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The Genus Hafnia: from Soup to Nuts

Microbial Diseases Laboratory, Division of Communicable Disease Control, California Department of Health Services, Richmond, California 94804

SUMMARY

INTRODUCTION

HISTORICAL PERSPECTIVES

NOMENCLATURE, TAXONOMY, AND CLASSIFICATION

Nomenclature

Biochemical and Numerical Taxonomy Studies

DNA Relatedness

Phylogenetic and Related Studies

EPIDEMIOLOGY

Environmental Distribution

Disease States Associated with H. alvei

Ecologic Structure

Clinical Frequency, Outbreaks, and Nosocomial Infections

CLINICAL SIGNIFICANCE

Historical Anecdotes Regarding the Association of Hafniae with Human Disease

Bacteremia

Gastroenteritis

A controversial issue.

Is H. alvei an enteropathogen?

Respiratory Tract Infections

Miscellaneous Infections

PATHOGENICITY

LABORATORY ISOLATION AND IDENTIFICATION

Cultural Characteristics

Identification

Commercial systems.

Conventional methods.

Specialized tests. (i) Hafnia-specific phage.

(ii) GAD.

(iii) LPA.

Biotypes. (i) Biotype 1 strains.

(ii) Barbe biotypes.

Distinguishing H. alvei complex from Escherichia albertii.

Phenotypic and genotypic separation of H. alvei DNA groups 1 and 2.

Molecular identification.

Serology

ANTIMICROBIAL SUSCEPTIBILITY

CONCLUSIONS

ADDENDUM

REFERENCES

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INTRODUCTION

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The genus Hafnia is one of more than 40 genera that currently comprise the family Enterobacteriaceae. Although Møller originally described this genus in 1954 (89), the legitimacy of this group was constantly challenged over the next two decades, often being referred to by such synonyms as "Enterobacter alvei" "Enterobacter aerogenes subsp. hafniae" and "Enterobacter hafniae" (www.bacterio.cict.fr/h/hafnia.html). Subsequent DNA-DNA relatedness investigations conducted by Don Brenner and the Centers for Disease Control and Prevention (CDC) conclusively established that these bacteria represented a unique taxon within the family and that at least two or more DNA groups existed within the genus (20, 21, 131).

Although this genus is over 50 years old, very little is known about these organisms in regard to the role(s) they may play as both human and veterinary pathogens. In one sense, these bacteria appear to be "orphan enterobacteria" in search of a medical and public health identity. Hafnia is fairly often isolated from clinical material, yet with the exception of its documented role as a rare cause of bacteremia (31, 104, 105), its relationship to other clinical infections is questionable. In fact, data from the early 1990s suggesting that hafniae were true enteropathogens (4, 5) now turn out to have been incorrectly attributed to hafniae rather than to the actual pathogen, Escherichia albertii (63, 69). The possible role of Hafnia in bacterial gastroenteritis is presently unknown, although one recent case report suggests a possible role in enteritis (see Addendum).

Over the past quarter of a century, there have been few studies that have systematically looked at the role of these bacteria in clinical microbiology. Greipsson and Priest (56) published the only taxonomic study on this genus in 1983, and Günthard and Pennekamp (59) reported on the clinical significance of the only large series of extraintestinal isolates. Recently there have been a number of studies describing and characterizing multiple genomospecies within the genus, phylogenetic relatedness, clinical infections, pathogenicity, and biochemical characteristics of genomovars. This review attempts to systematically review the pertinent literature on this genus, discuss current controversies surrounding these bacteria, and shed some light on the potential role these "orphan enterobacteria" may play in human disease.

HISTORICAL PERSPECTIVES

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It is the subject of some speculation and debate as to when organisms currently in the genus Hafnia were first isolated. In 1919, L. Bahr worked on a bacterium that he designated "Bacillus paratyphi-alvei," an organism reputedly pathogenic for bees but not mice or guinea pigs (121). One of Bahr's apparently authentic "Bacillus paratyphi-alvei" strains (referred to as "Paratyphus alvei") was subsequently characterized in 1954 as belonging to a new group of enteric bacteria for which Møller coined the name "Hafnia group" during a systematic investigation of amino acid decarboxylase patterns among members of the family Enterobacteriaceae (89). Some groups subsequently questioned the legitimacy of this name in light of the fact that Bahr's strains differed in some biochemical characteristics from those described by Møller. However, Møller considered that Bahr's strain should be regarded as the type species of Hafnia, and he suggested the name Hafnia alvei.

