Investigating Potential Virulence Factors in Enterobacter Sakazakii

NON-TECHNICAL SUMMARY: Enterobacter sakazakii is an emerging, powedered infant formula-borne pathogen with a mortality rate of 50-75 percent in neonates and children. The objective of this study is to determine the viability characteristics of E. sakazakii in dry, powdered, infant formula, and determine the efficacy of natural, food-grade antimicrobials for killing E. sakazakii in infant formula. Further, the proposed research will identify proteins in E. sakazakii that bind to fibronectin, an important constituent of the extracellular matrix found in all eukaryotic cell tissues.

<P>

APPROACH: <BR> Objective 1: Lyophilized cells of E. sakazakii will be added to 500 g portions of pathogen-free infant formula powder to obtain an inoculation level of 100 CFU or 10000 CFU of the pathogen per gram of the formula. The inoculated formula will be mixed thoroughly and stored in airtight containers at 4C and 23C (room temperature). The population of viable E. sakazakii will be determined on Violet Red Bile Glucose agar immediately after inoculation (day 0), day 3, day 5, day 7, and thereafter weekly up to 12 weeks of storage. <BR>Objective 2: The antibacterial effect of caprylic acid (CA) and monocaprylin (MC) will be determined on a 5-strain mixture of E. sakazakii. The effect of CA (0, 10, 25, and 50 mM), MC (0, 10, 25, and 50 mM) on E. sakazakii will be determined at 4, 8, 23 and 37C. The population of surviving pathogen in the treatment and control samples of the formula will be determined at 0, 12, 24, and 48 h at 4 and 8C, whereas the samples stored at 23 and 37C will be sampled at 0, 1, 6, and 12 h. <BR>Objective 3: The mechanism of killing E. sakaszakii by caprylic and monocaprylin will be determined by scanning electron microscopy. <BR>Objective 4: The binding of E. sakazakii strains to fibronectin will be assayed by a microtitre plate test. <BR>Objective 5: To identify potential proteins in E. sakazakii that bind to fibronectin, its outer membrane proteins will be extracted by Triton X-114 phase-partitioning. The proteins will be separated by SDS-PAGE and incubated with fibronectin to determine binding to fibronectin. The protein band(s) that bind to fibronectin will be excised, and their sequence will be determined by mass spectrometry.

<P>

PROGRESS: 2003/10 TO 2006/09<BR>
E. sakazakii is an emerging, food-borne pathogen causing severe meningitis, meningo-encephalitis, sepsis, and necrotizing enterocolitis in neonates and infants. Being transmitted exclusively through infant formula powder, the population susceptible to E. sakazakii is extremely vulnerable, with a high mortality rate. The Joint FAO/WHO workshop on E. sakazakii and other microorganisms in powdered infant formula recommends among other steps, promoting (a) use of internationally validated detection and molecular typing methods for E. sakazakii, (b) research on the ecology, taxonomy, and virulence of E. sakazakii and (c) ways to reduce its level in reconstituted powdered infant formula. Keeping these recommendations in mind, the following research was undertaken. The antibacterial effect of monocaprylin (monoglyceride ester of caprylic acid) on E. sakazakii in reconstituted infant formula was investigated. A 5-strain mixture of E. sakazakii was inoculated into 10 ml samples of reconstituted infant formula (at 6.0 log CFU/ml) followed by addition of 0, 25, or 50 mM (1%) monocaprylin. The samples were incubated at 37 or 23C for 0, 1, 6 and 24 h, and at 8 or 4C for 0, 6, 24 and 48 h, and the surviving populations of E. sakazakii at each sampling time were enumerated. The treatments containing monocaprylin significantly reduced (P < 0.05) the population of E. sakazakii, compared to the controls. Monocaprylin (50 mM) reduced the pathogen by >5 log CFU/ml by 1 h of incubation at 37 or 23C, and by 24 h of incubation at 8 or 4C. The outer membrane protein A (ompA) gene along with its flanking sequences from E. sakazakii (ATCC 51329) was cloned in pGEM-T Easy vector and sequenced. Comparison of the nucleotide sequence and the deduced amino acid sequence of the ompA gene with other sequences available in the GenBank revealed a high degree of homology with ompA of other gram-negative bacteria belonging to Enterobacteriaceae. Based on regions of the ompA gene unique to E. sakazakii, two primers were synthesized to develop and optimize an E. sakazakii-specific PCR. The PCR amplified a 469-bp DNA product from all E. sakazakii strains tested, but not from other bacteria. Experiments to determine the sensitivity of the PCR indicated that it could detect as low as 1000 CFU/ml of E. sakazakii in infant formula directly, and 1 CFU after an 8 h enrichment step. The outer membrane protein A (OmpA) was identified as a major fibronectin binding protein of E. sakazakii by ligand immuno blotting assays. Further, the contribution of OmpA to the invasion of human brain microvacular endothelial cells (BMEC) was investigated by creating an isogenic mutant in the ompA gene and comparing its invasive efficiency with the wild type strain. The mutant was significantly (P < 0.05) attenuated in its invasiveness and complementation of the mutant with ompA restored the invasive phenotype considerably, to a level approximately 3-fold higher than that of the mutant. These findings indicate that OmpA is one of the determinants that contributes to E. sakazakii invasion of BMEC in vitro, and may potentially play a role in the pathogenesis of neonatal meningitis caused by this organism.
<BR>
<BR>

