 4,217 CDSs, 125 RNAs, and 30 pseudogenes. Thiopeptide, beta-lactone, siderophore, and homoserine lactone biosynthetic gene clusters were also identified. Other gene clusters of interest include those associated with bioremediation, biocontrol, and plant growth promotion- all of which are reported for H. alvei for the first time. This dataset serves to expedite the exploration of the biosynthetic and metabolic potentials of the species. Furthermore, being the first published genome sequence of a soil isolate, this dataset enriches the comparative genomics study of H. alvei strains.

Identification of a non-pathogenic surrogate organism for chlorine dioxide (ClO2) gas treatment.

The identification of non-pathogenic surrogate microorganisms is beneficial for determining and validating the efficacy of antimicrobial treatments in food manufacturing environments. A surrogate organism was identified to aid in the decontamination process of fresh produce when treated with chlorine dioxide (ClO(2)) gas. Thirty-two known strains of pathogenic and non-pathogenic microorganisms and seven unknown microbial isolates from mushroom, tomatoes, and strawberries were evaluated. The primary goal was to find alternative non-pathogenic organisms that had an equal or higher resistance compared to Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes. Among the strains tested, MR1 (mushroom isolate), E. coli O157:H7 C7927, E. coli O157:H7 204P, STB2 (strawberry isolate), and vegetative cells of Bacillus cereus 232 in wet inoculum were found to be the most resistant to gaseous ClO(2) treatment at 0.3 mg/l for 1 min and D-values at 0.3 mg/l ClO(2) were 3.53, 1.95, 1.72, 1.68, and 1.57 min, respectively. For identification, the MR1 and STB2 strains were identified using a Ribotyper with the EcoRI restriction enzyme of 16S rDNA sequence. MR1 was identified as Hafnia alvei with a similarity value of 94% using the ribotype pattern and with a 93.6% similarity using an API 20E strip, and with a 99% similarity using 16S rDNA analysis. The Ped-2E9-based cytotoxicity assay was conducted for the MRI strain extracellular toxin and whole cell toxicity and did not show cytotoxicity. Analysis, using multiplex PCR, was performed to verify absence of the eaeA gene. H. alvei is a suitable non-pathogenic surrogate, with higher resistance to ClO(2) gas compared to pathogens studied, that may be useful to establish optimum conditions of ClO(2) gas decontamination systems.

Genetic Diversity of O-Antigens in Hafnia alvei and the Development of a Suspension Array for Serotype Detection.

Hafnia alvei is a facultative and rod-shaped gram-negative bacterium that belongs to the Enterobacteriaceae family. Although it has been more than 50 years since the genus was identified, very little is known about variations among Hafnia species. Diversity in O-antigens (O-polysaccharide, OPS) is thought to be a major factor in bacterial adaptation to different hosts and situations and variability in the environment. Antigenic variation is also an important factor in pathogenicity that has been used to define clones within a number of species. The genes that are required to synthesize OPS are always clustered within the bacterial chromosome. A serotyping scheme including 39 O-serotypes has been proposed for H. alvei, but it has not been correlated with known OPS structures, and no previous report has described the genetic features of OPS. In this study, we obtained the genome sequences of 21 H. alvei strains (as defined by previous immunochemical studies) with different lipopolysaccharides. This is the first study to show that the O-antigen gene cluster in H. alvei is located between mpo and gnd in the chromosome. All 21 of the OPS gene clusters contain both the wzx gene and the wzy gene and display a large number of polymorphisms. We developed an O serotype-specific wzy-based suspension array to detect all 21 of the distinct OPS forms we identified in H. alvei. To the best of our knowledge, this is the first report to identify the genetic features of H. alvei antigenic variation and to develop a molecular technique to identify and classify different serotypes.

Phenotypic and genotypic properties of the genus Hafnia.