There are no extant cultures of Bahr's strains. However, in the early 1940s Stuart and colleagues (135) biochemically characterized a large group of paracolon bacteria. Within the group named "Paracolon Aerobacter," two divisions were recognized based upon the results of indole-methyl red-Voges-Proskauer-citrate assays. Division 2 contained seven distinct biotypes, the largest of which was referred to as type 32011 (35 cultures). Type 32011 and related types had properties generally consistent with inclusion in the genus Hafnia, being indole, lactose, and sucrose negative; aerogenic; and Voges-Proskauer (VP) positive. Type 32011 strains were subsequently characterized by several independent groups, including Edwards and Ewing (29), who confirmed that the biochemical characteristics of type 30211 were identical or nearly identical to those reported for the "Hafnia group" by Møller and others.

From the 1940s until the mid- to late 1970s, when nomenclature and classification issues surrounding this taxa were resolved, H. alvei was known by a number of different names, including "Paracolon Aerobacter," "Paracolobactrum aerogenoides," Bacillus cadaveris, the Hafnia group, Enterobacter alvei, and Enterobacter hafniae (16, 120, 121). For one brief period of time it was even relegated to subspecies rank within Enterobacter aerogenes (32). However, subsequent taxonomic investigations demonstrated that it belonged to a unique lineage within the Enterobacteriaceae and deserved genus (species) rank (see "NOMENCLATURE, TAXONOMY, AND CLASSIFICATION" below). The name Hafnia alvei appears in the Approved Lists of Bacterial Names, and the type strain is ATCC 13337^T (129). Three of the oldest extant cultures of H. alvei are ATCC 13337^T (Stuart's type 32011 = NCTC 8105 = NCDC 434-68), ATCC 11604 ("Paracolobactrum aerogenoides" = 3565 [Conn 3565], Stuart's type 32011) of Borman et al. (16), and NCTC 6578 of Gale and Epps (43).

NOMENCLATURE, TAXONOMY, AND CLASSIFICATION

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Few studies have dealt with the genus Hafnia by employing numerical taxonomy principles. Bascomb and coinvestigators (12) studied members of the then-tribe Klebsiellae, including 15 strains of H. alvei. By using three different methods of numerical classification, all H. alvei strains were found to form a separate and distinct branch with a rank similar to that of the tribe Klebsiellae. Johnson et al. (73) studied 384 strains in the family Enterobacteriaceae for 216 independent characters, including morphological, biochemical, and physiologic traits; 10 of these enteric isolates were H. alvei. Based upon simple matching (S_M) and Jaccard (S_J) coefficients, H. alvei strains were found to cluster independently of other groups, joining members of the tribe Klebsiellae at 78% similarity. Those authors concluded that this justified the status of this taxon as an independent genus rather than being placed within the genus Enterobacter. The only study to specifically look at Hafnia by use of numerical taxonomic principles was that of Greipsson and Priest (56), who studied 47 strains from the United Kingdom, Japan, France, and the United States by using 101 different characters. All 47 strains exhibited high similarity values (>87%) by S_M coefficient when average-linkage cluster analysis was used with the type strain NCTC 8105^T (ATCC 13337^T), located near the center of the phenon. These results support previous conclusions reached by Johnson et al. (73) that all H. alvei isolates reside within a single phenon or cluster.

The first direct evidence proving the existence of more than one DNA group within Hafnia alvei was reported by Steigerwalt and coinvestigators (131) as part of a study of DNA relatedness between Enterobacter and Serratia species. In that study, using H. alvei ("E. hafniae") CDC 4360-67 as the source of labeled DNA, two distinct DNA groups could be discerned, one highly (85% to 100%) related to CDC 4360-87 and one less (51% to 55%) related; the latter contained the type strain of H. alvei, ATCC 13337^T. H. alvei was found to be only 16% to 24% related to other members of the Enterobacteriaceae by DNA-DNA hybridization in that study. In subsequent publications, Brenner (20, 21) reported the existence of two or more hybridization groups (HGs) within H. alvei, and this has been confirmed in subsequent studies (47). Furthermore, Brenner (21) found that the type strain of Obesumbacterium proteus (ATCC 12841^T) is 75% related to DNA group 2 (in some studies called DNA group 1) of H. alvei and is 52% related to the type strain of H. alvei, ATCC 13337^T. Thus, a valid type strain for O. proteus does not exist. Table 1 lists reference strains and their vernacular designations from surveys that studied both H. alvei DNA groups (HGs). By convention, the type strain for the nomenspecies (ATCC 13337^T) and related reference strains should be classified as DNA group 1 (HG1), since this group represents H. alvei sensu stricto. DNA group 2 (HG2, unnamed) is represented by several reference strains, including ATCC 29927. CDC 4360-67 (DNA group 2) was not retained in the permanent culture collection of CDC and is no longer available as a reference strain for DNA group 2 (HG2) (C. O'Hara, personal communication).

