Faster Detection of Staphylococcus aureus in Milk and Milk Powder by Flow Cytometry

Abstract

A flow cytometry (FCM)-based method was developed for the faster detection of Staphylococcus aureus in milk and milk powder. Viable S. aureus cells were recognized by highly selective, fluorescently labeled antibodies and Propidium Iodide, and then analyzed by FCM. Using a 5-h pre-enrichment period, the method could detect low numbers of S. aureus cells in 6 h, with a limit of detection of 7.50 cells/mL in milk and 8.30 cells/g in milk powder. The established method was compared with the plate-based method using 75 ultra-high-temperature-treated milk samples, 25 pasteurized milk samples, 66 raw milk samples, and 123 milk powder samples. The two methods yielded similar results for the detection of the pathogen in all sample types. The FCM-based method allows effective and faster monitoring of S. aureus contamination and can be applied to the rapid detection of microorganisms in milk and dairy products.

Introduction

S taphylococcus aureus is one of the most common pathogens responsible for diverse disease symptoms in humans and animals (Hansen et al., 2019). As one of the major causal agents of bovine mastitis, S. aureus is considered high-risk contamination in raw milk (Varela-Ortiz et al., 2018). Although the use of pasteurization during production processes has become common, this microorganism might still contaminate pasteurized, ultra-high-temperature-treated (UHT), and powdered milk in the postprocessing (Wang et al., 2012; Meesilp and Mesil, 2018; Dai et al., 2019).

European Commission regulations (EC, 2007) state that quantitative determination of S. aureus in milk and dairy products is with a limit of detection (LOD) of 10–100 colony-forming units (CFU)/mL or CFU/g. Notably, one of the main consumers of milk powders is infant, which may be at greater risk of disease from products with a low level of S. aureus contamination because of its lower immunity. In consequence, for a more accurate and effective detection, faster methods that can efficiently detect low numbers of viable S. aureus cells are urgently needed (Templier and Roupioz, 2017).

The conventional culture and plate-based method is the gold standard and the most common method used for S. aureus detection in standardized laboratories given its high sensitivity and ideal specificity (Banada et al., 2009), nevertheless, it is excessively time-consuming, with a long time-to-results (TTR) (it can take up to a few days to obtain a result), and is labor intensive. In the past few decades, numerous efforts have been made to develop rapid methods for S. aureus detection, including immunoassay methods (Yan et al., 2017; Alamer et al., 2018), PCR-based assays (Liu et al., 2019a; Moezi et al., 2019), biosensors (Ghali et al., 2016; Templier and Roupioz, 2017; Zheng et al., 2019), and flow cytometry (FCM) (Bassoe, 1984; Meng et al., 2017; Shan et al., 2019). Unfortunately, immunoassay methods suffer from poor sensitivity and/or lack quantitative results (Schaumburg et al., 2019), while biosensor-based methods have difficulty in distinguishing between viable and nonviable bacterial cells. In addition, there are certain limitations in simplicity and rapidity in the PCR-based methods (Wang et al., 2018).

FCM is a good option as it allows rapid and automatic quantification of viable cells among nonviable cells (Ananta et al., 2005). Previous studies have reported FCM-based methods for the rapid detection of single bacterial cells of species such as Escherichia coli (Liu et al., 2019b) and Salmonella (Wang et al., 2020) in dairy food matrices, while that of viable S. aureus cells in low numbers in dairy, although of great importance, has not been explored.

Therefore, in this study, we combined FCM with a pre-enrichment procedure to achieve faster detection of viable S. aureus cells in low numbers in milk and milk powder.

Materials and Methods

Bacterial strains and culture conditions

The information of all bacterial strains used in this study is listed in Table 1. Moreover, S. aureus ATCC 6538P was the target strain. Cells of all strains were grown at 37°C in nutrient broth (Oxoid, Basingstoke, UK), centrifuged at 10,000 × g for 5 min, and then resuspended in phosphate-buffered saline (PBS; pH 7.2).

Table 1.

