Abstract
Providencia heimbachae was first described in 1986. It has been isolated from penguin feces and an aborted bovine fetus. To date, there has been no reported isolation of this organism from human specimens. We now report the isolation of P. heimbachae from the stool of a 23-year-old woman with idiopathic diarrhea. The identity of the human strain was determined biochemically and by DNA relatedness to the type strain of P. heimbachae.
Providencia heimbachae was first described in 1986 by Müller et al. (7) using 13 strains isolated from the feces of healthy penguins in Germany and from an aborted bovine fetus in the United States. No human isolates have been previously reported. We report the first isolation of this organism from a human, obtained from the stool of a 23-year-old woman in Washington who was suffering from unexplained diarrhea.
Case report.
A 23-year-old woman presented to the Seattle-King County, Washington, county health department with complaints of four to five watery stools per day over the previous 60 days. She denied any abdominal cramping, fever, or malaise. She did not drink well water or unpasteurized milk and had not been traveling either within the United States or outside of the country. She had no history of irritable bowel syndrome, and the diarrhea improved when the patient ate a bland, starchy diet. There was no blood or mucus present in the stools. Tests for ova and parasites, occult blood, and fecal leukocytes were all negative. Her complete blood count and erythrocyte sedimentation rate were both within normal limits. The only medication that the patient was receiving was sertraline hydrochloride (Zoloft) (50 mg) one to two times per day. Approximately 30 days after the patient reported to the county health department, the symptoms resolved and the patient reported that she was asymptomatic. There is no evidence that the patient received antibiotics.
A culture of the stool specimen on only one occasion yielded a gram-negative rod which grew better when incubated in 5% carbon dioxide. Initial conventional biochemical tests performed at the county health department were unable to identify the organism. The strain was tested in the Sherlock System (MIDI, Inc., Newark, Del.) using the CLIN database, version 3.9. It should be noted that P. heimbachae is not included in this database. This system identified the organism as Yersinia pseudotuberculosis at a discrimination level of 0.527, signifying a moderately good identification.
The strain was sent to the Centers for Disease Control and Prevention for confirmation. Identification was performed by conventional biochemical methods as previously described (3–5). While the results are not identical to those of the type strain (Table 1), they were sufficiently similar to indicate a high probability that the organism was P. heimbachae. Important differences include the fact that the type strain is positive for phenylalanine deaminase, gas production from glucose, and fermentation of maltose and d-mannitol, and is positive for motility only after delayed incubation of 4 days, while the case isolate is negative in these tests and is motile only after 6 days. The type strain gives a negative reaction in Jordan’s tartrate and ONPG (o-nitrophenyl-β-d-galactopyranoside), while the case isolate is positive for tartrate and delayed positive for ONPG.
TABLE 1.
Biochemical profiles of the clinical isolate and the Providencia heimbachae type strain
| Test | Reactionb
|
|
|---|---|---|
| Clinical isolate (ATCC 700659) | Type strain (ATCC 35613) | |
| Indolea | − | − |
| Methyl red | + | + |
| Voges-Proskauer | − | − |
| Citrate (Simmons) | − | − |
| H2S production on Kliger or triple sugar iron agar | − | − |
| Urea | − | − |
| Phenylalanine | − | + |
| Lysine (Moeller) | − | − |
| Arginine (Moeller) | − | − |
| Ornithine (Moeller) | − | − |
| Motility | − | + (4) |
| Gelatin (22°C) | − | − |
| Growth in the presence of KCN | − | − |
| Malonate | − | − |
| d-Glucose, acid | + | + |
| d-Glucose, gas | − | + (4) |
| Acid from: | ||
| Adonitol | + | + |
| l-Arabinose | − | − |
| d-Arabitol | + | + |
| Cellobiose | − | − |
| Dulcitol | − | − |
| Erythritol | − | − |
| d-Galactose | + | + |
| Glycerol | + (3) | + (6) |
| myo-Inositol | + (3) | + (7) |
| Lactose | − | − |
| Maltose | − | + (6) |
| d-Mannitol | − | + (6) |
| d-Mannose | + | + |
| Melibiose | − | − |
| α-CH3-glucoside | − | − |
| Raffinose | − | − |
| l-Rhamnose | + | + |
| Salicin | − | − |
| d-Sorbitol | − | − |
| Sucrose | − | − |
| Trehalose | − | − |
| d-Xylose | − | − |
| Esculin hydrolysis | − | − |
| Acid from mucate | − | − |
| Tartrate (Jordan) | + | − |
| Acetate utilization | − | − |
| Lipase (corn oil) | − | − |
| Deoxyribonuclease | − | − |
| NO3−→NO2− | + | + |
| Oxidase | − | − |
| Catalase | + | |
| o-Nitrophenyl-β-d-galactopyranoside | + (3) | − |
| Tyrosine clearing | + | + |
See text.