Hedegaard and colleagues (62) investigated the utility of partial sequencing of the infB gene (encoding translation initiation factor 2) as a phylogenetic tool for studying 39 Enterobacteriaceae strains. By using a clinical strain of H. alvei and the distance matrix method, the closest neighbors to hafniae were found to be members of the tribe Proteeae. Phylogenetic investigations using gyrB gene (encoding the ATPase domain of DNA gyrase) and protein sequence data yielded mixed results (27). Dauga (27) noted that there was atypical codon usage (third-codon G+C content) in the gyrB gene of H. alvei relative to other Enterobacteriaceae, which suggested foreign acquisition of this gene. If this is true, analysis of the gyrB sequences would be indicative only of the origin of the gene and not the species. Wertz et al. (139) studied five housekeeping gene and 16S rRNA gene sequences in an analysis of a limited series of species in the Enterobacteriaceae. Cladograms generated by maximum-likelihood analysis found that H. alvei's nearest neighbor was Serratia plymuthica for four genes (groEL, gyrA, pgi, and ompA), while by gapA sequencing it was Enterobacter cloacae and by 16S rRNA analysis K. pneumoniae. Phylogenetic data to date are inconclusive regarding where the genus belongs. One study on the evolution of aromatic amino acid biosynthesis genes and enzymes suggests clustering of H. alvei with Edwardsiella, Yersinia enterocolitica, and Proteus vulgaris (2). However, to date phylogenetic data regarding where the genus should be rooted are inconclusive.

EPIDEMIOLOGY

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The gastrointestinal tracts of animals, and in particular mammals, appear to be a very common ecologic habitat for hafniae. Paleomicrobiology investigations have identified H. alvei originating from intestinal mass samples and sediment collected from 12,000-year-old mastodon remains in Michigan and Ohio (110). In a study of 642 Australian mammals, Gordon and FitzGibbon (53) found that H. alvei was the third most common enteric species identified, following Escherichia coli and E. cloacae. The isolation of H. alvei was significantly associated with recovery from marsupial carnivores and murid rodents. Hafniae have also been recovered sporadically from pack animal manure samples collected from national park trails (28) and from 7% of grizzly and black bear specimens tested (51). Among avian species, H. alvei has been frequently isolated from birds of prey, including falcons, owls, and turkey vultures; even high-altitude alpine accentors that have virtually no contact with humans have had Hafnia isolated from them, at frequencies ranging from 3% to 16% (9, 78, 142). Other sources for H. alvei include reptiles (snakes and skinks), invertebrates, insects, fish, and bats (24, 52, 98).

Food products commonly yield hafniae. Meats, including minced and vacuum-packed beef and pork products, ground beef chubs, and retail packages, harbor H. alvei (82, 113). One Finnish study found almost 50% of all enteric isolates recovered from refrigerated meats to be H. alvei (113). A subsequent study identified 34% of minced meat samples, 14% of milk and cream products, and 12% of freshwater fish as containing hafniae (44). Counts of H. alvei in these samples were as high as 7.5 x 10¹⁰ to 8.0 x 10¹⁰ CFU/g (82). Some isolates of H. alvei recovered from albacore produced histamine, although the relevance of this finding to scrombroid poisoning is unknown (76). Other consumables that are reported to yield H. alvei include cheese and honey (90). Vegetables do not appear to be a frequent reservoir for these bacteria, although H. alvei was recovered from nine Finnish and imported vegetable samples in one study (100). There are only a few anecdotal reports describing the isolation of hafniae from water (98).

H. alvei has been implicated in a variety of other animal infections. H. alvei has been reported as the cause of an epizootic outbreak of septicemia in rainbow trout in Bulgaria (47). Hafnia was recovered in pure culture from parenchymal organs during the acute phase of disease and was also isolated from necrotic tissues later in the course of infection. A 1963 French publication describes an epizootic outbreak of disease in white laboratory rats 3 to 8 weeks old (8). A number of the rats were lethargic and died unexpectedly. Autopsy demonstrated splenic hypertrophy (four to five times normal size) with congestion in visceral organs, including the brain and cerebellum. H. alvei was recovered from samples of organ tissues and heart blood. This strain possessed phenotypic properties that are consistent with inclusion in the genus, except that these were VP negative (85% of H. alvei isolates are positive) and produced acid from dulcitol (<1% of H. alvei isolates are positive). Other animal diseases linked to Hafnia infection include caprine pneumonia (127) and equine abortion (91).

View larger version (25K): [in this window] [in a new window]   FIG. 1. Schematic representation of major disease associations associated with the genus Hafnia and their environmental distribution.

CLINICAL SIGNIFICANCE

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The first credible report linking H. alvei to human illness came from a 1967 case study by Jennis and McCarthy (72). A 58-year-old man who underwent multiple medical procedures for a duodenal ulcer developed Hafnia bacteremia with fever (40.5°C) and hypotension (80/50) 6 days after surgery. Two blood culture specimens were positive for H. alvei, and he was treated with oxytetracycline, to which he responded favorably. Two years later, a second case of H. alvei bacteremia was described; this was in another 58-year-old male with suspected myocardial infarction (31). H. alvei was repeatedly isolated from blood cultures for over a month despite multiple antibiotic regimens, leading to the suspicion of a hidden nidus of infection. No such focus was ever identified, and it was postulated that the bacteremia continually seeded from the genitourinary tract and retention catheter. These two publications provided strong evidence that H. alvei could cause extraintestinal infections. A study from the Mayo Clinic in 1971 confirmed that hafniae could cause secondary illnesses associated with wounds/abscesses and respiratory problems, including bronchopneumonia (138).

