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* Department of Applied Biological Science, Faculty of Science and Technology, and
Division of Immunobiology, Research Institute for Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan
Department of Veterinary Internal Medicine, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034-8628, Japan
Kohiruimaki Animal Medical Service, Tohoku, Aomori 039-2683, Japan
|| Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda, Chiba 278-8510, Japan
1 Corresponding author: rabe{at}rs.noda.tus.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: mastitis • separation • Staphylococcus aureus • somatic cell
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During the course of bacterial mastitis, active inflammations result in massive migration of inflammatory cells. In addition, the number of endothelial cells also increases sharply. Such a large number of somatic cells often interferes with the PCR assay by making the agarose gel bands smear. As a result, it becomes difficult to identify the infecting bacteria by the PCR method. To solve this problem, we attempted to establish a method of separating bacterial cells from somatic cells in mastitic milk by removing somatic cells from the milk samples.
We have been focusing on silica beads for the separation of microbes by using them as a chromatographic carrier. Silica beads are easily obtainable, stable, and inexpensive compared with other chromatographic carriers such as sepharose. In addition, the surfaces of microbial cells are negatively charged because of the presence of carbonyl and phosphate groups (Black, 1996), and amino groups are known to adsorb microbes by electrostatic forces (Kubota et al., 2005). Thus, we assumed that the affinity for amino-silica would be different between somatic cells and bacteria, and that it should be possible to separate these 2 populations by using silica that was modified with amino groups. In this study, we tested the efficiency of this new separation method with a PCR-based method for detecting and identifying Staph. aureus and demonstrated that with this process, we could detect Staph. aureus from heavily contaminated mastitis milk by PCR.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Genomic DNA Extraction
One milliliter of sample milk (Figures 1
and 2) or cell suspension (Figure 3) was centrifuged at 1,500 x g for 15 min at 4°C. The pellet was resuspended in 1 mL of lysis buffer (0.5% N-laurylsarcosine, 50 mM Tris-HCl, 25 mM EDTA) and centrifuged again at 1,500 x g for 15 min at 4°C. Two hundred microliters of lysostaphin (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was added to the pellet. It was mixed well and incubated for 60 min at 55°C. Genomic DNA was extracted with 200 µL of phenol and phenol-chloroform. After centrifugation at 15,000 x g for 5 min at 4°C, the upper liquid phase was transferred to a new microcentrifuge tube and 1 mL of ice-cold ethanol was added. After centrifugation at 15,000 x g for 5 min at 4°C, the supernatant was removed and dried. Extracted DNA was resuspended in 50 µL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and incubated for 10 min at 65°C.
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Purification of Leukocytes
Healthy Holstein cow blood was obtained from Tokyo Shibaura Zouki (Tokyo, Japan). A 500-mL quantity of blood was centrifuged at 600 x g for 30 min. The buffy coat of blood was obtained after centrifugation and the same quantity of ACK lysing buffer (NH4Cl, 8.29 g/L; KHCO3, 1.00 g/L; EDTA, 0.037 g/L; pH 7.3) was added for 2 min. A large quantity of PBS buffer was added and the sample was centrifuged at 300 x g for 5 min. After removing the supernatant, cells were washed twice with PBS buffer and once with 10 mM MOPS and 150 mM NaCl (pH 7). The cells were used in the following separation experiments.
The population of cells was treated as described below. Cells were washed with wash buffer (PBS containing 1% BSA and 0.05% NaN3) and treated with either anti-GM1 (Veterinary Medical Research and Development Inc., Pullman, WA) or nothing. They were then stained with fluorescent conjugated antibodies [Goat F(ab')2 Anti-Mouse IgG(H+L) FITC Conjugate, Caltag Laboratories Inc., Carlsbad, CA]. A flow cytometer (FACS Calibur, Becton Dickinson Co. Ltd., Sparks, MD) equipped with an argon ion laser was used for measurement of the differential leukocyte count. The isolated cells were nearly all leukocytes (92.86%).
Adsorption of Bacterial Cells and Leukocytes onto Amino-Silica and Pure Silica
Bacterial cells and the leukocyte pellet were resuspended in adsorption buffer (150 mM NaCl, 10 mM MOPS, pH 7). Cell suspensions were adjusted to various densities and 1 mL was incubated with 25 mg of amino-silica (NH-DM1020, Fuji Silysia Chemical Ltd., Kasugai, Japan) or pure silica (MB5D, Fuji Silysia Chemical Ltd.) in a microcentrifuge tube on a tube rotator for 60 min at room temperature. After incubation, the silica settled to the bottom of the tube immediately without centrifugation. The amount of adsorbing cells was determined by counting the number of cells remaining in the supernatant.
