The source of contamination of leafy green produce with Escherichia coli O157:H7 has become a serious national concern. A state and federal collaborative investigation of watershed contamination was conducted in the Salinas Valley of California from October 2005 to September 2006. Surface water from creeks, agricultural drainage ditches, sloughs, and a river, modified Moore swabs placed in flowing surface water, and creek sediment and soil samples were obtained and examined for E. coli O157:H7 (3, 6).
Soil and sediments were suspended in modified buffered peptone water with sodium pyruvate (mBPWp). Surface water was mixed with an equal volume of 2× mBPWp. Modified Moore swabs were immersed in mBPWp. Pre-enrichment broths were subjected to recirculating immunomagnetic separation using the Pathatrix system (Matrix Microscience, Golden, CO) (6). Paramagnetic beads were washed, resuspended, and plated onto tellurite-cefixime-sorbitol-MacConkey agar (TC-SMAC) and CHROMagar O157 (CHROMagar, Paris, France). Up to eight colonies per sample were confirmed using eosin-methylene blue agar, motility, the 4-methylumbelliferyl-β-d-glucuronic acid (MUG) reaction, Vitek 2 Compact biochemical identification (bioMerieux, Inc., Durham, NC), O157:H7 serology (Denka Seiken, Japan), and real-time PCR (8).
Twenty-three strains of a variant saccharose/sucrose-negative (SAC) E. coli O157:H7 biotype were recovered from seven samples of water, soil, and modified Moore swabs. The 23 isolates were MUG negative and O157:H7 serology positive and contained the stx1, stx2, and uidAm genes. Of the 23 isolates, 15 were misidentified by Vitek 2 Compact version 01.02 0.01.04 as Escherichia fergusonii with 99% probability and no contraindications, requiring 5 h to complete analysis. Eight were identified as “low discrimination,” with “analysis organisms and tests to separate” given as Escherichia coli O157 and Escherichia coli. The “contraindicating typical biopattern(s)” listed negative SAC at 99% probability. Results required an average of 10.25 h to completion. Multiple isolates from the same sample fell into both categories.
Many factors influence the results provided by an automated biochemical identification system: age of the culture, the medium, saline diluent concentration, pH, cell suspension density, card lots, and the database and algorithm of the machine (Vitek 2 user manual; bioMerieux, Inc., Durham, NC). Subsequently, 32 strains, including E. coli O157 of both high and low probability, previously misidentified E. coli O157, and E. fergusonii from human and animal sources were run on Vitek 2 Compact on the same day, controlling other factors. The 47 biochemical reactions on the gram-negative (GN) card were analyzed. Relevant biochemical reactions are given in Table 1.
TABLE 1.
E. coli and E. fergusonii isolates and biochemical reactions from Vitek 2 Compact
| Identification | Vitek identification | Isolate | Source | Biochemical reaction (GN card location no.)a
|
||||||
|---|---|---|---|---|---|---|---|---|---|---|
| ADO (3) | PyrA (4) | d-CEL (7) | ProA (23) | SAC (33) | PHOS (45) | lMLTa (61) | ||||
| O157 | O157, low discrimination | 262-C1 | Water | − | − | − | + A | − | + A | + A |
| 303-1 | MSb | − | − | − | + A | − | + A | + A | ||
| 308A | MS | − | − | − | + A | − | (+) A | + A | ||
| 421-1 | Soil | − | − | − | + A | − | (−) A | + A | ||
| E. fergusonii | 304-2 | MS | − | − | − | − A | − | − A | − A | |
| 308C | MS | − | − | − | − A | − | − A | − A | ||
| 389D | Water | − | − | − | − A | − | − A | − A | ||
| 420-1 | Soil | − | − | − | − A | − | − A | − A | ||
| 420-9 | Soil | − | − | − | − A | − | − A | − A | ||
| 421-9 | Soil | − | − | − | − A | − | − A | − A | ||
| O157, 97-99% probability | 162-P1 | MS | − | − | − | − | + B | (−) | − | |
| 162-P7 | MS | − | − | − | − | + B | + | − | ||
| 166-P2 | MS | − | − | − | − | + B | (−) | − | ||
| 276-2 | Water | − | − | − | + | + B | + | − | ||
| 315D | MS | − | − | − | − | + B | − | − | ||
| 319E | MS | − | − | − | − | + B | − | − | ||
| 322D | MS | − | − | − | − | + B | − | − | ||
| 43888 | ATCC | − | − | − | + | + B | + | − | ||
| 43889 | ATCC | − | − | − | + | + B | (+) | − | ||
| 43890 | ATCC | − | − | − | − | + B | + | − | ||
| 43894 | ATCC | − | − | − | + | + B | + | − | ||
| 700375 | ATCC | − | − | − | + | + B | (+) | − | ||
| E. fergusonii | E. fergusonii | 85A5953 | Human | + C | + C | + C | − | − | − | − |
| 87A1710 | Human | + C | + C | + C | − | − | + | − | ||
| 87A3837 | Human | + C | + C | + C | + | − | + | − | ||
| 50003-14a | Goose | + C | + C | + C | − | − | − | − | ||
| 50003-12a | Goose | + C | + C | + C | − | − | − | − | ||
| 50002-B | Pig | + C | + C | + C | + | − | (−) | − | ||
| 35469 | ATCC | + C | + C | + C | + | − | + | − | ||
| 35471 | ATCC | + C | + C | + C | − | − | + | − | ||
| 35472 | ATCC | + C | + C | + C | − | − | + | − | ||
| 35473 | ATCC | + C | + C | + C | − | − | + | − | ||
Significant characteristics establishing biotype are highlighted by letters: A, SAC negative; B, SAC-positive and variable or negative for ProA, PHOS, and I-MLTa; C, ADO, PyrA, and d-CEL positive (see the text).
MS, modified Moore swab.
All misidentified E. coli O157:H7 strains and the “low-discrimination” strains shared the SAC-negative phenotype. The “low-discrimination” strains were proline arylamidase (ProA) positive, phosphatase (PHOS) positive or variable, and l-malate assimilation (lMLTa) positive. The misidentified strains were negative for these three biochemicals. The E. coli O157 strains with 97 to 99% probabilities were all SAC positive and were variable or negative in the above three reactions. All E. fergusonii strains were positive for adonitol (ADO) and d-cellobiose (d-CEL) fermentation, the reactions used to characterize this species (4, 5). None of the misidentified strains was ADO or d-CEL positive.
E. fergusonii has been associated with pathogenesis in humans and animals (2, 9), so this identification cannot be discounted. Confirmation that the organism is ADO and d-CEL positive is critical.
A biotype is defined as a group of strains that have a common biochemical reaction pattern that is unusual for the particular species (4, 7). Even within a given biotype there appears to be some diversity. Several authors have noted the pitfalls of automated biochemical identification systems with increasing biodiversity (1, 10, 11). The consequences of misidentifying E. coli O157:H7 are severe. Multiple identification tools, such as serology and molecular methods, should be used for confirmation of identifications made by automated systems.
Footnotes
Published ahead of print on 30 December 2008.
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