To date there have been three reports describing a series of cases involving infections or asymptomatic colonization with H. alvei; in each of these studies the number of patients ranged from 8 to 61 (59, 105, 138). In one investigation of 17 patients where most hafniae isolated were judged to be commensals, 59% of all isolates were determined to be have been transmitted nosocomially (138), while in a smaller study of 8 patients, most or all of whom had true H. alvei infections, 63% of the infections were found to be community acquired (105). Most individuals (63% to 93%) colonized or infected with hafniae have had underlying diseases, the most common of which are cancer (in particular hematologic malignancies), surgery, trauma, chronic or acute pulmonary disease, cirrhosis/hepatitis, and pancreatitis. In the largest series of cases, 66% of the patients were intubated, including all those from whom H. alvei was recovered from the respiratory tree (59). Two studies found that 37% to 55% of persons colonized/infected with Hafnia had received antibiotics prior to the isolation of this microorganism. No deaths attributable to H. alvei were recorded in any of these three studies.

From 12% to 75% of clinical specimens yield H. alvei in pure culture (59, 105, 138). Sites most likely to yield hafniae as the sole pathogens include urine and blood. When found as part of mixed microbial flora, they are often associated with other enteric bacteria, staphylococci, streptococci, and yeast (59). When hafniae have been isolated in pure growth or as predominant flora, there is a better correlation with disease status (e.g., bronchopneumonia) than when H. alvei is recovered in small numbers (138).

There have been relatively few cases of Hafnia bacteremia reported in the medical literature. Of those cases, 18 were published between 1967 and 2004 with enough data to characterize the series to some extent, although many of these reports lack laboratory information, including methods of identification of hafniae and biochemical characteristics of the infective strain. Of the 18 cases reported in Table 3, 12 occurred in adults, 4 in pediatric patients (2 to 13 years of age), and 2 in neonates (8 to 20 days old). Neonatal infections may be linked to frequent carriage of hafniae by women as part of the vaginal flora prior to birth (92). The symptoms associated with Hafnia sepsis are typical of gram-negative septicemias in general and include fever (38.6 to 40.5°C), occasional chills, and abdominal pain (14, 23, 26, 31, 37, 57, 72, 87, 105, 145). Diarrhea, either during or preceding the septic crisis, is not a consistent feature of H. alvei bacteremia but has been reported on several occasions. Fecal samples have been described as being bloody, melenic, or tarry in consistency (26, 31, 48, 104, 105). Additional laboratory studies, when reported, include an elevated level of C-reactive protein (6.8 to 37.3 mg/dl) and leukocytosis (14,200 to 32,600 mm³) (23, 26, 37, 57, 105). A majority of these blood-borne infections occur in males (71%) and are community acquired. The times of first positive Hafnia blood cultures in hospitalized patients have ranged from 1 to 41 days after admission. Half of all bacteremic patients were immunocompromised, with malignancies, liver disease, or human immunodeficiency virus (HIV) infection being the most common underlying conditions responsible for their impaired immune status. Several persons have also developed Hafnia bacteremia subsequent to liver transplants (10), and in one case Hafnia bacteremia occurred in a soldier following a gunshot wound and surgery (14). Two cases of pneumatosis intestinalis accompanying H. alvei bacteremia have also been described, one in a 6-year-old boy with nonlymphocytic leukemia (145) and another in a 20-day-old infant with necrotizing enterocolitis (48).

Most cases of H. alvei bacteremia are monomicrobic, although rare instances of polymicrobic septicemia involving Pantoea agglomerans (55) and Enterococcus faecalis (10) have been reported; the number of positive blood cultures in each case report ranged from one to seven. Besides blood, H. alvei has been recovered from other anatomic sites in approximately 33% of cases, including hepatic abscesses, pancreatic pseudocyst fluid, sputum, pleural fluid, feces, and a central venous catheter (10, 37, 48, 59). Although the source of H. alvei bacteremias is unknown in most instances, the presumed origin of infections is the gastrointestinal or respiratory tract.

Hafnia alvei ("Enterobacter hafnia") meningitis in a 1-year-old Iranian child with intermittent fevers of 1 month in duration has been reported (88). A cerebrospinal fluid tap grew H. alvei (no blood culture results were reported), and she was treated with gentamicin, chloramphenicol, and tetracycline. Although she started to improve, she died of nosocomially acquired Pseudomonas aeruginosa sepsis.

