Detection of Enterobacter Sakazakii in Dried Infant Milk Formula by Cationic-Magnetic-Bead Capture | Salmonella

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Detection of Enterobacter Sakazakii
   A  PPLIED AND  E NVIRONMENTAL   M ICROBIOLOGY , Sept. 2006, p. 6325–6330 Vol. 72, No. 90099-2240/06/$08.00  0 doi:10.1128/AEM.03056-05Copyright © 2006, American Society for Microbiology. All Rights Reserved. Detection of   Enterobacter sakazakii  in Dried Infant Milk Formula byCationic-Magnetic-Bead Capture N. R. Mullane, 1 J. Murray, 2 D. Drudy, 1 N. Prentice, 2 P. Whyte, 1 P. G. Wall, 3  A. Parton, 2 and S. Fanning 1 * Centre for Food Safety, School of Agriculture, Food Science and Veterinary Medicine, 1  and School of  Public Health & Population Science, 3 University College Dublin, Belfield, Dublin 4, Ireland, and Matrix Microscience Ltd., Lynx Business Park, Fordham Road, Newmarket,Cambridgeshire CB8 7NY, United Kingdom 2 Received 28 December 2005/Accepted 28 June 2006  Enterobacter sakazakii  has been associated with life-threatening infections in premature low-birth-weightinfants. Contaminated infant milk formula (IMF) has been implicated in cases of   E. sakazakii  meningitis.Quick and sensitive methods to detect low-level contamination sporadically present in IMF preparations wouldpositively contribute towards risk reduction across the infant formula food chain. Here we report on thedevelopment of a simple method, combining charged separation and growth on selective agar, to detect  E. sakazakii  in IMF. This protocol can reliably detect 1 to 5 CFU of   E. sakazakii  in 500 g of IMF in less than 24 h.  Enterobacter sakazakii  is a gram-negative rod that was for-merly known as “yellow-pigmented  Enterobacter cloacae ” until1980 (8). This bacterium is an emerging opportunistic patho-gen predominantly associated with bacterial meningitis in im-munocompromised neonates (2, 9, 14, 20). Other clinical pre-sentations of the infection include bacteremia and necrotizingenterocolitis (14, 18). While it appears that the frequency of   E. sakazakii  infections is low, progress to understand the epide-miology and institute effective control measures has been poorto date. Reported case-mortality meningitis rates vary from 40to 80% among infected infants, with the majority of those whosurvive  Enterobacter  -associated meningitis (94%) developingan irreversible neurological sequela (20).The bacterium has been cultured from a variety of foodmatrices, including cheese, meat, vegetables, grain, bread,herbs, and spices (12, 14). Although the natural habitat of   E. sakazakii  has yet to be identified, infant milk formula (IMF)has been epidemiologically linked to cases of neonatal menin-gitis (1, 10, 19). A protocol previously established by the U.S. Food andDrug Administration is being used to screen IMF for the pres-ence of   E. sakazakii  (17). No validation studies with this pro-tocol have been reported and, furthermore, no detection limithas been established. Briefly, this method requires at least 5days to complete and consists of an initial preenrichment step,followed by a second enrichment step and subsequent isolationof pure colonies on violet red bile glucose agar. Several iso-lated colonies are selected and restreaked onto tryptone soyagar (TSA). Typical yellow-pigmented colonies are detectedafter an overnight incubation for 48 to 72 h at 25°C. Finally,these presumptive colonies are identified biochemically (17).This approach provides only a generic test for  Enterobacteria- ceae  and lacks the necessary capability to specifically identify  E. sakazakii . Recently, a number of selective agars have becomeavailable to aid identification. One of these is the chromogenicDFI agar (Druggan-Forsythe-Iversen formulation), which canbe used to identify and enumerate  E. sakazakii  (11, 12).In this report we describe the development and applicationof a simple capture and detection protocol for  E. sakazakii  indried infant formula. This method requires a short enrichmentperiod, followed by capture of the bacteria and subsequentidentification after plating onto DFI agar. The protocol candetect between 1 and 5 CFU in 500 g of powdered formula,following sample pooling, in less than 24 h. MATERIALS AND METHODSPathatrix principle.  