Bacterial Strains Tested for the Reactivity with Anti-Staphylococcus aureus Antibody Using Flow Cytometry

Bacteria strain	Source	FITC
Staphylococcus aureus strains (n = 10)
S. aureus	ATCC 6538P	P
S. aureus	ATCC 27217	P
S. aureus	ATCC 25923	P
S. aureus	ATCC 6385	P
S. aureus	ATCC 29213	P
S. aureus	CICC 10786	P
S. aureus	CICC 10787	P
S. aureus	CICC 10788	P
S. aureus	CICC 10789	P
S. aureus	CICC 10790	P
Non-S. aureus strains (n = 37)
Staphylococcus epidermidis	ATCC 12228	N
S. albus	ATCC 8032	N
S. chromogenes	GDMCC 1.915	N
S. xylosus	BNCC 337649	N
S. haemolyticus	BNCC 139485	N
S. simulans	CICC 24064	N
Bacillus subtilis	ATCC 6633	N
B. subtilis var. niger	ATCC 9372	N
B. cereus	CICC 10352	N
Listeria monocytogenes	CICC 21540	N
L. monocytogenes	ATCC 11911	N
Rhodococcus equi	ATCC 6939	N
Enterococcus faecalis	ATCC 19433	N
Serratia marcescens	ATCC 8039	N
Escherichia coli	ATCC 25922	N
E. coli	ATCC 44102	N
E. coli	ATCC 10798	N
E. coli	ATCC 13706	N
Escherichia coli O157:H7	CICC 21530	N
Salmonella typhimurium	ATCC 13311	N
S. typhimurium	ATCC 19585	N
S. heidelberg	ATCC 8326	N
S. choleraesuis	ATCC 10708	N
S. braenderup	ATCC 700136	N
S. enteritidis	ATCC 13076	N
S. newington	ATCC 29628	N
S. senftenberg	ATCC 8400	N
S. abaetetuba	ATCC 35640	N
S. cubana	ATCC 12007	N
S. adelaide	ATCC 10718	N
Enterobacter cloacae	ATCC 13047	N
E. aerogenes	ATCC 13048	N
E. sakazakii	ATCC 25944	N
Citrobacter freundii	ATCC 43864	N
Vibrio cholerae	CICC 23794	N
V. parahaemolyticus	ATCC 17802	N
Pseudomonas aeruginosa	CICC 10419	N

ATCC, American Type Culture Collection; BNCC, BeNa Culture Collection; CICC, China Center of Industrial Culture Collection; FITC, Fluorescein isothiocyanate; GDMCC, Guangdong Microbial Culture Collection Center; N, negative, means that the strain could not be recognized by the FITC-labeled antibody; P, positive, means that the strain could be recognized by the FITC-labeled antibody.

Dual staining of S. aureus and FCM

The FCM used was an A50-Micro model (Apogee, Hemel Hempstead, UK). Fluorescein isothiocyanate (FITC)-labeled anti-S. aureus antibody was prepared by labeling goat anti-S. aureus polyclonal antibody (Cat. No. 5310-0314; SeraCare Life Sciences, Milford, MA) using a FITC Labeling Kit (Cat. No. ARL0021K; Frdbio Science & Technology, Wuhan, China). Propidium Iodide (PI) was purchased from Molecular Probes (Cat. No. P3566; Eugene, OR).

A S. aureus ATCC 6538P suspension of ∼10⁷ cells/mL in PBS was mixed with the antibody (final concentration: 1 μg/mL) and PI (final concentration: 1 μg/mL), and this reaction was allowed to proceed at room temperature in the dark for 15 min. Then, the suspension was analyzed by the FCM. According to principle of the dual labeling with FITC-labeled antibody and PI (Wang et al., 2020), the gating strategy can be set as follows (Supplementary Fig. S1): Set up an forward scatter (FSC) versus side scatter (SSC) plot with a gate around the bacterial population Region 1 (R1). On the fluorescence (FL)1 versus FL3 dot plot gated on R1, viable cells were usually enumerated by counting events that scatter the incident blue light at the expected intensities and emit green, but not red fluorescence.

Inclusivity/exclusivity testing

To assess the inclusivity and exclusivity of the FCM-based method, the 10 S. aureus and 37 non-S. aureus strains were incubated with FITC-labeled anti-S. aureus antibody and analyzed by FCM.

Preparation of artificially contaminated samples

UHT milk was obtained from Monmilk Dairy (Hohhot, Inner Mongolia, China). Milk powder was purchased from Friesland Campina Domo B.V. (Frisolac, Friesland, Netherlands). In initial tests, no bacterial cells were detected in the milk and milk powder by the plate-based method (FDA, 2016). Artificially contaminated milk and milk powder samples were prepared as follows: 1 mL of the suspension of viable S. aureus cells was centrifuged at 10,000 × g for 5 min, and added into 25 mL of milk or 25 g of milk powder.