Desorption of Cells from Amino-Silica
Cell suspensions were prepared [Staph. aureus (optical density 600 = 0.5), 4.3 x 108 cells/mL; leukocytes, 7.0 x 105 cells/mL]. Cells in adsorption buffer were adsorbed onto amino-silica for 60 min at room temperature and the supernatants were removed. After washing with adsorption buffer, cells were incubated with solutions of various compositions (shown in Table 1) for 15 min. The supernatants were removed and cell recovery was examined by spectrometry at 600 nm (Staph. aureus) or cell counting (leukocytes) of the supernatant.
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Determination of Zeta Potential
The zeta potential of cells was measured with a Ni-comp 380 ZLS instrument (Particle Sizing Systems Co., Santa Barbara, CA) at room temperature. The Nicomp 380 ZLS instrument was equipped with a 20-mW laser diode and photomultiplier tube detector with one optical fiber set at 19.8°. The zeta potential value was calculated from the electrophoretic mobility of the suspensions according to the Helmholtz-Smoluchowski equation (Jan and Chien, 1973; Van Loosdrecht et al., 1987; Wang et al., 2004; Dittrich and Sibler, 2005). Leukocytes and Staph. aureus were washed with adsorption buffer (150 mM NaCl, 10 mM MOPS, pH 7), and suspended in adsorption buffer. The suspensions were then transferred to the electrophoresis cell for measurements. The experimental reproducibility was tested 5 times, and the average values are reported here.
Separation of Staph. aureus from an Artificially Mixed Cell Suspension with Amino-Silica
Cell suspensions of Staph. aureus (8.0 x 107 cells/ mL) and leukocytes (4.0 x 105 cells/mL) were mixed in adsorption buffer. One milliliter of the cell mixture was incubated with 25 mg of amino-silica in adsorption buffer for 60 min in a microcentrifuge tube. After adsorption, the supernatant (supernatant fraction) was transferred to another tube. Cell-adsorbed amino-silica was washed with 1 mL of adsorption buffer for 5 min (wash fraction). The supernatant was removed, and desorption solution (100 mM citric acid, 0.05% pluronic F-68, 100 mM NaCl, pH 6) was added. After a 15-min incubation, the supernatant was removed (desorption fraction) and added to the first supernatant tube (recovery fraction).
Cells in each fraction of Staph. aureus and leukocytes were counted, and the percentage of cells present in each fraction was calculated. The initial cell count ratio was assumed to be 100 (%). In addition, each fraction was used for DNA extraction and PCR assays.
Separation of Staph. aureus from Milk Samples with Amino-Silica
Milk samples were collected from cows with clinical or subclinical udder infections within the first 3 wk after delivery. The samples were assessed by the California Mastitis Test (Schalm and Noorlander, 1957) and selected for possible mastitic milk. Staphylococcal mastitis was judged by Staph. aureus-specific PCR assays or colony isolation on selective agar plate medium (X-SA medium, Nissui Co., Tokyo, Japan). From these experiments, we selected 2 clinical samples and 1 subclinical sample that were infected with Staph. aureus. Samples were stored at –30°C until further examination.
One milliliter of milk sample was centrifuged at 1,500 x g for 15 min at 4°C and the supernatant was removed. The pellet was mixed with 12.5, 25, or 50 mg of amino-silica and 1 mL of adsorption buffer and incubated for 60 min. After adsorption, the supernatant was transferred to a new tube and centrifuged at 1,500 x g for 15 min. After removing the supernatant, the amino-silica was incubated with desorption solution for 15 min. The supernatant was added to the tube containing the pellet from the first adsorption buffer supernatant and centrifuged at 1,500 x g for 15 min. The pellet was used for DNA extraction and PCR assays.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 6 out of 8 samples showed clear bands (lanes 5 to 12) with the Staph. aureus-specific primers, and 2 samples had smeared bands (lanes 3 and 4). We found that the latter 2 samples contained numerous somatic cells and phagocytized bacterial DNA from somatic cells, whereas the others had significantly less. These results suggested that the smeared bands were due to the presence of somatic cells in the milk.
Cell Adsorption Capacity of Amino-Silica and Pure Silica
Because it seemed that the presence of somatic cells interfered with the PCR assay, we attempted to separate somatic cells from bacteria in the milk samples. To this end, we tried several beads that were expected to have different abilities to absorb bacteria and somatic cells. Among them, silica appeared to be the most suitable for these purposes. Thus, we tested the adsorption of Staph. aureus and leukocytes onto 2-types of silica, amino-silica and pure silica, to determine whether any differences were apparent (Figure 3).
