The Genus Hafnia: from Soup to Nuts

NomenclatureThe specific epithet in the name Hafnia alvei is derived from the Latin noun alveus, meaning beehive, with “alvei” meaning “of a beehive.” Ewing (33) questioned the epithet “alvei,” stating that the name implied that these bacteria had something to do with bees or beehives although they did not. However, H. alvei has been recovered on occasion from the intestines of honeybees (Apis mellifera) as well as from honey, and several of these strains are included in the BCCM (Brussels, Belgium) collection (125). The genus name Hafnia is the historical name (Havn) for the city of Copenhagen, Denmark (122).

Biochemical and Numerical Taxonomy StudiesPrior to the advent of molecular biology, bacterial nomenclature and taxonomy were dependent primarily upon characters or phenotypes of cultures expressed under defined conditions. In their most sophisticated form, such analyses employed the principles of Adansonian taxonomy (numerical taxonomy or numerical phenetic analysis), but often studies simply compared large data sets of biochemical characteristics between two apparently different groups. In that vein, members of the family Enterobacteriaceae (17-19, 60, 75, 119) were grouped into tribes consisting of genera with similar characteristics. The tribe Klebsiellae contained four such genera (Klebsiella, Enterobacter, Hafnia, and Serratia), with most but not all of the tribe's members sharing a number of phenotypic features. These features included formation of acetylmethylcarbinol (VP), growth on Simmon's citrate agar and in potassium cyanide broth, urea negativity (48 h), and failure to produce hydrogen sulfide (33).

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.

DNA RelatednessDespite the general conclusions drawn from several phenotypic studies regarding the homogeneity of Hafnia isolates, other studies provided indirect evidence that genetic diversity exists within this nomenspecies. A study by Gavini and colleagues (45) identified two distinct Hafnia phenons, designated I₁ and I₂, in their analysis of 32 strains originating from feces and different water sources. These two clusters could not be unambiguously separated from one other, although they differed in the frequency of certain characters, including production of acetylmethylcarbinol at 37°C and utilization of malonate and d-tartrate. Another clue suggesting diversity within H. alvei involves the moles percent G+C content of hafniae (66). Two very different G+C contents for Hafnia have been reported; the more common estimate is 48.7 mol%, while a higher value of 52.6 to 57.7 mol% has also been published (56).

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).

Other recent studies employing different techniques also support the division of the genus Hafnia into two or more groups. 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). As in the previous study, two populations were detected, one consisting of 83 strains (including ATCC 13337^T and ATCC 29926) and a second group of 81 isolates (including ATCC 29927). These studies indicate that 16S rRNA sequencing and multilocus enzyme electrophoresis also detect HGs that were previously recognizable only through DNA pairing studies.

Phylogenetic and Related StudiesOnly a limited number of phylogenetic investigations have included one or more strains of H. alvei. The utility of 16S rRNA sequencing to study the family Enterobacteriaceae has been questioned due to the high intrafamily DNA relatedness values and limitations on the resolution of 16S rRNA for this group based upon the number of informative sites (27, 130). Spröer et al. (130) found that the type strain of H. alvei exhibited 99.5% sequence similarity to the type strain for O. proteus, DSM 2777^T (ATCC 12841^T), supporting previous observations by Brenner (21) regarding the DNA relatedness of these two species. In that study, the nearest neighbors to H. alvei were two distinct Serratia clusters containing nine species. Dauga (27) also looked at the phylogenetic position of H. alvei ATCC 13337^T relative to other Enterobacteriaceae. By using the neighbor-joining method, H. alvei was rooted by the tribe Proteeae (containing the genera Proteus, Providencia, and Morganella); by maximum-likelihood analysis, H. alvei clustered with Serratia, while by the distance method its nearest neighbors were the Escherichia-Salmonella-Citrobacter group. A 2003 study employing only a few enteric genera found the closest relative to H. alvei to be Klebsiella pneumoniae based upon 16S rRNA gene sequencing (139).

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.

Environmental DistributionMost standard microbiology texts list mammals, birds, reptiles, fish, soil, water, sewage, and foods as sources from which hafniae can be recovered (35, 36). However, there are surprisingly little hard data in the literature on this topic, and no systematic environmental surveys on the genus Hafnia have been published. Most of the data regarding the environmental distribution of hafniae come from investigations on the frequency, incidence, concentration, and distribution of members of the family Enterobacteriaceae. Host and geographical factors appeared to influence the thermal niche of H. alvei isolated from Australian mammals in one study (97).

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 × 10¹⁰ to 8.0 × 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).