The present study characterised 73 Hafnia alvei isolates and five Escherichia isolates (originally identified as H. alvei) isolated from cases of diarrhoeal disease by the International Centre for Diarrhoeal Disease Research Branch (ICDDRB) in Bangladesh. Based upon the hydrolysis of arbutin and aesculin and the fermentation of salicin and D-arabinose, four distinct biotypes could be recognised among the 73 H. alvei isolates tested; biotype 1 (D-(-)-arabinose-positive only) accounted for 75% of all isolates analysed. Hydrolysis of aglycone compounds such as arbutin, salicin and aesculin appeared to be associated with expression of beta-glucosidase activity. ICDDRB isolates, when compared with type or reference strains of H. alvei, were shown not to belong to the genus Hafnia based upon resistance to Hafnia-specific bacteriophage 1672, possession of the phoE gene, expression of glutamate decarboxylase activity and significant 16S rDNA sequence divergence (approximately 8%) from the type strain, ATCC 13337T. True H. alvei strains, implicated in outbreaks of diarrhoeal disease in Canada, lacked the eaeA gene in contrast to ICDDRB isolates. Twenty-two H. alvei isolates were selected for further study. Based upon partial 16S rDNA sequencing, these 22 isolates fell into two genomic groups (genomospecies), identical to DNA groups previously established by DNA hybridisation studies. Markers such as motility, biotype, or enzymic or carbohydrate fermentation patterns did not correlate totally with DNA grouping, although malonate utilisation appeared to be the single best discriminatory phenotype. The results indicate that the genus Hafnia is heterogeneous and there do not appear to be any laboratory data available specifically linking these organisms to gastro-enteritis.

Structural and serological studies on Hafnia alvei O-specific polysaccharide of alpha-D-mannan type isolated from the lipopolysaccharide of strain PCM 1223.

On the basis of chemical and methylation analyses, one- and two-dimensional (1)H- and (13)C-NMR spectroscopy, including COSY, TOCSY, NOESY and (1)H, (13)C HSQC experiments, a neutral O-specific polysaccharide isolated from Hafnia alvei strain PCM 1223 lipopolysaccharide (LPS) was found to be an alpha-mannan composed of pentasaccharide repeating units having the following structure:-->3)-alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->. Immunoblotting showed a strong cross-reactivity between anti-H. alvei PCM 1223 serum and LPSs of Escherichia coli O9 and Klebsiella pneumoniae O3. The serological relationship of the LPSs of these bacteria is due to the structural identity of their O-specific polysaccharides, though the LPSs differ in their core regions.

Genetic and ecological structure of Hafnia alvei in Australia.

Multilocus enzyme electrophoresis of 161 Hafnia alvei isolates from 158 hosts and 3 water column samples collected in Australia revealed that this species consists of two genetically distinct groups. The two groups of H. alvei differed significantly in their genetic structure and host distribution. The taxonomic class of the host but not geographic locality explained a significant proportion of the observed genetic and biochemical variation among strains within each genetic group.

Degradation of phytate by the 6-phytase from Hafnia alvei: a combined structural and solution study.

Phytases hydrolyse phytate (myo-inositol hexakisphosphate), the principal form of phosphate stored in plant seeds to produce phosphate and lower phosphorylated myo-inositols. They are used extensively in the feed industry, and have been characterised biochemically and structurally with a number of structures in the PDB. They are divided into four distinct families: histidine acid phosphatases (HAP), ß-propeller phytases, cysteine phosphatases and purple acid phosphatases and also split into three enzyme classes, the 3-, 5- and 6-phytases, depending on the position of the first phosphate in the inositol ring to be removed. We report identification, cloning, purification and 3D structures of 6-phytases from two bacteria, Hafnia alvei and Yersinia kristensenii, together with their pH optima, thermal stability, and degradation profiles for phytate. An important result is the structure of the H. alvei enzyme in complex with the substrate analogue myo-inositol hexakissulphate. In contrast to the only previous structure of a ligand-bound 6-phytase, where the 3-phosphate was unexpectedly in the catalytic site, in the H. alvei complex the expected scissile 6-phosphate (sulphate in the inhibitor) is placed in the catalytic site.

Characterization of the brewery spoilage bacterium Obesumbacterium proteus by automated ribotyping and development of PCR methods for its biotype 1.

AIMS: To study the ability of automated ribotyping to characterize Obesumbacterium proteus and Hafnia alvei, to design primers and to evaluate standard end-point and real-time PCR for the detection of O. proteus biotype 1 in beer and in brewers's yeast-containing samples. METHODS AND RESULTS: Automated ribotyping was carried out using the standard method with EcoRI and PvuII. The digestions with both enzymes clearly differentiated O. proteus biotypes 1 and 2 and H. alvei. PCR primers were designed according to the 16S rRNA gene sequence of the O. proteus type strain. Two primer sets (Obs137-Obs558 and Obs137-Obs617) detected O. proteus biotype 1 and H. alvei but not O. proteus biotype 2 or other tested beer spoilage bacteria (40 species) in the end-point and real-time PCR, indicating their high specificity. The detection limit for O. proteus was 160-1600 CFU 100 ml(-1) beer in the end-point PCR reactions and < or =160 CFU 100 ml(-1) beer in the real-time PCR reactions. More cells (from 16 to 3200) were needed for detection in the presence of brewer's yeast cells. CONCLUSIONS: Automated ribotyping is a useful tool to characterize and identify O. proteus and H. alvei isolates. The designed primers are suitable for the rapid detection of O. proteus biotype 1 and H. alvei in brewery samples by PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: Automated ribotyping and PCR could improve microbiological quality control in breweries by facilitating the detection, identification and tracing of spoilage bacteria.