In 1991, renewed interest in hafniae as bona fide enteropathogens was reawakened by a report of Albert et al. (4) describing diarrhea thought to be caused by H. alvei in a 9-month-old girl. This strain elicited diarrhea in rabbits and was found to contain the attaching-and-effacing gene (eae) of enteropathogenic E. coli. Other reports by the same authors followed, describing similar H. alvei strains linked to diarrhea and possessing the eae gene (5). These publications further triggered a series of case histories (109, 141) documenting Hafnia as a cause of acute gastroenteritis, with each of these studies citing the prior work of Albert et al. (4, 5), and a number of other studies and reviews cited accumulating evidence regarding the reputed pathogenicity of H. alvei (59).

Although the strains described by Albert et al. were initially identified as H. alvei by using the API 20E strip, subsequent studies by independent research teams suggested that these strains might not in fact belong to this genus. These conclusions were reached on the basis of 16S rRNA gene sequencing, the failure of the isolates described by Albert et al. to be lysed by a Hafnia-specific bacteriophage, and other atypical biochemical properties (see "LABORATORY ISOLATION AND IDENTIFICATION" below) (65, 69, 114). Further study indicated that the strains described by Albert et al. belonged to the genus Escherichia (69), and a proposal to include them in the genus Escherichia as a new species, E. albertii, was formally made by Huys and associates in 2003 (63).

Is H. alvei an enteropathogen? Given the fact that the most credible recent information supporting the case for Hafnia as an enteric pathogen was actually based upon studies performed on strains that are not hafniae but rather members of the genus Escherichia, where does this leave us with isolates of H. alvei sensu stricto?

The single best indicator of the enteropathogenicity of any organism is the isolation of only that bacterium from multiple patient samples during the acute phase of an outbreak. When such isolates are related to one another, and hopefully to strains recovered from a vehicle of infection such as consumable products, the incriminating evidence becomes even stronger. In the case of H. alvei, several outbreaks attributed to this microorganism have been described (Table 2), but none of these completely meet the definition listed above. For the outbreaks reported to date, the best and most detailed report is that of Ratnam and others (106, 107). In support of the pathogenicity of these strains were the recovery of H. alvei as predominant or heavy pure growth from 13 of 23 stools (57%) involved in these two outbreaks, the fact that strains from both outbreaks possessed the same biotype (biotype 1) and serotype (O3:HNM), and the fact that of over 1,000 stools subsequently tested after these outbreaks, only one yielded H. alvei. However, in this study, no virulence factor was identified and there was no human immune response recorded or pathological data provided to corroborate culture results, and subsequent studies by an another laboratory testing reference strains from each outbreak found that they belonged to different genomospecies (71). Food and food handlers were the suspected sources, but this could not be proven.

Not much is known regarding the carrier state, although it is known to exist. In 1963, Matsumoto (85) screened 1,913 stools from asymptomatic inhabitants of Nagano Prefecture in Japan and found that 251 of these persons (13%) carried H. alvei. These numbers crossed all age groups and occupations. However, very different conclusions were drawn from a more recent study of Finnish travelers to Morocco, where no H. alvei was detected in the stools of 321 healthy persons (115). In that same study, H. alvei was recovered from 2% to 16% of persons with diarrhea in two distinct settings. However, a majority of these H. alvei isolates (57%) were recovered in conjunction with other recognized enteropathogens (Salmonella, Campylobacter, Aeromonas, or Shigella), thus limiting the conclusions that could be drawn from this survey.

Apart from these observations, there are few additional data regarding the possible enteropathogenicity of hafniae. Several case reports have been published (109, 141), and resolution of symptoms was accompanied by eradication or significant reduction of H. alvei in the gastrointestinal tract. However, antimicrobial therapy prescribed to eliminate Hafnia could also have been effective against other possible bacterial agents. Several potential virulence factors (other than eae) which could play a role in gastroenteritis have been also described, but either these reports are very old, the factor has only been found in a few strains, or the activity cannot be faithfully reproduced (see "PATHOGENICITY" below). Thus, at present, there are very few epidemiologic, clinical, and laboratory data to support Hafnia as a cause of gastroenteritis. This does not mean that hafniae are not enteric pathogens, just that much more work and evidence need to be accumulated to support such a position.