Disease States Associated with H. alveiThere are only a limited amount of data presently available on disease states associated with H. alvei. There are two well-described outbreaks of H. alvei infection associated with the poultry industry. Real et al. (108) described an outbreak of disease in commercial laying hens in Spain, characterized by loss of appetite, decreased egg productivity, catarrhal enteritis, and fulminant septicemia. Prominent histopathologic features of the disease included hepatosplenomegaly, multifocal nectrotizing hepatitis, and splenitis. H. alvei was recovered in pure culture from infected tissues at necropsy. A very similar disease presentation could be reproduced using wild-type H. alvei isolated from ill hens experimentally infected by either the intraperitoneal or oral route. In 2004, a large outbreak of H. alvei disease in 12-week-old pullets was reported from Italy (103). These pullets initially suffered from anorexia, depression, ruffled feathers, and diarrhea. The overall mortality rate was 20.7%. Autopsy findings were remarkably similar to those observed by Real and colleagues (108), including catarrhal enteritis and hepatosplenomegaly. Again, the disease could be experimentally reproduced in healthy pullets challenged with the infective strain.

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).

Ecologic StructureThe first evidence suggesting that there may be a nonrandom association of distinct genetic populations of H. alvei with specific ecologic niches comes from a recent study by Okada and Gordon (98). In that investigation, genetic group 2 strains (corresponding to DNA group 2 in Table 1) were predominantly associated with freshwater fish, while strains from genetic group 1 (corresponding to DNA group 1 in Table 1) were associated with birds, frogs, invertebrates, and water isolates. Both groups were equally represented in mammals and reptiles.

Clinical Frequency, Outbreaks, and Nosocomial Infections H. alvei is an uncommon human pathogen, although this species has received increased attention from the medical community over the past decade due to its possible association with gastroenteritis (Fig. 1) (see “CLINICAL SIGNIFICANCE” below). Hafniae are commonly recovered from oropharyngeal specimens, from the gastrointestinal tract, less often from blood, and from a variety of other anatomic sites, including tissue, urine, and catheters (59, 138). In a study of 17 Hafnia alvei (“Enterobacter hafniae”) isolates recovered by the Mayo Clinic from 1968 to 1970, only 5 isolates (29%) were judged to be clinically significant (138). In all five of these cases, H. alvei was determined to be a secondary pathogen (respiratory, n = 2; abscess, n = 3). Overall, the mean age of persons infected/colonized with H. alvei was 52.9 years, with a male/female ratio of 1:1.1. Slightly over 50% of all isolates were acquired via a nosocomial route, including all five associated with infection (138). In only two instances was H. alvei recovered in pure culture, and in neither case was the strain clinically significant. A 1996 study by Günthard and Pennekamp (59) reviewed 80 H. alvei isolates isolated from 61 patients over a 30-month period (1992 to 1995). As in the previous report, the mean age of all patients was 53.5 years; however, almost two-thirds of the patients were male. In only 6 of these 61 patients (9.8%) was H. alvei was thought to be the etiologic agent of the disease, three of which were proven cases (septicemia, n = 2; peritonitis, n = 1) and three of which were probable cases (septicemia, n = 2; pneumonia, n = 1).

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FIG. 1.

Schematic representation of major disease associations associated with the genus Hafnia and their environmental distribution.

There are no well-described outbreaks of disease caused by H. alvei in which the epidemiologic, clinical, and laboratory correlates are overwhelmingly in support of an unquestionable role for hafniae in these illnesses. Table 2 lists five outbreaks of gastrointestinal disease, affecting between 2 and 72 individuals, in which H. alvei was suspected as the possible etiologic agent. Two Canadian outbreaks of gastroenteritis that occurred 1 month apart in the same institution in inpatients suggest a possible role for hafniae in the disease process. However, only 32% of the patients had H. alvei recovered in large numbers from their stools, and one symptomatic person continued to grow large numbers of hafniae from feces even after the person became asymptomatic (107). A report from Russia describes the possible association of H. alvei with diarrhea in 72 hospitalized persons and provides some microbiologic and immunologic data to support this hypothesis; however, a number of questions remain (54). These include a short incubation period, unusual symptoms (possibly neurotoxicosis), and the presence of rotavirus in at least some of these samples (54). Clearly, more definitive work in this area needs to be performed.

Since H. alvei is presently an uncommon human pathogen, incidence data such as those derived from the National Nosocomial Infection Surveillance System for many enteric pathogens are not available (112). From three separate studies, the percentage of documented H. alvei nosocomial infections ranged from 38% to 59%, although the absolute number of cases was relatively small (8 to 80) and they spanned a number of years (59, 105, 138).