Pathatrix (Matrix Microscience Ltd., Newmarket, UnitedKingdom) is a patented capture system that is based on the use of coatedparamagnetic beads. These cationic (positively charged) magnetic beads electro-statically attract the negatively charged lipopolysaccharide on the surface of gram-negative bacteria.This protocol requires a preprogrammed workstation (Matrix MicroscienceLtd.), generic consumables, and (in this study) positively charged (cationic)paramagnetic beads (ZCCB-CAT; Matrix Microscience, Newmarket, UnitedKingdom) (a general schematic overview of the operation of Pathatrix is shownin Fig. 1). Unlike other particle-based separation techniques, Pathatrix facilitatesthe sampling and subsequent analysis of a complete homogenate. In the protocolreported here, a weighed IMF sample is homogenized for 1 min and enriched(for 6 h) in buffered peptone water (BPW; Difco Laboratories, Le Pont de Claix,France) at 42°C, in a thermally controlled static incubator. Following transfer of the stomacher bag to the workstation and connection of the circulatory system,the cationic beads are introduced and a 30-minute capture sequence is initiated(Fig. 1). The complete sample homogenate is circulated (flow rate of 450 ml perminute) over the charged cationic beads, to which the bacteria become attachedeach time they pass over the magnet. Upon completion of this capture phase,bound organisms are washed and eluted to a clean Eppendorf tube. Captured  E. sakazakii  organisms are plated directly onto DFI agar (Oxoid CM1055; Oxoid,Hampshire, United Kingdom) and incubated at 37°C for 18 h. All experimentalanalyses were performed in triplicate, and data presented represent the means of those independent evaluations. Bacterial strains and culture conditions.  Enterobacter sakazakii  type strainNCTC 11467 was used throughout this study for all optimization and sensitivityexperiments. In addition, previously characterized  Salmonella enterica  serotypesTyphimurium DT104 and Enteritidis (4) were used in competition experimentsto determine the selectivity of the cationic beads. Other  Enterobacteriaceae known to cause meningitis were also evaluated with the Pathatrix system. Table 1provides a list of these bacteria. All bacterial strains were cultured overnight in tryptic soy broth (TSB; Oxoid, * Corresponding author. Mailing address: Centre for Food Safety,School of Agriculture, Food Science and Veterinary Medicine, Uni- versity College Dublin, Belfield, Dublin 4, Ireland. Phone: 353-1 7166082. Fax: 353-1 716 6091. E-mail:  Hampshire, United Kingdom) at 37°C in an orbital shaker (Thermo ElectronCorp., Ohio). Overnight cultures were subcultured after 18 h into fresh pre- warmed TSB, and growth was continued to late log phase (optical density at 610nm, 0.8). Cell densities were determined by spectrophotometry (Biomate 5;ThermoSpectronic, Cambridge, United Kingdom). To obtain an initial low cellnumber, 10-fold serial dilutions were performed in phosphate-buffered saline(Oxoid, Hampshire, United Kingdom). Viable cell numbers were determined bydirect plating to TSA following incubation at 37°C for 18 to 24 h. Freeze-drying of bacterial cells.  E. sakazakii  NCTC 11467 cells were grown onTSA at 37°C for 24 h and suspended in double-strength skimmed milk. One mlof the suspension was dispensed into a sterile ampoule and connected to amanifold freeze-dryer (EF03; Edwards, Sussex, United Kingdom) for 24 h. Theampoules were sealed using a glass burner and stored at room temperature.Freeze-dried cells were rehydrated in sterile deionized H 2 O, and the number of CFU were determined by the most-probable-number (MPN) assay (15), usingserial dilutions in BPW after aliquoting into 96-well microtiter plates. Based onthe growth observed at higher dilutions, the MPN of survivors was calculatedusing an “eight-tube” technique (3). IMF.  