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 β-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 (∼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.