Historical Anecdotes Regarding the Association of Hafniae with Human DiseaseThere are numerous narrative comments in the medical and scientific literature regarding the potential pathogenicity of H. alvei. In most instances these comments center on presumed associations between this bacterium and diarrheal disease. In 1943, Stuart and Rustigian (134) reported on one outbreak of mild to acute gastroenteritis that involved a fairly sizeable number of persons in Rhode Island, with the common thread of consumption of milk or milk-related products from the same supplier. The implicated organism was biotype 32011 (“H. alvei”) (Table 2). In the same study, the authors also described two apparent laboratory-acquired gastrointestinal infections, in a research student and in a glassware technician, both of whom handled biotype 32011. Other reports similar to this have surfaced from time to time, such as the case reports of Harada et al. (61) in 1957 describing the isolation of H. alvei from two siblings, who developed diarrhea 3 weeks apart. However, most of these data are extremely sketchy, and Matsumoto (85) in 1963 stated that the pathogenicity of Hafnia, at least for humans, was not well clarified.

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).

BacteremiaThere are no national figures on the incidence or number of cases of H. alvei bacteremia in the United States. A 2004 publication from the United Kingdom reports the number of cases of Hafnia bacteremia varying from 19 to 42 per year between 2001 and 2003 (6). If one compares the 2002 population of the United Kingdom (roughly 59 million) to that of the United States (287 million), this translates into somewhere between 92 and 204 cases of Hafnia bacteremia occurring in the United States annually, and this no doubt is an underestimate given the lack of an active surveillance system for this uncommon pathogen.

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).

Although all 18 H. alvei septicemia cases listed in Table 3 appear to have resolved successfully with medical intervention, including one case with septic shock and disseminated intravascular coagulation (84), an unusual finding in several reports was the chronic nature of the bacteremia. In addition to the report of Englund (31), cited above, Grajwer and associates (55) reported a second case of chronic polymicrobic bacteremia involving H. alvei in a healthy 13-year-old girl. H. alvei was isolated from seven blood culture bottles (six pure culture) 6 weeks after clearance of her initial septic episode. Her infection resolved, but only after a prolonged course of treatment that included aminoglycosides and an antipseudomonal penicillin. A third case of chronic septicemia has been reported by Conte et al. (26); this was in an HIV-positive 8-year-old boy who had two separate bacteremic episodes 18 days apart that were apparently caused by the same strain. The authors could find no apparent reason for the septic relapse after appropriate antimicrobial chemotherapy for the initial episode.

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.

Respiratory Tract InfectionsNext to the gastrointestinal tract, hafniae are most commonly recovered from respiratory secretions such as sputum, tracheal and bronchial aspirates, nasal smears, and bronchoalveolar lavage fluid and from the mouth and throat (59, 138). The vast majority of these isolates are not clinically significant. Washington et al. (138) isolated eight strains of H. alvei over a 2-year period at the Mayo Clinic. Of these eight isolates, only two (25%), from sputum and trachea, were determined to be clinically relevant as secondary pathogens; both were associated with cases of pneumonia, with one of these accompanied by pulmonary abscesses. Both of these significant isolates were the predominant flora and were associated with two fatal cases of bronchopneumonia. Günthard and Pennekamp (59) reported similar findings, where eight respiratory isolates were found to represent colonization rather than infection. Of seven H. alvei respiratory isolates recovered at a community hospital in New York City from 1989 to 1992, only one patient had Hafnia-associated disease (77). That singular case involved a 68-year-old female with chronic obstructive pulmonary disease and a left lower lung infiltrate with cardiomegaly and renal failure. H. alvei was recovered from her sputum and in pure culture from a fiberoptic bronchoscopy performed with a protected sheath brush. She was treated with aztreonam but subsequently died, and it is unclear how much hafniae contributed to her eventual demise. Collectively, these seven patients had a mean age of 60 years and had a variety of underlying diseases, including chronic obstructive pulmonary disease, lung cancer, and HIV infection. A case of nosocomial pneumonia due to Hafnia in a person on mechanical ventilation for 12 days duration has also been reported from Switzerland; H. alvei was again recovered in pure culture from a bronchoscopy specimen (40).

Miscellaneous Infections H. alvei has been reported to be a rare cause of a variety of other extraintestinal infections. The analysis of these case reports is complicated by two facts. First, hafniae are often recovered from such syndromes as part of a mixed infection, and it is difficult to assess how much H. alvei contributes to the overall disease process. Second, in other instances there is only circumstantial evidence at best that Hafnia caused the illness or syndrome in question. Often this circumstantial evidence involves the recovery of these bacteria in large concentrations from gastrointestinal samples. Whether such a circumspect analysis is appropriate is unclear.