Eight commercially available dried IMF powders were reconstituted andused in the course of this study. The brands consisted of different formulationsand blends, including organic varieties, powders supplemented with prebiotics,and others tailored for specific age groups. Confirmation by real-time PCR.  A real-time PCR assay (16) targeting the  dnaG  gene on the macromolecular synthesis operon of   E. sakazakii  was modifiedand used to confirm identification of presumptive colonies (6). Briefly, thermalamplification of the target region was performed in a Rotor-Gene RG-300instrument (Corbett Research, Cambridge, United Kingdom) in a final volumeof 20  l containing 200  M deoxynucleoside triphosphates, 4.0 mM MgCl 2 , 2.0  l of 10   reaction buffer, 500 nM of each primer, 250 nM of probe, 1 U  Taq DNA polymerase, and 100 ng of purified template DNA. A presumptive  E. sakazakii  colony was taken from each DFI agar plate andsuspended in 50  l sterile water (Sigma, Ayrshire, United Kingdom). Cells werelysed by heating to 80°C for 10 min, and DNA was separated from cellular debrisby centrifugation for 2 min at 10,000    g  . Real-time PCR was performed byadding 2   l (containing approximately 100 ng of DNA template) of this super-natant to 18  l of PCR master mix (as outlined above). Defining the limits of detection.  To determine the sensitivity of the method,Pathatrix was operated using three different culture variations. From these data,the limit of detection was defined. Recovery in BPW without enrichment.  To determine the lower limits of de-tection using the Pathatrix protocol, mid-log-phase  E. sakazakii  cells were seriallydiluted in phosphate-buffered saline to establish a range from 10 2 through 10 6 CFU/ml. One-ml volumes were added directly into 249 ml BPW without IMF,prewarmed to 42°C. (No enrichment step was carried out on this occasion.)Following homogenization in a stomacher for 1 min, a 30-min capture overcharged paramagnetic beads was performed and  E. sakazakii  cells were plateddirectly onto DFI. These plates were incubated for 18 h at 37°C, and the colonies were counted. Recovery in BPW and IMF without enrichment.  The previous experiment wasrepeated using the same range of cell concentrations as before, with inocula of 1 ml in 25 g of IMF in 224 ml BPW. No enrichment step was performed. Recovery in BPW and IMF with enrichment.  Varied cell numbers, from 1 to15 CFU, were added into 100 g of dry IMF powder, and the mixture was addedto 900 ml of prewarmed BPW. Eight IMF formulations were evaluated. Follow-ing homogenization, the culture of each IMF was incubated statically in a plasticstomacher bag (Seward Ltd., London, United Kingdom) for 6 h at 42°C. EachIMF sample was individually captured as described previously after 250 ml of the FIG. 1. Schematic diagram showing the Pathatrix system. All relevant features are indicated with arrows. The temperature-controlled pot isindicated.TABLE 1. Pathatrix recovery of other  Enterobacteriaceae  in100 g of IMF diluted with 900 ml BPW  a Species Inoculum(CFU/100 g)Recovery onDFI agar  Enterobacter cloacae  1–5    Enterobacter cloacae  6–10   Citrobacter freundii  1–5   Citrobacter freundii  6–10   Citrobacter diversus  1–5   Citrobacter diversus  6–10    Klebsiella pneumoniae  1–5    Klebsiella pneumoniae  6–10    Klebsiella oxytoca  1–5    Klebsiella oxytoca  6–10    a  A 250-ml volume of the enriched culture was analyzed with the Pathatrix system. Recovery results:   , 1 to 10 CFU;   , 10 to 100 CFU;   ,   100CFU. 6326 MULLANE ET AL. A  PPL  . E NVIRON . M ICROBIOL  .  