Possible Misidentification of Haemophilus aphrophilus as Pasteurella gallinarum

Sir—Recent reports by Al Fadel Saleh et al. [1] and Arashima et al. [2] have drawn attention to Pasteurella gallinarum as a possible cause of disease in humans. Since, to our knowledge, there have been no previous reports of isolation of P. gallinarum from humans, these 2 reports raise suspicion that misidentification of a strain has occurred. Several years ago, a strain (P 961) isolated from a blood sample obtained from a patient with probable endocarditis was sent to an experienced veterinary (poultry) bacteriologist as part of an exchange of strains. The veterinary bacteriologist identified the strain as catalase-negative P. gallinarum. However, an experienced nonveterinary medical bacteriologist (W.F.) later identified the strain as Haemophilus aphrophilus.P. gallinarum is well known to veterinary bacteriologists, whereas H. aphrophilus is better known to nonveterinary medical bacteriologists; therefore, it is probable that an unknown isolate will be identified as belonging to the taxon best known to the particular bacteriologist who is making the identification. The misidentification of P. gallinarum as H. aphrophilus may have occurred because the 2 strains have few differentiating characteristics, and because they are seldom included in the same identification table in textbooks and in other reference publications (the work of Kilian and Frederiksen [3] is one exception). Catalase and gas from glucose are the main findings that allow for differentiation of P. gallinarum and H. aphrophilus (table 1). Findings that allow for differentiation of the Haemophilus aphrophilus and Pasteurella gallinarum strains. Al Fadel Saleh et al. [1] indicated that they characterized the investigated strain as P. gallinarum by use of the API 20E and API 20NE systems (BioMerieux), and on the basis of a positive result for catalase and a negative result for gas from glucose. Arashima et al. [2] indicated that they identified the P. gallinarum bacteria on the basis of “their biochemical properties.” The phenotypical description of the strain mentioned by Al Fadel Saleh et al. is compatible with that of P. gallinarum; however, P. gallinarum is not included in the API 20NE system database, which makes use of this system of dubious benefit. We have been told by BioMerieux (K. Astrup, personal communication) that strains of P. gallinarum have produced reactions that have led to the octal codes 5020004 and 5000004 (i.e., on the basis of a reduction in NO3, a positive result for acid from glucose, a positive or negative result for p-nitrophenyl-β-D-galactopyranoside [PNPG], and a positive result for oxidase), reactions that are characteristic of both P. gallinarum and H. aphrophilus. To elucidate the matter, we tested 3 strains of both species (including the type strains), by means of conventional testing and by use of the API 20NE system. We also tested 3 strains of Actinobacillus actinomycetemcomitans, a species that is considered to be similar to H. aphrophilus. The results of these tests are shown in tables 2 and 3 (only tests that gave different results among the strains or that contributed to the octal code are shown). Differentiation of strains of Haemophilus aphrophilus, Pasteurella gallinarum, and Actinobacillus actinomycetemcomitans, according to the results of conventional tests. Differentiation of strains of Haemophilus aphrophilus, Pasteurella gallinarum, and Actinobacillus actinomycetemcomitans, according to the results of testing done by use of the API 20NE system. When conventional testing was done, the o-nitrophenyl-β-D-galactopyranoside (ONPG), glucose, and lactose tests distinguished P. gallinarum from H. aphrophilus, and when testing was done with use of the API 20NE system, the PNPG test allowed for differentiation of the 2 strains. A. actinomycetemcomitans can be distinguished from H. aphrophilus according to the results of tests for ONPG, sucrose, and lactose; the CO2 dependency of A. actinomycetemcomitans is an extra aid for identification of the species. When testing was done with use of the API 20NE system, A. actinomycetemcomitans strains produced the same reactions as did P. gallinarum strain P 190. Because some P. gallinarum strains produce acid from lactose and are positive for β-galactosidase, use of the conventional ONPG test or the PNPG test in the API 20NE system are not sufficient for differentiation of P. gallinarum and H. aphrophilus, regardless of whether we found our 3 P. gallinarum strains to test negative for these features. It seems plausible that the strain described by Al Fadel Saleh et al. could be a P. gallinarum strain, since it was reported to test positive for catalase and negative for gas from glucose. Because Arashima et al. provided no information about the strain that they reported, it remains unclear whether their strain really was a P. gallinarum strain. We conclude that use of the API 20NE system does not allow for differentiation between P. gallinarum, H. aphrophilus, and A. actinomycetemcomitans. The 3 species can be identified by use of conventional tests, although with difficulty, since presence of catalase may be the only finding that distinguishes P. gallinarum from H. aphrophilus. When characterized by 16S rRNA sequencing, P. gallinarum and H. aphrophilus are widely separated (dissimilarity, >7%) [5], which indicates that they are indeed separate species. When unusual strains are found in unusual circumstances, we strongly recommend that either (1) the strains be sent to a competent reference laboratory, where they can be compared with authentic strains of suspected species, or (2) the investigators obtain reference strains and make this comparison themselves. H. aphrophilus is well known as an organism that may be found in humans and that may cause endocarditis; it has not been found in other hosts. P. gallinarum is well known to poultry bacteriologists, and its isolation from human clinical material should be questioned until such isolation can be rigorously proven. If these strains are kept, they should be sent to an experienced reference laboratory (e.g., the Centers for Disease Control and Prevention [Atlanta] or Statens Seruminstitut [Copenhagen]). We thank Annemarie Hesselbjerg, Statens Seruminstitut, Copenhagen, for providing subcultures of the strains.