Genetic environment of acquired bla(ACC-1) beta-lactamase gene in Enterobacteriaceae isolates.

We studied the genetic organization of bla(ACC-1) in 14 isolates of Enterobacteriaceae from France, Tunisia, and Germany. In a common ancestor, ISEcp1 was likely involved in the mobilization of this gene from the Hafnia alvei chromosome to a plasmid. Other genetic events involving insertion sequences (particularly IS26), transposons (particularly Tn1696), or sulI-type integrons have occurred, leading to complex genetic environments.

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.

Virulence factors and pathogenicity of Hafnia alvei for gilthead seabream, Sparus aurata L.

Virulence factors (eae gene, haemolytic capacity, fimbriae, resistance to the bactericidal effect of serum, siderophore production) and pathogenicity for gilthead seabream, Sparus aurata L., were analysed for 23 Hafnia alvei strains. None of the strains used in LD50 studies were lethal for seabream at doses as high as >10(8) cfu mL(-1). In chronic challenge studies differences in severity of the inflammatory response were observed between strains. On the basis of correlation of the inflammatory response to different strains of H. alvei in seabream with those virulence factors studied, it was only possible to establish a positive correlation between pathogenicity and resistance to the bactericidal effect of fish serum. Gilthead seabream is thus a species with considerable resistance to experimental infection with H. alvei. The bacterium does, however, have the capacity to remain viable in seabream for up to 3 months, without any clinical signs. Hafnia alvei is a well-recognized human and animal pathogen. Thus, as the pathogen can coexist with aquaculture operations, cultured gilthead seabream could represent a risk to human health as a carrier in some circumstances.

Evolutionary genetics of a new pathogenic Escherichia species: Escherichia albertii and related Shigella boydii strains.

A bacterium originally described as Hafnia alvei induces diarrhea in rabbits and causes epithelial damage similar to the attachment and effacement associated with enteropathogenic Escherichia coli. Subsequent studies identified similar H. alvei-like strains that are positive for an intimin gene (eae) probe and, based on DNA relatedness, are classified as a distinct Escherichia species, Escherichia albertii. We determined sequences for multiple housekeeping genes in five E. albertii strains and compared these sequences to those of strains representing the major groups of pathogenic E. coli and Shigella. A comparison of 2,484 codon positions in 14 genes revealed that E. albertii strains differ, on average, at approximately 7.4% of the nucleotide sites from pathogenic E. coli strains and at 15.7% from Salmonella enterica serotype Typhimurium. Interestingly, E. albertii strains were found to be closely related to strains of Shigella boydii serotype 13 (Shigella B13), a distant relative of E. coli representing a divergent lineage in the genus Escherichia. Analysis of homologues of intimin (eae) revealed that the central conserved domains are similar in E. albertii and Shigella B13 and distinct from those of eae variants found in pathogenic E. coli. Sequence analysis of the cytolethal distending toxin gene cluster (cdt) also disclosed three allelic groups corresponding to E. albertii, Shigella B13, and a nontypeable isolate serologically related to S. boydii serotype 7. Based on the synonymous substitution rate, the E. albertii-Shigella B13 lineage is estimated to have split from an E. coli-like ancestor approximately 28 million years ago and formed a distinct evolutionary branch of enteric pathogens that has radiated into groups with distinct virulence properties.

Biochemical-genetic characterization and regulation of expression of an ACC-1-like chromosome-borne cephalosporinase from Hafnia alvei.