IMPACT: 2003/10 TO 2006/09<BR>
Monocaprylin could potentially be used to inactivate E. sakazakii in reconstituted infant formula. The PCR combined with enrichment culturing could potentially be used as a rapid tool for detecting the presence of E. sakazakii in infant formula. This study identified a virulence factor in E. sakazakii that potentially contributes to invasion of the central nervous system resulting in brain infection.

<BR><BR>

PROGRESS: 2005/01/01 TO 2005/12/31<BR>
The outer membrane protein A (ompA) gene along with its flanking sequences from E. sakazakii (ATCC 51329) was cloned in pGEM-T Easy vector and sequenced. Comparison of the nucleotide sequence and the deduced amino acid sequence of the ompA gene with other sequences available in the GenBank revealed a high degree of homology with ompA of other gram-negative bacteria belonging to Enterobacteriaceae. Based on regions of the ompA gene unique to E. sakazakii, two primers were synthesized to develop and optimize an E. sakazakii-specific PCR. The PCR amplified a 469-bp DNA product from all E. sakazakii strains tested, but not from other bacteria. Experiments to determine the sensitivity of the PCR indicated that it could detect as low as 1000 CFU/ml of E. sakazakii in infant formula directly, and 1 CFU after an 8 h enrichment step.
<BR><BR>
IMPACT: 2005/01/01 TO 2005/12/31<BR>
The PCR combined with enrichment culturing could potentially be used as a rapid tool for detecting the presence of E. sakazakii in infant formula.
<BR><BR>PROGRESS: 2004/01/01 TO 2004/12/31<BR>
The antibacterial effect of monocaprylin (monoglyceride ester of caprylic acid) on Enterobacter sakazakii in reconstituted infant formula was investigated. A 5-strain mixture of E. sakazakii was inoculated into 10 ml samples of reconstituted infant formula (at 6.0 log CFU/ml) followed by addition of 0, 25, or 50 mM (1%) monocaprylin. The samples were incubated at 37 or 23C for 0, 1, 6 and 24 h, and at 8 or 4C for 0, 6, 24 and 48 h, and the surviving populations of E. sakazakii at each sampling time were enumerated. The treatments containing monocaprylin significantly reduced (P < 0.05) the population of E. sakazakii, compared to the controls. Monocaprylin (50 mM) reduced the pathogen by >5 log CFU/ml by 1 h of incubation at 37 or 23C, and by 24 h of incubation at 8 or 4C.
<BR><BR>
IMPACT: 2004/01/01 TO 2004/12/31<BR>
Monocaprylin could potentially be used to inactivate E. sakazakii in reconstituted infant formula, however sensory studies are warranted before recommending its use.