Figure 3A and 3B show the adsorption of Staph. aureus and leukocytes, respectively. We examined adsorption saturation for amino-silica and pure silica in adsorption buffer. Both types of silica adsorbed Staph. aureus equally well (Figure 3A). In contrast, for leukocytes, amino-silica adsorbed better than pure silica (Figure 3B). These experiments also indicated that 25 mg of amino-silica could completely adsorb less than 109 cells/mL for Staph. aureus and 106 cells/mL for leukocytes, and that a linear curve was obtained according to the number of cells present in the samples. Thus, in the following experiments, the cell densities used were less than 109 cells/mL for Staph. aureus and 106 cells/mL for leukocytes.
Next, we evaluated several different conditions (pH 5 to 9, 0 to 500 mM NaCl) to increase the adsorption efficiency for each type of silica, but we did not observe a notable difference in adsorption (data not shown). Based on these results, we decided to use the original conditions for adsorption of both types of cells onto amino-silica (pH 7, 150 mM NaCl).
Desorption of Cells from Amino-Silica
Using the previously determined cell numbers and amount of amino-silica, we tested many desorption conditions for Staph. aureus and leukocytes; the results are summarized in Table 1. First, we tried 0.1 and 1 M NaCl solutions. Indeed, the 1 M NaCl solution showed good recovery of cells (66.5%) compared with the 0.1 M solution (14.3%), but the recovery of cells with 1 M NaCl was still too low. We then tried using various reagents such as detergents, PEG, EDTA, and organic acids to identify more effective conditions. The use of PEG or pluronic F-68 did not result in good recovery of bacteria. However, the use of EDTA or citric acid was almost as effective in recovering bacteria as was 1 M NaCl. We then tried mixing these solutions with the detergent pluronic F-68. Very high collection rates of 83.2 and 89.4% were obtained with desorption solutions containing either EDTA or citric acid with pluronic F-68 and 100 mM NaCl. The combination of citric acid or EDTA and the detergent pluronic F-68 significantly improved the recovery rate.
In addition, it should be noted that no leukocytes were desorbed from amino-silica under any of the tested conditions (Table 1). In the following experiments, this desorbing solution [0.1 M NaCl and 0.1 M citric acid, 0.05% (wt/vol) pluronic F-68, 0.1 M NaCl] was used for Staph. aureus recovery.
Separation of Staph. aureus from Artificially Mixed Cell Suspensions
The characteristic features of adsorption and desorption of pure suspensions of either Staph. aureus or leukocytes with amino-silica were determined from these experiments. Next, we conducted experiments to separate Staph. aureus from cell mixtures containing Staph. aureus and leukocytes by using an adsorption buffer.
First, we evaluated the cell recovery ratios by counting cells in each fraction (supernatant, adsorption, and desorption) after adsorption and desorption. After adsorption, leukocytes (99.6%) and Staph. aureus (43.5%) were adsorbed onto amino-silica. Staphylococcus aureus (56.5%) was then contained in the supernatant fraction. In the desorption fraction, leukocytes (0.4%) and Staph. aureus (29.5%) were gained. Using this method, we collected well-purified Staph. aureus (86.0%) with very few contaminating leukocytes (0.8%).
Next, we tested for the presence of Staph. aureus in each fraction by PCR. As shown in Figure 4, Staph. aureus-specific PCR products were detected in both the supernatant of the cell mixture containing Staph. aureus and leukocytes after adsorption (Figure 4, lane 4) and in the desorption solution (Figure 4, lane 6). These results confirmed that we could separate Staph. aureus from the cell mixture without leukocyte contamination.
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). We used 3 samples of mastitis milk (A, B, and C), without a purification step (lanes 2, 3, and 4, respectively, in Figure 1). Although we obtained a clear band from the Staph. aureus-specific PCR for sample A, samples B and C gave a smeared bands on the agarose gel. Next, we applied the new purification procedure for these 3 samples with various quantities of amino-silica. In this experiment, different amounts of amino-silica were added to the samples to completely adsorb all of the somatic cells. Although changing the amount of amino-silica had no effect on sample A, the smearing of the PCR band for sample B was eliminated when more than 25 mg of amino-silica was used. Moreover, in the case of sample C, which showed more strongly smeared bands without treatment, 50 mg of amino-silica was needed to generate clear bands of Staph. aureus PCR product. To confirm the adsorbing effect of amino-silica on somatic cells, we counted somatic cell numbers in samples before and after adsorption. In particular, 50 mg of amino-silica adsorbed 4.5 x 106 of 6.3 x 106 somatic cells in sample B and 6.0 x 106 of 8.3 x 106 cells in sample C.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Thus, we attempted to establish a new method for the selective separation of Staph. aureus from mastitis milk by using amino-silica before DNA extraction for PCR. We found that although amino-silica could efficiently adsorb both somatic cells and Staph. aureus, somatic cells were much more strongly adsorbed than bacterial cells. We identified specific conditions under which most of the somatic cells adsorbed and only Staph. aureus cells were desorbed from the amino-silica. We tested the efficacy of this new separation method for detection and identification of Staph. aureus by a PCR-based method and demonstrated that after this process, we could dramatically improve detection of Staph. aureus from heavily contaminated mastitis milk by PCR.