A naturally occurring AmpC beta-lactamase (cephalosporinase) gene was cloned from the Hafnia alvei 1 clinical isolate and expressed in Escherichia coli. The deduced AmpC beta-lactamase (ACC-2) had a pI of 8 and a relative molecular mass of 37 kDa and showed 50 and 47% amino acid identity with the chromosome-encoded AmpCs from Serratia marcescens and Providentia stuartii, respectively. It had 94% amino acid identity with the recently described plasmid-borne cephalosporinase ACC-1 from Klebsiella pneumoniae, suggesting the chromosomal origin of ACC-1. The hydrolysis constants (k(cat) and K(m)) showed that ACC-2 was a peculiar cephalosporinase, since it significantly hydrolyzed cefpirome. Once its gene was cloned and expressed in E. coli (pDEL-1), ACC-2 conferred resistance to ceftazidime and cefotaxime but also an uncommon reduced susceptibility to cefpirome. A divergently transcribed ampR gene with an overlapping promoter compared with ampC (bla(ACC-2)) was identified in H. alvei 1, encoding an AmpR protein that shared 64% amino acid identity with the closest AmpR protein from P. stuartii. beta-Lactamase induction experiments showed that the ampC gene was repressed in the absence of ampR and was activated when cefoxitin or imipenem was added as an inducer. From H. alvei 1 cultures that expressed an inducible-cephalosporinase phenotype, several ceftazidime- and cefpirome-cross-resistant H. alvei 1 mutants were obtained upon selection on cefpirome- or ceftazidime-containing plates, and H. alvei 1 DER, a ceftazidime-resistant mutant, stably overproduced cephalosporinase. Transformation of H. alvei 1 DER or E. coli JRG582 (ampDE mutant) harboring ampC and ampR from H. alvei 1 with a recombinant plasmid containing ampD from E. coli resulted in a decrease in the MIC of beta-lactam and recovery of an inducible phenotype for H. alvei 1 DER. Thus, AmpR and AmpD proteins may regulate biosynthesis of the H. alvei cephalosporinase similarly to other enterobacterial cephalosporinases.

Chimeric nature of two plasmids of Hafnia alvei encoding the bacteriocins alveicins A and B.

The complete nucleotide sequences of two bacteriocin-encoding plasmids isolated from Hafnia alvei (pAlvA and pAlvB) were determined. Both plasmids resemble ColE1-type replicons and carry mobilization genes, as well as colicin-like bacteriocin operons. These bacteriocins appear to be chimeras consisting of translocation domains from Tol-dependent colicins, unique binding domains, and killing and immunity domains similar to those of the pore-forming colicin Ia. Just as is found for colicin Ia, these H. alvei bacteriocins (alveicins) lack lysis genes. The alveicins are unusually small at 408 and 358 amino acids for alveicin A and B, respectively, which would make alveicin B the smallest pore-forming bacteriocin yet discovered. The pattern of nucleotide substitution in the alveicins suggests that the dominant forces in the evolution of their killing domains and immunity genes are neutral mutation and random genetic drift rather than diversifying selection, which has been implicated in the evolution of other colicins. Five of six bacteriocinogenic isolates of H. alvei were found to carry plasmids identical to pAlvA. Comparisons of the levels of nucleotide divergence in five housekeeping genes to the levels of divergence in their respective plasmids led us to conclude that pAlvA is transferring laterally through the H. alvei population relatively rapidly.

Conjugative plasmid mediated inducible nickel resistance in Hafnia alvei 5-5.

Hafnia alvei 5-5, isolated from a soil-litter mixture underneath the canopy of the nickel-hyperaccumulating tree Sebertia acuminata (Sapotaceae) in New Caledonia, was found to be resistant to 30 mM Ni(2+) or 2 mM Co(2+). The 70-kb plasmid, pEJH 501, was transferred by conjugation to Escherichia coli, Serratia marcescens, and Klebsiella oxytoca. Transconjugant strains expressed inducible nickel resistance to between 5 and 17 mM Ni(2+), and cobalt resistance to 2 mM Co(2+). A 4.8-kb Sal- EcoRI fragment containing the nickel resistance determinant was subcloned, and the hybrid plasmid was found to confer a moderate level of resistance to nickel (7 mM Ni(2+)) even to E. coli. The expression of nickel resistance was inducible by exposure to nickel chloride at a concentration as low as 0.5 mM Ni(2+). By random Tn phoA'-1 insertion mutagenesis, the fragment was shown to have structural genes as well as regulatory regions for nickel resistance. Southern hybridization studies showed that the nickel-resistance determinant from pEJH501 of H. alvei 5-5 was homologous to that of pTOM9 from Alcaligenes xylosoxydans 31A.

The eaeA gene is not found in Hafnia alvei from patients with diarrhea in Aragón, Spain.

A total of 102 Hafnia alvei clinical strains isolated from different patients with diarrhea has been tested, using polymerase chain reaction and dot-blot hybridization, for the enteropathogenic Escherichia coli attaching and effacing A (eaeA) gene to establish their role as a causative agent of diarrhea in our environment. None of them was positive for the eaeA gene. We cannot consider the eaeA gene as the virulence-associated factor implicated in the H. alvei strains isolated from diarrheal feces in our region.