Optimization of the pre-enrichment time

Six artificially contaminated samples of milk and milk powder with S. aureus at a final concentration of ∼10¹ CFU/mL and 10¹ CFU/g, respectively, were prepared as follows: A suspension of S. aureus at a final concentration of (1.2 ± 0.2) × 10³ CFU/mL was prepared by the dilution method. The populations of the S. aureus suspension were confirmed using a plate-based method by inoculating the samples onto Baird–Parker agar (Land Bridge, Beijing, China) plates (n = 3) (FDA, 2016). Then 250 μL of the S. aureus suspension was transferred to a sample using a pipette, so that the sample could contain (12 ± 2) CFU/mL or CFU/g of S. aureus, which is enough to be of clinical significance, as prescribed by the European Commission regulations (EC, 2007).

Each sample (25 mL or 25 g) was added to 225 mL of sterilized 7.5% sodium chloride broth (SCB; Land Bridge, Beijing, China) and incubated at 37°C in a shaker incubator set at 180 rpm. After 0, 3, 4, 5, and 6 h of pre-enrichment, 6 mL of the mixture was collected and analyzed by the plate-based method. All samples were analyzed in sextuplicate. The coefficient “K,” which is used to evaluate S. aureus growth, was calculated using Eq. (1): $K t = \frac{N_{t}}{N_{0}}$ (1)

where K_t is the ratio of the number after t h of pre-enrichment to the initial number, N_t is the concentration of S. aureus cells after t h of pre-enrichment, N ₀ is the initial number of S. aureus cells, and t is the pre-enrichment time.

Assessment of the relationship between the initial cell number and the number after 5 h of pre-enrichment

The optimal pre-enrichment time in this study was determined to be 5 h. To determine the relationship between the initial number and the number after 5 h of pre-enrichment, four groups of artificially contaminated samples were prepared (milk or milk powder with S. aureus ATCC 6538P cells or S. aureus ATCC 25923 cells). Detailed information on the samples is given in Table 2. The use of two different S. aureus strains was for evaluating whether they would have similar reproduction rates under the optimized pre-enrichment conditions. Each sample (25 mL or 25 g) was added into 225 mL of SCB, and 6 mL of each mixture was collected immediately and after incubation in the shaker incubator at 37°C for 5 h, and analyzed by the plate-based method in sextuplicate.

Table 2.

Artificially Contaminated Samples Used for Validating the Relationship Between the Initial Number and the Number After 5 h of Pre-Enrichment and Regression Equations

Bacteria strain	Concentration (CFU/mL or CFU/g)^a	Sample	Number of samples^b	Regression equation^c
Staphylococcus aureus ATCC 6538P	10¹	25 mL of milk	6	$L o g N_{5 h} = 1 . 0428 L o g N_{0} + 2 . 6703$ R ² = 0.9955
	10²		6
	10³		6
	10⁴		6
	10⁵		6
S. aureus ATCC 6538P	10¹	25 g of milk powder	6	$L o g N_{5 h} = 1 . 0127 L o g N_{0} + 2 . 8377$ R ² = 0.9962
	10²		6
	10³		6
	10⁴		6
	10⁵		6
S. aureus ATCC 25923	10¹	25 mL of milk	6	$L o g N_{5 h} = 1 . 0060 L o g N_{0} + 2 . 8287$ R ² = 0.9710
	10²		6
	10³		6
	10⁴		6
	10⁵		6
S. aureus ATCC 25923	10¹	25 g of milk powder	6	$L o g N_{5 h} = 1 . 0242 L o g N_{0} + 2 . 8322$ R ² = 0.9958
	10²		6
	10³		6
	10⁴		6
	10⁵		6

Concentration, the approximate initial number of S. aureus cells.

A total of 120 individual samples were divided into four groups. Each of the four groups contains 30 samples with five different initial numbers.

N _5h, the number of S. aureus cells after 5 h of pre-enrichment; N ₀, the initial number of S. aureus cells; The fitting regression equation was carried out from the plate-based results of the 30 samples in each of the four groups.

CFU, colony-forming unit.

Sample analysis by the FCM-based method

Each sample (25 g or 25 mL) was added to 225 mL of SCB and incubated at 37°C for 5 h for the pre-enrichment of S. aureus. One milliliter of the mixture was used for the isolation procedure of S. aureus cells as described in the previous study (Gunasekera et al., 2000). The recovery of the isolation procedure ranged from 90% to 99% in our preliminary experiment (Supplementary Table S1), which was confirmed to be effective for the following detection.