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.

Cultural CharacteristicsNot much attention has been given to optimizing cultural conditions for the specific recovery of hafniae. This can be attributed mostly to two facts: the lack of any substantial evidence that H. alvei is indeed a bona fide enteropathogen and the infrequent occurrence of hafniae as primary or secondary pathogens in other highly contaminated specimens, such as the respiratory tract and wounds. Even classic reference texts in the field of microbiology deal little with the isolation and cultural characteristics of H. alvei (86, 121, 122). Hafnia grows in media containing 2% to 5% NaCl, over a pH range of 4.9 to 8.25, and over thermal gradients of 4°C to 44°C (56); the optimum temperature for growth has been reported as 35°C (89). Some investigations have found growth at 44°C to be variable (73). There is general agreement that almost 100% of Hafnia strains grow on MacConkey, Hektoen enteric, eosin-methylene blue, and xylose-lysine-deoxycholate agars, all of which are differential to moderately selective media (56, 123, 124). For other media, such as desoxycholate-citrate agar (DCA), there are conflicting data, in that Greipson and Priest (56) reported that all 47 strains they tested grew on DCA, while Sakazaki (123) found that DCA was inhibitory for many hafniae. Regarding more inhibitory selective media, from 25% to 60% of strains fail to grow on Salmonella-Shigella (SS) agar, while 75% to 100% of isolates are inhibited on brilliant green medium (68, 73).

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).

IdentificationThe genus Hafnia consists of strains that conform to general characteristics for inclusion in the family Enterobacteriaceae, being gram-negative, peritrichously flagellated rods that are oxidase negative, are nonsporulating, produce nitrate reductase, and are positive for enterobacterial common antigen. They are facultatively anaerobic, producing acid with or without gas from the metabolism of d-glucose and other carbohydrate or carbohydrate-like compounds (35). Major distinguishing phenotypic features of the Hafnia complex include being indole negative, lysine and ornithine decarboxylase positive, arginine dihydrolase negative, and H₂S and d-sorbitol negative, with production of acid from the fermentation of d-glucose, l-arabinose, and l-rhamnose. Most strains are also VP positive and are motile (35). Strains that deviate from this ideal phenotype do exist but are uncommon (70, 120). Often they can be mistaken for H₂S-negative Salmonella on primary plating media or can be confused with/misidentified as either Enterobacter or Serratia. On occasion, members of the genus Hafnia can also be mistaken for E. coli or Yokenella regensburgei (123). Tests useful in distinguishing H. alvei from Y. regensburgei include VP and glycerol fermentation (both of which are positive for hafniae) and acid production from melibiose (which is negative for hafniae) (79, 133).

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).

SerologyThe immunochemistry of Hafnia lipopolysaccharide (LPS) is extremely complicated. All H. alvei LPS appears to contain glucose, glucosamine, heptose, and 3-deoxyoctulosonic acid (117). Some LPS also contains other amino sugars or carbohydrates such as mannose, galactose, galactosamine, and mannosamine. The core oligosaccharide structure of some strains consists of an identical hexasaccharide structure composed of two d-glucose residues, three ld-heptose residues, and one 3-deoxyoctulosonic acid residue. Other strains have an additional core fragment (117). There is extensive serologic and immunologic diversity in this genus, and active research continues in this arena (74).

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 (“H”) types (123, 124). Unfortunately, this typing system is no longer available because of the loss of many test strains for specific O- and H-antigen groups (123). A second, independent scheme proposed by the Laboratory of Opportunistic Enteric Pathogens at the Mechnikov Research Institute for Vaccines and Sera in Moscow, Russia, recognizes 39 O and 36 H types (13, 117). The availability of this second typing scheme is unknown, and we are unaware of any recently published studies reporting Hafnia serotypes or serogroups. Some Hafnia antigens are shared with other enteric groups, potentially producing serologic cross-reactions. These groups include Salmonella, Shigella, E. coli, Citrobacter freundii, and E. cloacae (123, 124, 137). Some H. alvei strains have been reported to agglutinate in O157 antisera (123).

Several other antigens in hafniae have been described. Several studies have reported that some H. alvei strains produce K antigens which may actually be a slime antigen (124). Other strains produce the alpha antigen described by Stamp and Stone (30). This antigen consists of a complex of three factors, and Hafnia strains that carry this antigen either contain all three or two of these three factors. Other enteric groups, such as Providencia, occasionally carry the alpha antigen as well.