J. Agric. Food Chem. 2007, 55, 4617−4623
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Differentiation of Enterobacter sakazakii from Closely Related Enterobacter and Citrobacter Species Using Fatty Acid Profiles PAUL WHITTAKER,* CHRISTINE E. KEYS, ERIC W. BROWN, FREDERICK S. FRY
AND
Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 5100 Paint Branch Parkway, College Park, Maryland 20740-3835
Capillary gas chromatography with flame ionization detection (GC-FID) was used to determine the cellular fatty acid (CFA) profiles of 134 Enterobacter sakazakii strains, and these were compared to the CFA profiles of other closely related Enterobacter and Citrobacter species. For GC-FID analysis, whole cell fatty acid methyl esters (FAMEs) from cells cultured on brain heart infusion (BHI) agar at 37 °C for 24 h were obtained by saponification, methylation, and extraction into hexane/methyl tertbutyl ether. A database for E. sakazakii was prepared using fatty acid profiles from the 134 strains. Major fatty acids of E. sakazakii strains evaluated in this study were straight-chain 12:0, 14:0, and 16:0, unsaturated 18:1 ω7c, and 17:0 ωcyclo 7-8. Principal component analysis (PCA) based on CFA profiles for E. sakazakii strains shows separation of E. sakazakii subgroups A and B. The CFA profiles for E. sakazakii and Enterobacter cloacae show that there are several fatty acids, 14:0, 17:0 ωcyclo 7-8, 18:1 ω7c, and summed 16:1 ω6c/16:1 ω7c, that differ significantly between these two species. A PCA model based on CFA profiles for E. sakazakii strains clearly shows separation of E. sakazakii from closely related Enterobacter and Citrobacter species. Analysis of FAMEs from E. sakazakii strains grown on BHI agar by a rapid GC-FID method can provide a sensitive procedure for the identification of this organism, and this analytical method provides a confirmatory procedure for the differentiation of E. sakazakii strains from closely related Enterobacter and Citrobacter species.
KEYWORDS: Gas chromatography; fatty acids; Enterobacter sakazakii
INTRODUCTION
Enterobacter sakazakii is a Gram-negative, rod-shaped pathogen that has been implicated in foodborne diseases. In 1929, Pangalos reported that a yellow-pigmented coliform was the cause of septicemia in an infant (1). Subsequently, Urmenyi and Franklin (1961) reported two cases of terminal neonatal meningitis that occurred in 1958 during an outbreak in England, and Joker et al. (1965) in Denmark reported an infection caused by E. sakazakii in a child who survived meningitis but developed severe mental and neurological impairment (1-3). In the later reports, the pathogen was described as a yellow-pigmented Enterobacter cloacae. It was not until 1977 that E. sakazakii was distinguished from E. cloacae (4). E. sakazakii has emerged as a rare cause of life-threatening neonatal meningitis, necrotizing enterocolitis, and septicemia (5). The bacterium has been * Corresponding author (telephone 301 4361797; fax 301 4362665; e-mail
[email protected]).
detected in dry infant milk formula products and, with improper handling, has been implicated in several clinical investigations. Clark et al. (6) in 1990 were the first to correlate E. sakazakii isolated from two neonatal patients and dried infant formula by epidemiologic typing. E. sakazakii infections are rarely reported but have been implicated in causing illness in neonates and children aged 3 days to 4 years with at least 76 cases of infections and 19 deaths in infants and children (7, 8). The organism has been detected in either prepared formula, the environment in which it was prepared, or unopened products. The first report in the United States of E. sakazakii infection associated with infant formula was in 2001, prompting recall of a commercial product (9). A letter was sent by the U.S. Food and Drug Administration (FDA) informing health care professionals that E. sakazakii infections are associated with the use of milk-based powdered infant formulas (10). In this study, a rapid GC-FID method was used to determine if it could identify E. sakazakii strains from food, the environ-
10.1021/jf070193a This article not subject to U.S. Copyright. Published 2007 by the American Chemical Society Published on Web 05/02/2007
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J. Agric. Food Chem., Vol. 55, No. 11, 2007
Whittaker et al. Table 2. Comparison of Cellular Fatty Acid Profiles for E. sakazakii
Table 1. Bacterial Agents Analyzed by GC
and E. cloacaea microorganism
Enterobacter sakazakii
Enterobacter sakazakii that tested as Enterobacter cloacae Enterobacter cloacae
Enterobacter agglomerans Enterobacter intermedius Citrobacter freundii Enterobacter amnigenus Enterobacter aerogenes Enterobacter asburiae Enterobacter gergoviae Enterobacter hormaechei Citrobacter koseri Enterobacter dissolvens Enterobacter cancerogenus a
designation
source of strain
613 1424 1425 1426−1428 1429 1430 1431 1432 1433 1434 1435 1445 1447 1449 1508−1509 1511−1517 1519−1520 1523−1531 1533−1534 1536 1537 1538 1542 1543 1545 1547−1561 1563−1565 1566 1568−1569 1570 1571 1573 1578 1584 1585−1586 1587−1591 1593−1595 1596 1597−1598 1600 1602−1605 1632 1638 1640 1642−1645 1662−1665 1666 1668−1687 1891−1894 2148−2157 1532 1544 1667 1895 1437 1646 2140 2141 2142 2134 2135 2136 2137 2138 2139 2143 2144 2145 2146 2147
food clinical unknown environmental clinical unknown clinical unknown clinical unknown food clinical clinical clinical environmental environmental environmental environmental environmental food clinical food clinical unknown unknown unknown clinical environmental food clinical food food food clinical unknown nonclinicala nonclinical environmental nonclinical nonclinical nonclinical unknown unknown unknown unknown food environmental food unknown clinical environmental environmental environmental unknown food unknown unknown clinical clinical food environmental unknown unknown clinical clinical clinical clinical clinical food clinical
Nonclinical is an unknown food or environmental source.
ATCC no. 29544 51329
cellular fatty acid
E. sakazakii (%)
E. cloacae (%)
P value
12:0 13:0 unknown 13.951 14:0 unknown 14.502 14:0 2OH 15:0 16:1 ω5c 16:0 15:0 3 OH 17:1 ω8c 17:0 ω cyclo 7−8 17:0 18:1 ω7c 18:0 19:0 cyclo ω8c 19:0 iso 19:0 summed 13:0 3OH/15:1 iso summed 14:0 3OH/16:1 iso summed 16:1 ω6c/16:1 ω7c similarity index
1.58 ± 0.53 0.00 ± 0.00 0.04 ± 0.14 9.61 ± 0.78 0.82 ± 0.19 0.00 ± 0.00 0.09 ± 0.17 0.04 ± 0.06 31.21 ± 1.85 0.00 ± 0.00 0.00 ± 0.04 3.74 ± 1.59 0.18 ± 0.22 24.02 ± 2.09 0.45 ± 0.14 0.49 ± 0.36 0.00 ± 0.00 0.02 ± 0.05 0.00 ± 0.00 9.01 ± 0.95 18.63 ± 2.19 0.802 ± 0.118
2.66 ± 1.34 0.35 ± 0.31 0.20 ± 0.30 7.82 ± 1.61 0.94 ± 0.18 0.25 ± 0.50 1.03 ± 1.38 0.07 ± 0.07 30.55 ± 2.54 0.07 ± 0.09 0.14 ± 0.26 11.70 ± 5.20 2.05 ± 1.55 16.30 ± 2.44 0.50 ± 0.10 1.60 ± 1.30 0.08 ± 0.17 0.08 ± 0.09 0.21 ± 0.42 8.67 ± 0.86 14.46 ± 6.15 0.743 ± 0.104