In brief, 2.5 mg of proteinase K (Molecular Probes, Eugene, OR) and 500 μL of 0.1% Triton X-100 (Sigma, St. Louis, MO) were added to 1 mL of the mixture, and the samples were incubated at 37°C for 30 min. After incubation, 0.5 mL of PBS was added to the sample and mixed thoroughly by inverting the tube 100 times for the reaction to complete. The mixtures were centrifuged at 12,000 × g for 5 min to remove lipid components, and the pellets containing S. aureus cells were collected.

After the isolation procedure, S. aureus cells were resuspended in 1 mL of PBS, and stained with the FITC-labeled antibody and PI. Finally, the stained cells were centrifuged again, resuspended in 0.1 mL of PBS, and analyzed by FCM. The number of S. aureus cells could be calculated using Eq. (2), as described in Section Relationship between the initial cell number and the number after 5 h of pre-enrichment.

Sample analysis by the plate-based method

Each sample (25 g or 25 mL) was added to 225 mL of SCB. Before pre-enrichment, 6 mL of the mixture was collected and analyzed by the plate-based method (FDA, 2016) in sextuplicate. The remainder of the mixture was further incubated for 5 h and then analyzed by the FCM-based method.

Detection of S. aureus in artificially contaminated samples

Artificially contaminated milk and milk powder samples with S. aureus at final concentrations of 10⁰ to 10⁵ CFU/mL and 10⁰ to 10⁵ CFU/g were prepared, respectively (Table 3). The samples were examined by the FCM-based method after 5 h of pre-enrichment. All enumeration experiments were performed in triplicate.

Table 3.

Detection of Staphylococcus aureus in Artificially Contaminated Samples by the Flow Cytometry-Based Method

Sample	Addition (n = 3) (CFU/mL or CFU/g)^a	Detection (n = 3) (cells/mL or cells/g)^b
25 mL of milk	(7.50 ± 1.33) × 10⁰	(11.30 ± 2.58) × 10⁰
	(1.30 ± 0.21) × 10¹	(1.77 ± 0.33) × 10¹
	(1.30 ± 0.21) × 10²	(1.61 ± 0.38) × 10²
	(1.47 ± 0.26) × 10³	(1.57 ± 0.20) × 10³
	(1.47 ± 0.26) × 10⁴	(1.73 ± 0.22) × 10⁴
	(2.22 ± 0.38) × 10⁵	(2.30 ± 0.27) × 10⁵
25 g of milk powder	(8.30 ± 1.19) × 10⁰	(9.50 ± 3.01) × 10⁰
	(1.07 ± 0.17) × 10¹	(1.42 ± 0.25) × 10¹
	(1.07 ± 0.17) × 10²	(1.23 ± 0.10) × 10²
	(1.70 ± 0.19) × 10³	(1.86 ± 0.18) × 10³
	(2.78 ± 0.31) × 10⁴	(2.71 ± 0.20) × 10⁴
	(2.78 ± 0.31) × 10⁵	(2.49 ± 0.19) × 10⁵

Addition, the number of S. aureus cells determined by the plate-based method.

Detection, the number of S. aureus in the artificially contaminated sample determined by the FCM-based method.

FCM, flow cytometry.

Detection of S. aureus in real samples

In total, 75 retail UHT milk samples, 25 retail pasteurized milk samples, and 123 retail milk powder samples from different brands and different lot numbers were purchased from local markets. In addition, 66 raw milk samples were obtained from a local dairy farm. All samples were analyzed by the FCM- and plate-based methods.

Results

Effective FCM detection of S. aureus by dual staining

FITC-labeled antibody and PI were used to distinguish viable S. aureus cells from dead cells and other bacteria. Viable S. aureus cells emit only green fluorescence, whereas dead S. aureus cells emit green and red fluorescence (Fig. 1). The FCM allowed an efficient and accurate detection of viable S. aureus cells (Supplementary Fig. S2).

FIG. 1.

Staining of Staphylococcus aureus cells. (A) Unstained S. aureus cells. (B) S. aureus cells with FITC-labeled antibody. (C) Viable S. aureus cells with PI. (D) Dead S. aureus cells with PI. (E) Viable S. aureus cells with FITC-labeled antibody and PI. (F) Dead S. aureus cells with FITC-labeled antibody and PI. CFU, colony-forming unit; FITC, fluorescein isothiocyanate; FL, fluorescence; PI, propidium iodide.