We tried many conditions for adsorption and desorption of Staph. aureus and leukocytes to amino-silica. Amino-silica adsorbed both leukocytes and Staph. aureus well (Figure 3). To consider the interaction of cells and amino-silica, the cell surface electrical charge was very important. Therefore, we measured the zeta-potential of cells to evaluate their electrostatic surface charges. Their zeta-potential values were as follows: leukocytes –9.29 ± 6.72; Staph. aureus cells, –1.09 ± 3.45 (±SD). This indicates that both Staph. aureus cells and leukocytes are negatively charged. On the other hand, the surface of the amino-silica has many amino-groups; therefore, its surface is positively charged. This information suggests that the attraction force between cells and amino-silica was mainly based on electrostatic interactions. Pure silica could also adsorb both types of cells, but the attraction forces in this case were thought to be mainly due to hydrophobic interactions.
Desorption experiments for Staph. aureus and leukocytes were carried out under various conditions (Table 1). A solution of high ionic strength was thought to detach cells from silica if the adsorption of cells onto silica was caused only by ionic interactions. We found that 1 M NaCl could detach more Staph. aureus cells (66.5%) than 0.1 M NaCl (14.3%), but more efficient recovery was needed. Accordingly, the most appropriate condition to recover Staph. aureus was the combination of citric acid or EDTA and the detergent pluronic F-68. Small molecular weight compounds such as citric acid and EDTA appeared to have stronger affinity for the surface of amino-silica than Staph. aureus cells, so their addition resulted in efficient recovery of Staph. aureus. Moreover, the detergent would weaken the hydrophobic interactions between Staph. aureus and amino-silica. The combination of an organic acid and a detergent was reported to be an effective condition for detaching several bacterial species from silicate material containing amino groups (Kubota et al., 2005). In contrast, no leukocytes were desorbed from amino-silica under any of the tested conditions (Table 1). Furthermore, leukocytes are more negatively charged than Staph. aureus, as mentioned above, and because the cells are larger than bacterial cells, leukocytes would have many more points of contact with the amino-silica compared with Staph. aureus cells. For this reason, we suggest that the attraction between leukocytes and amino-silica was very strong and that the interaction was irreversible in nature.
It should be noted that in the artificial cell mixture, adsorption was different from our expectation, as shown in the results in Figure 3. Leukocytes were well adsorbed even in the mixture, but Staph. aureus cells did not adsorb as strongly (Figure 4). The reason was the difference in affinity between the cells and amino-silica. The leukocytes adsorbed more rapidly onto the amino-silica than did Staph. aureus (data not shown). Therefore, the adsorption sites for Staph. aureus were thought to be blocked by bound leukocytes. Moreover, this could be explained by the zeta-potential results. For this reason, the electrostatic attraction for amino-silica was thought to be stronger for leukocytes than for Staph. aureus.
Finally, we tried to detect Staph. aureus from naturally occurring mastitis milk samples by our PCR-based method (Figure 2). It was clear that detection of Staph. aureus from samples that were heavily contaminated with somatic cells (Figure 2, samples B and C) was successful, and clear PCR bands were obtained. Moreover, we could find no adverse influence of the pretreatment with amino-silica on detection compared with untreated samples (Figure 2, sample A). This suggests that milk samples in any condition, whether heavily contaminated with somatic cells or not, could be used in this separation system before the PCR assay.
Typically, a concentration of somatic cells of approximately 106 cells/mL is thought to be the highest contaminating cell level present in mastitis milk, and samples B and C in this study had leukocyte concentrations in this range. We could remove almost all of the somatic cells from these samples and succeeded in detecting Staph. aureus by PCR with our method. This suggests that 50 mg of amino-silica was an appropriate amount for use with a real sample of heavily contaminated milk.
The separation system in this study was very effective for samples that may contain polymerase reaction inhibitors (untargeted cells, nucleic acids, or macromolecular compounds such as protein). Moreover, preliminary experiments suggested that other mastitis-causing bacteria, including Escherichia coli, Streptococcus dysgalactiae, Streptococcus agalactiae, and Streptococcus uberis, could also be selectively obtained by this method (data not shown).
In conclusion, this cell separation system was easy, rapid, and inexpensive, and used readily available amino-silica as the primary reagent for selective cell separation. We expect that in the future, the technology developed in this study could be applicable for identifying other bacterial contaminants in the food and dairy industries as well as in the medical field.
Received for publication October 13, 2006. Accepted for publication May 25, 2007.
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, cells in supernatant from amino-silica; 
, cells adsorbed onto pure silica; 
, cells in supernatant from pure silica.