enriched culture was circulated for 30 min. Captured  E. sakazakii  cells weresubsequently eluted and plated directly onto DFI agar. Competition experiments with  Salmonella  and other  Enterobacteriaceae .  Var-ied cell numbers of   E. sakazakii  along with defined numbers of either  S. enterica serotype Typhimurium DT104 or serotype Enteritidis were spiked simulta-neously into 100 g of IMF in 900 ml BPW. Enrichment and capture steps wereperformed as described previously. The ability of the Pathatrix protocol tocapture the target organism in the presence of low to high (1 to 50 CFU)numbers of   Salmonella  cells was determined.Other  Enterobacteriaceae  known to sporadically contaminate IMF and whichhave been linked as etiological agents of meningitis in infants were also includedfor analysis (Table 1). The Pathatrix protocol was applied to these bacteria toassess its ability to capture and detect them (as previously described). Detection by pooling.  A pooling protocol was developed to facilitate thesimultaneous screening of large sample weights of IMF. This strategy was appliedpostenrichment and used a sampling plan previously approved by the AmericanOrganization of Analytical Chemistry Research Institute (AOAC-RI approvedfor  Salmonella  spp. [090203B],  Listeria  spp. [090201B], and  Escherichia coli  O157[070502]), based on the analysis of a defined portion of each individual sample. Detection of   E. sakazakii  by pooling.  Dry preweighed IMF samples containing various numbers of   E. sakazakii  cells were enriched as outlined previously.One-hundred-gram IMF samples were placed into a 3.5-liter stomacher bag andmixed with 900 ml BPW. This was replicated an additional four times, equivalentto the analysis of 500 g of powder (Fig. 2). An  E. sakazakii  inoculum of 1 to 5CFU was added to one stomacher bag only. All mixes were homogenized for 1min and incubated statically for 6 h at 42°C. A pooling strategy was employed(Fig. 2) to facilitate the simultaneous screening of all five IMF samples. Eachstomacher bag was removed from the incubator, and a 50-ml sample was takenfrom each and combined to give a final volume of 250 ml. The entire 250-mlsample volume was circulated for 30 min (as before). After capture, the washedcationic-bacteria suspension was concentrated, spread plated directly onto DFIagar, and incubated at 37°C for 18 h. If the 500-g pooling protocol generated apositive result, each 100-g sample was retested to identify the contaminatedsample. Detection of desiccated  E. sakazakii  by pooling.  Using the pooling protocol(described above), the performance of Pathatrix to detect dry-stressed  E. saka- zakii  with an extended enrichment time (8 h) was assessed. Desiccated cells wererehydrated in deionized H 2 O, serially diluted in BPW, and added immediately tothe reconstituted IMF. Suspect, contaminated acid casein powders were alsoevaluated for recovery of stressed  E. sakazakii  cells. Fifty randomly acquired acidcasein dry powdered samples representing part of a microbiological qualityassurance program undertaken by a dairy products company supplying ingredi-ents to an IMF manufacturing facility were tested. Levels of   E. sakazakii  con-tamination (if any) in these acid casein samples were unknown. RESULTS The time required for enrichment of samples was indepen-dently determined by evaluating different incubation periods(data not shown). Sufficient cell numbers were generated after6 h, and these could be successfully captured. The enrichmenttime can be extended to 8 h when severely stressed  E. sakazakii cells are suspected. Similarly, the optimum enrichment tem-perature was determined (data not shown) by investigating arange of temperatures (including 35, 37, 39, and 42°C). Theoptimum enrichment temperature was 42°C, although the vari-ation in doubling time was negligible between 39 and 42°C.The upper temperature was chosen for the purpose of confer-ring a minor selective advantage for the enrichment of   E. sakazakii  in IMF. All presumptive  E. sakazakii  colonies wereconfirmed by real-time PCR. Defining the limits of detection. (i) Recovery in BPW with-out enrichment.  