Although wounds would be thought to be common sites from which to recover hafniae, there are surprisingly few publications in the literature on this topic (40). Berger et al. (14) recovered H. alvei along with P. aeruginosa from a nosocomial wound infection of a 19-year-old soldier who had sustained a gunshot wound. What if any role hafniae played in the infectious process is unclear. H. alvei has also been isolated from a psoas abscess, but this gram-negative bacterium was isolated simultaneously with Streptococcus bovis (105). In a series of 17 cases of Hafnia colonization/infection, 3 were associated with wounds or abscesses (138). In all three cases they were thought to be secondary pathogens, although again they were recovered as part of a mixed microbial flora. Detailed information on these cases was not provided. The only convincing case of a wound infection attributed to H. alvei was reported by Augustin and Cunha (7) and involved a 44-year-old male who sustained a penetrating trauma to his right buttock by carpet nails. A subsequent abscess developed, and 50 ml of serosanguinous fluid grew a pure culture of H. alvei. H. alvei has infrequently been linked to hepatobiliary disease, including two cases of hepatic abscess (mixed cultures and bacteremia after liver transplantation) (10). Two cases of cholecystitis associated with Hafnia infection have also been published (105, 144). In one of these cases, that of a 67-year-old woman who apparently developed biliary disease after handling fish, hafniae were recovered from the infected gall bladder in small numbers in comparison to the predominant Aeromonas. Most recently, a single case of spontaneous bacterial peritonitis caused by H. alvei in a 60-year-old Saudi man with underlying mesothelioma has been reported (3).

The urinary tract occasionally harbors hafniae. Ramos and Dámaso (105) isolated H. alvei in pure culture from urine specimens of three different patients and felt they were clinically significant, while Washington et al. isolated hafniae in small numbers from a similar number of persons without signs of disease and determined that they were commensals (138). Septic arthritis associated with the recovery of both H. alvei and Klebsiella oxytoca from joint fluid of a 29-year-old woman after anterior cruciate ligament reconstruction has been reported (83). A case of reactive arthritis of greater than 10 weeks in duration has been linked to H. alvei enteritis (93). Although this pathogen was recovered in large numbers from feces, the joint fluid, blood, and urine were culture negative; the role of hafniae in this illness is debatable. Hafnia endophthalmitis has been reported in two instances. One was as part of a coinfection with Salmonella enterica subsp. arizonae (22). In this specific case, involving a 55-year-old Hispanic woman, the source of infection appeared to be snake powder from Mexico used to season food. A second case has recently been reported from Spain (118). In both instances, visual perception was lost despite aggressive medical intervention.

PATHOGENICITY

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Understanding of which virulence factors play a role in the pathogenicity of Hafnia in regard to both human and animal infections is presently in its infancy. The limited amount of research in this area is most likely attributable to the low frequency of occurrence of this species in human illnesses and the fact that there are no clear-cut disease syndromes specifically associated with H. alvei. A further obstacle has been the unfortunate association of the eae gene with enteritis-producing strains that were thought to be H. alvei (4) but that were determined years later to actually be members of a new species, E. albertii (63).

What is presently known regarding Hafnia pathogenicity can be looked at from two perspectives, that is, virulence factors potentially operative in extraintestinal infections and those restricted primarily to the intestine. With all of these data there are numerous "gaps" in information, and many of the original findings date back 2 decades or more, where the description of strains and techniques is often incomplete.

Lethality studies, such as determinations of 50% lethal dose, to compare the overt pathogenicity of H. alvei to those of other members of the family, including E. coli, Shigella, and Salmonella, have not been performed. There is some sketchy and anecdotal information that hafniae can produce morbidity and mortality in certain animal models. One study found that 60% to 100% of rainbow trout injected subcutaneously and white mice and guinea pigs injected intravenously with aliquots of broth cultures containing hafniae died within 24 h (mice) to 10 days (fish); mice injected subcutaneously or orally challenged did not die (46). Unfortunately, the inoculum used in these experiments was not quantified, although the challenge dose was likely very high. One other study mentions that mice injected intrapertioneally with hafniae did not succumb to infection (91).

Approximately 35% to 55% of H. alvei strains are resistant to the bactericidal activity of human or bovine serum (67, 102). The classical pathway appears to be the major route by which serum-susceptible strains are killed; preincubation of bovine serum with H. alvei O-specific polysaccharide reduces or eliminates the bactericidal activity (67). Iron-scavenging mechanisms can also play important roles in pathogenesis. H. alvei strains produce siderophores as detected by a universal chemical assay (71, 102). One study found that the H. alvei siderophore was of neither the hydroxamate (aerobactin) nor the catechol (enterobactin) type (67). The siderophores of H. alvei can, however, take up ferric enterobactin and ferrichrome via an E. coli FepA-like outer membrane protein (119). Other factors described for hafniae, which may or may not play a role in their pathogenicity, include mannose-sensitive and mannose-resistant (MR/K, type 3 fimbria) hemagglutinins (102). Enzymatic properties associated with some pathogenic bacteria, including hemolysin, protease, elastase, and lecithinase activities, have not been detected in hafniae (71).

As early as the 1950s some strains of H. alvei were thought to be enteropathogenic, based upon results in animal studies. Sakazaki (120) found that 15 H. alvei strains produced enteritis in the ligated rabbit intestine model. However, no credible data evolved over the next 30 years. Numerous studies in the 1990s confirmed the fact that true H. alvei did not contain the eae gene as had been previously reported (65, 69), and in fact one Spanish study found all 102 H. alvei strains tested to be eae negative by both PCR and dot blot analysis (126). Other fecal isolates associated with outbreaks of gastroenteritis have uniformly been negative for heat-stable and heat-labile enterotoxins, invasiveness, and production of Vero cell cytotoxins (106).