−, negative at the end of the 48-h incubation period or at the time of the test; +, positive after 48 h or at time of the test. The numbers in parentheses indicate the day on which the reaction became positive.
Radiolabeled DNA from the clinical isolate was hybridized to unlabeled DNAs from the P. heimbachae type strain and another P. heimbachae strain, using the hydroxyapatite method as previously described (2). A species consists of strains whose DNAs are 70% or more related at 60°C and 55% or more related at 75°C and whose DNAs contain 6% or less divergence in related nucleotide requences (1, 2). The high species level of DNA relatedness, 94% and 96%, respectively, with the two other strains of P. heimbachae at 60°C and 93% and 97%, respectively, at 75°C with corresponding divergences of 1.5 and 1.0 confirmed that the clinical isolate was P. heimbachae (Table 2).
TABLE 2.
Levels of DNA relatedness of the clinical isolate to species of Providencia and Proteus
| Source of unlabeled DNA | Relatedness to labeled DNA from the clinical isolate (strain CDC 4562-98)
|
||
|---|---|---|---|
| Relative binding ratio at 60°C (%)a | Divergence (%)b | Relative binding ratio at 75°C (%)a | |
| Clinical isolate CDC 4562-98 (ATCC 700659) | 100 | 0.0 | 100 |
| Providencia heimbachae ATCC 35613T | 96 | 1.5 | 97 |
| Providencia heimbachae CDC 8027-83 | 94 | 1.0 | 93 |
| Providencia alcalifaciens CDC 3370-67 | 43 | 17.5 | 4 |
| Providencia rettgeri CDC 1163T (ATCC 29944) | 42 | 17 | 8 |
| Providencia stuartii CDC 2896-68T (ATCC 29914) | 42 | 16 | 6 |
| Providencia rustigianii CDC 132-68T (ATCC 33673) | 40 | 13.5 | 5 |
| Proteus mirabilis CDC PR-14T (ATCC 29906) | 29 | 19.5 | 2 |
| Proteus vulgaris CDC PR-1 (ATCC 29905) | 28 | 13.5 | 8 |
Relative binding ratios were determined as follows: (percentage of DNA bound to hydroxyapatite in heterologous reaction/percentage of DNA bound to hydroxyapatite in homologous reaction) × 100.
Divergence was calculated on the assumption that a 1% decrease in the thermal stability of a heterologous DNA duplex compared with the thermal stability of the homologous DNA duplex was caused by 1% of the bases within the duplex that were unpaired; divergence was calculated to the nearest 0.5%.
When the organism was inoculated into additional commercial identification systems, it was reported either as unidentified or as Providencia species because P. heimbachae is not contained in the databases. The API 20E (bioMérieux Inc., Hazelwood, Mo.) yielded a profile number of 0044210, resulting in no identification. Inoculation of a GNI+ card (bioMérieux Inc.) yielded bionumber 7002103420, resulting in an “unidentified organism” message. The MicroScan Rapid Neg ID 3 panel (Dade Behring, Inc., West Sacramento, Calif.) identified the organism as Providencia species at a probability level of 99.61%.
Susceptibility to antimicrobial agents was determined by broth microdilution using the standardized methods described by the National Committee for Clinical Laboratory Standards (8). This organism was susceptible to the penicillins (ampicillin, piperacillin, piperacillin-tazobactam, and ticarcillin-clavulanic acid), cephalosporins (cefazolin, cefoxitin, cefotetan, cefotaxime, ceftriaxone, ceftazidime, cefepime, and cefpodoxime), beta-lactams (imipenem and aztreonam), aminoglycosides (gentamicin, tobramycin, and amikacin), quinolones (ciprofloxacin), chloramphenicol, and trimethoprim-sulfamethoxazole. It was resistant to tetracycline (MIC = 32).
We do not know if this organism in this setting was clinically significant. The Physicians’ Desk Reference, in its clinical trial data, suggests that 2% of patients taking sertraline hydrochloride may experience diarrhea (6). We do not know if this had any significance in this patient. Even if this was the cause of the diarrhea, it would not explain the presence of the organism. Hospital laboratories would probably not expect to identify this organism without using a reference lab. Key biochemical test results that might lead to suspicion of this organism include negative reactions for indole, citrate, and KCN and its positive fermentation reactions for adonitol, d-arabitol, d-galactose, and l-rhamnose (7).
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