To establish the capture efficiency of the cat-ionic beads in the absence of a food matrix, predetermined FIG. 2. Pooling protocol approved by the American Organization of Analytical Chemistry for the simultaneous analysis of 500-g quantities of IMF. The dark box in the individual enrichment of samples represents an  E. sakazakii -positive sample.TABLE 2. Pathatrix detection of   E. sakazakii  in 100 g of IMF diluted with 900 ml BPW  a IMFsample no.Inoculum(CFU/100 g)Recovery onDFI agar 1 1–5   2 1–5   3 1–5   4 1–5   5 1–5   6 1–5   7 1–5   8 1–5   8 6–10   8 11–15    a  A 250-ml volume of the enriched culture was analyzed with the Pathatrix system. Recovery results:   , 1 to 10 CFU;   , 10 to 100 CFU;   ,   100CFU. V OL  . 72, 2006 CAPTURE AND DETECTION OF  E. SAKAZAKII   IN POWDERED IMF 6327  (low) numbers of   E. sakazakii  cells were added directly toBPW. On this occasion no enrichment was performed. Thelimit of detection was determined to be 10 CFU/ml (equivalentto 2.5  10 3 CFU in 250 ml). (ii) Recovery in BPW and IMF without enrichment.  Toinvestigate possible interference effects of a food matrix, theprevious experiment was repeated but with the additionIMF. Again, no enrichment was performed. These data es-tablished the limit of detection to be 20 CFU/ml (5    10 3 CFU in 250 ml), indicating a minimal interference effect bythe addition of IMF to the system. Epidemiological studieshave revealed that initial  E. sakazakii  cell numbers between0.36 and 66 CFU/g may be present in contaminated batchesof powder (13). (iii) Recovery in BPW and IMF with enrichment.  Followingthe confirmation of the test’s ability to capture  E. sakazakii  inIMF, low numbers of   E. sakazakii  were spiked into IMF andenriched for 6 h at 42°C. Table 2 summarizes the findings of these experiments. This method reliably detected between 1and 5 CFU  E. sakazakii  in 100 g IMF, with higher inoculaproducing a higher recovery of   E. sakazakii  (Table 2). Minor variations in recovery were observed between some IMFblends, which was possibly due to individual compositions en-hancing the growth of   E. sakazakii  or through backgroundinterference by other bacteria. Competition experiments with  Salmonella  and capture of other  Enterobacteriaceae .  IMF is not a sterile product and maycontain other related organisms (12, 13). These other bacteriacould potentially interfere with the detection of   E. sakazakii .To test this hypothesis, competition studies were undertaken with two  Salmonella enterica  serotypes (Typhimurium DT104and Enteritidis). Table 3 provides a summary of these data.  S. enterica  serotypes were easily discriminated on DFI agar fromthe blue-green colonies of   E. sakazakii , as the former appearedas a distinct black colony (Fig. 3). Although the competing Salmonella  organisms reduced the recovery of   E. sakazakii , FIG. 3. DFI agar plate showing mixed colony types containing  E. sakazakii  and  S. enterica  serotype Typhimurium DT104 (black).TABLE 3. Interference study with two  S. enterica  serotypes and100 g IMF diluted with 900 ml BPW  a S. enterica  serotypeand IMF sample no.Inoculum (CFU/100 g) Recovery on DFI agar S. enterica E. sakazakii S. enterica E. sakazakii Typhimurium1 10 1–5    2 20 1–5    3 50 1–5    Enteritidis1 1–5 1–5    2 1–5 1–5    2 10–15 1–5    3 1–5 1–5    3 10–15 1–5     a  A 250-ml volume of the enriched culture was analyzed with the Pathatrix system. For each IMF sample tested, a 100-g aliquot was used. Recovery results:  , 1 to 10 CFU;  , 10 to 100 CFU;  ,  100 CFU. 6328 MULLANE ET AL. A  PPL  . E NVIRON . M ICROBIOL  .
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