Approximately 70% of Hafnia strains carry one or no plasmids (128). However, plasmid-bearing strains often harbor very large extrachromosomal elements ranging from 128 to 256 kb (128). Not much is known regarding what genes or functions are encoded by these plasmids, although atypical properties, such as lactose fermentation, have been attributed to plasmid carriage by rare strains (81). Recently, two small plasmids designated pAlvA and pAlvB were found to encode bacteriocins, or alveicins (140). These bacteriocin-carrying plasmids are very small (5.1 to 5.2 kb), and the protein products produced from these genes range from 358 to 408 amino acids in size. The alveicins are active only against their own species; about 15% of H. alvei strains screened appear to produce alveicins.

LABORATORY ISOLATION AND IDENTIFICATION

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Classic strains of H. alvei are lactose and sucrose negative and as such appear as nonfermenting colonies on enteric isolation media. On moderately selective agars, they typically appear as large, smooth, convex, translucent colonies of 2 to 3 mm in diameter with an entire edge (68, 123); some may exhibit an irregular border. Several media have been employed on rare occasions to recover hafniae from fecal specimens. Using SS agar, Matsumoto (85) was able to isolate H. alvei from 13% of the 1,913 stools tested in 1961; this number probably represents a minimum value, since SS agar is known to be inhibitory to many Hafnia strains. Sakazaki (121) took advantage of the inability of hafniae to ferment D-sorbitol to recover H. alvei from 42% of 598 stools by using desoxycholate-lactose-sucrose-sorbitol agar, while comparable recovery rates on MacConkey agar were only 2%. H. alvei has also been isolated on sorbitol-MacConkey agar, a medium designed for the isolation E. coli O157:H7 (38). For environmental samples such as food, both violet red bile lactose and fecal coliform agars have been successfully used to isolate hafniae from consumables, including cheese and sausage (56, 80). Enrichment broths in which some but not all hafniae grow include selenite and tetrathionate broths (123).

Several additional cultural characteristics of H. alvei bear mentioning. Because H. alvei strains are H₂S negative, they can occasionally be mistaken for H₂S-negative Salmonella on media such as enteric agar (123). Similarly, some hafniae also produce red or pink colonies on xylose-lysine-desoxycholate agar (121). Some Hafnia strains or variants are lactose positive, a trait that in some instances has been determined to be plasmid mediated (81). Atypical H. alvei colonies on violet red bile lactose and fecal coliform agars have also been described (80).

Temperature can play a prominent role in phenotype expression in H. alvei. Strains typically incubated at lower (22°C) rather than higher (35°C) temperatures are more likely to produce acetylmethylcarbinol, form gas, and be more actively motile (89, 120, 123), while conversely, the methyl red test is positive at elevated temperatures. Early studies suggested that Hafnia strains were H₂S positive (89), although these strains are invariably H₂S negative on traditional screening media such as TSI (35). As with many Enterobacteriaceae that are H₂S negative on TSI, H. alvei strains are moderately to weakly H₂S positive on thiosulfate-containing media such as gelatin-cysteine-thiosulfate medium at 48 h (unpublished data).

Commercial systems. Most recent published studies involving the identification of Enterobacteriaceae on commercial systems have had little trouble identifying H. alvei. These systems include both MicroScan conventional and rapid panels (11, 42, 94, 116) and bioMérieux Vitek GNI+ and Vitek 2 systems (41, 42, 95). In five of these studies, 61 of 62 isolates (98%) were correctly identified as H. alvei (11, 41, 42, 94, 116). The single exception was an atypical strain that was esculin positive and L-rhamnose negative and that was identified as a rare biotype on MicroScan conventional panels (116). A sixth study conducted by the CDC found that 6 of 10 H. alvei strains yielded excellent identifications on Vitek ID-GNB, 3 of 10 strains produced a probability of a good identification, and 1 isolate was unidentified (95). An earlier study by the CDC found that four of nine H. alvei strains were identified as inactive E. coli by Vitek GNI+ (96). The converse situation can also occur, where species such as Salmonella enterica subsp. arizonae and Y. regensburgei have been misidentified as hafniae (96). A more recent observation concerns the recently described species E. albertii. Originally identified as H. alvei on the API 20E system by Albert et al. (4), this observation has subsequently been confirmed by other groups, and such misidentifications occur not only on the API 20E but also on Vitek and MicroScan rapid panels (1). Conventional biochemical tests are needed to distinguish between these two taxa at present (see "Conventional methods" below).

Agreement between commercially available systems and conventional biochemical formats varies with each test. Rodríguez et al. (116) found that there was generally good agreement between MicroScan Combo Negative type IS panels, conventional biochemical test results, and recorded character traits in Bergey's Manual of Systematic Bacteriology (122) for most phenotypic results. However major differences were noted in test correlation between commercial and conventional formats for o-nitrophenyl-ß-D-galactopyranoside (47.6%), urease (23.8%), VP (14.2%), and citrate (4.7%). For arginine dihydrolase, correlation of assays improved from 80.9% to 100% when test results were read manually rather than by using the WalkAway.

Conventional methods. Despite the fact that most strains of H. alvei can be easily identified using commercial systems, conventional biochemical test results can be necessary under a number of circumstances. These include when probability of a correct identification is low, when H. alvei strains with two or more atypical properties (e.g., VP negative and urease positive) are identified, when hafniae are isolated from an anatomic site not traditionally associated with this species (e.g., blood), for unusual disease syndromes linked to Hafnia infection, for chronic or recurring illnesses, and for publication purposes. One study assessing the reproducibility of results from 22 laboratories in the United Kingdom to identify a series of enteric bacteria in a multipoint identification scheme found H. alvei to be one of the most difficult organisms to identify, with 37% of strains reported with either an incorrect identification or no identification at all (143).

Specialized tests. (i) Hafnia-specific phage. In 1967, Guinée and Valkenburg (58) described a lytic bacteriophage that was specific for the genus Hafnia. Phage 1672 lysed all 100 Hafnia strains tested against it and none of over 200 other enteric bacteria, including Salmonella, Citrobacter, Enterobacter, Klebsiella, and Serratia strains. The test is performed by inoculating a 24-h broth culture onto the surface of a nutrient agar plate and, after removal of excess fluid, adding a drop of bacteriophage suspension to the plate. After overnight incubation at 37°C, a clear lytic zone surrounded by growth is recorded as positive (123). This is an excellent test to definitively identify members of the genus Hafnia and has been used extensively in strain characterization (71, 108). Phage 1672 is available from the American Type Culture Collection (Manassas, Va.) (catalogue no. 51873-B1). The propagating host strain for this phage is H. alvei ATCC 51873 (HER 1272 [1672]).

(ii) GAD. The assay for glutamate decarboxylase (GAD) activity can be performed in a single-tube format, as described by Rice and colleagues (111). A change in color from blue to yellow is considered to be a positive test. Escherichia species, including E. coli and E. albertii, are GAD positive, while hafniae are GAD negative (71, 111).

(iii) LPA. Among enteric bacteria, strong L-prolineaminopeptidase (LPA) activity is detectable only in H. alvei, Serratia marcescens, and Serratia liquefaciens (25, 34). The assay can be performed in tube or microtiter format or using commercially available kits (Aminopeptidase Wee-Tab; Key Scientific Products, Round Rock, Tex.) (71). This assay needs to be read within 30 min to 1 h, as weak LPA activity can be detected in other species upon prolonged incubation (34). LPA is useful in distinguishing hafniae from E. cloacae and related groups and from some Serratia species.

Biotypes. (i) Biotype 1 strains. Farmer and colleagues at CDC defined biotype 1 strains in 1985 as "H. alvei biogroup 1" or "H. alvei—brewery biogroups" (36). These strains are also synonymous with the previous epithet name Obesumbacterium proteus and the H. alvei biogroup 1 strains listed in Table 1 of reference 35 (J. J. Farmer, personal communication). Biogroup 1 strains have not been recovered from clinical specimens (36). Major phenotypic differences distinguishing "brewery strains" from typical H. alvei (Table 1 of reference 35), which includes both DNA groups, are listed in Table 4.

There were no major studies on this subject for over 20 years. In 2002, a study conducted by the Microbial Diseases Laboratory found that 22 Hafnia isolates could be assigned to their correct DNA relatedness groups by 16S rRNA gene sequencing (71). Biochemical characterization of these strains showed that while motility did tend to correlate with genomospecies (DNA group 1, 20% positive; DNA group 2, 71% positive), as in the study by Brenner (21), a better differential test appeared to be malonate utilization (DNA group 1, 100% positive; DNA group 2, 17% positive). Okada and Gordon (97) subsequently confirmed the discriminatory value of the malonate test in their ecologic study of hafniae in Australia. These collective data were reinforced by a 2005 study in which malonate and several other tests were found to be useful in separating these two relatedness groups (70). The proposal was also made in latter study to recognize four biogroups with H. alvei sensu stricto (DNA group 1), based upon the fermentation of D-arabinose and salicin as well as esculin hydrolysis. Table 7 provides cumulative data from several studies on the best tests currently available for separation of H. alvei DNA groups 1 and 2.

Two formal serologic typing schemes for H. alvei have been published. Riichi Sakazaki and colleagues at the National Institute of Health in Tokyo, Japan, pioneered the older of these two schemes. This scheme officially recognized 68 somatic ("O") and 34 flagellar ^<