Inclusivity/exclusivity testing

A total of 47 bacterial strains (Table 1) were stained with the FITC-labeled anti-S. aureus antibody to evaluate the inclusivity and exclusivity of the FCM-based method. All 10 S. aureus strains were recognized by the FITC-labeled antibody, whereas none of the 37 non-S. aureus strains was. This result suggested that the FCM-based method showed a high specificity for S. aureus.

Pre-enrichment time

Six artificially contaminated samples of milk and milk powder with S. aureus at a final concentration of ∼10¹ CFU/mL and 10¹ CFU/g, respectively, were prepared and analyzed by the plate-based method after 0, 3, 4, 5, or 6 h of pre-enrichment. The number of S. aureus cells in milk and milk powder samples continuously and steadily increased between 3 and 6 h (Fig. 2). Cells could be detected by FCM after 5 h, but not after 4 h of pre-enrichment. Hence, the optimal pre-enrichment time was determined to be 5 h.

FIG. 2.

Relationship between the coefficient “K” and the time of the pre-enrichment. A total of 6 milk samples (25 mL) with ∼10 CFU/mL of S. aureus and 6 milk powder samples (25 g) with ∼10 CFU/g of S. aureus were prepared. Each of all samples was incubated in 225 mL of SCB, and then analyzed by the plate-based method after 0, 3, 4, 5, and 6 h of pre-enrichment. Each sample was analyzed in six replicates. The coefficient “K” was calculated using the equation: $K_{t} = N_{t} ∕ N_{0}$ .

Relationship between the initial cell number and the number after 5 h of pre-enrichment

One hundred twenty artificially contaminated milk and milk powder samples were prepared, using two different S. aureus strains (n = 30 each). The initial number and the number after 5 h of pre-enrichment of each sample were determined by the plate-based method. There was an obvious linear relationship between initial and final number for each of the four groups of samples (Table 2), and there was no statistically significant difference between the four regression equations. Therefore, a fitting regression equation was established by combining the data for all four groups of samples: $L o g N_{5 h} = 1 . 0197 L o g N_{0} + 2 . 7986$ , R ² = 0.9886 (Fig. 3). The initial number of S. aureus cells in milk or milk powder could be calculated using Eq. (2).

FIG. 3.

Relationship between the initial number (N ₀) and the number after 5 h of pre-enrichment (N _5h). Four groups of artificially contaminated samples (Table 2) were prepared with different populations of S. aureus (S. aureus ATCC 6538P or S. aureus ATCC 25923) cells. The initial number and the number after 5 h of pre-enrichment of each sample were tested by the plate-based method, respectively. The fitting regression equation was carried out by combination of all the data from the four groups of samples. N ₀, the initial number of S. aureus cells in milk or milk powder; N _5h, the number of S. aureus cells in milk or milk powder after 5 h of pre-enrichment.

L o g (\frac{N_{F C M}}{10}) = 1 . 0197 L o g N_{0} + 2 . 7986

(2)

where NFCM is the output of the FCM, and $N_{0}$ is the initial number of S. aureus in milk or milk powder.

Detection of S. aureus in artificially contaminated samples

Raw and powdered milk samples with S. aureus at final concentrations of 10⁰–10⁵ CFU/mL or CFU/g, respectively, were prepared, and then analyzed by the FCM-based method. The results are presented in Table 3. Both FCM-based method and plate-based method yielded similar results, with a regression equation of $L o g N_{P l a t e} = 0 . 9695 L o g N_{F C M} + 0 . 1483$ , which indicated that FCM-based method has a good practical application potential for the detection of S. aureus in milk and milk powder. The LOD of the FCM-based method was 7.50 cells/mL in milk and 8.30 cells/mL in milk powder.

Detection of S. aureus in real samples

To confirm the performance of FCM-based method in real samples, 75 UHT milk, 25 pasteurized milk, 66 raw milk, and 123 milk powder samples were analyzed by the FCM-based and plate-based methods. Both methods yielded negative test results for all UHT milk, pasteurized milk, and milk powder samples. As for the raw milk samples, 21 tested positive and 45 negative according to the two methods (Table 4). In each of the 21 S. aureus-positive raw milk samples, similar numbers were reported by both methods (Supplementary Fig. S3).

Table 4.

Detection of Staphylococcus aureus in Real Samples

Sample	Number of samples	FCM-based method		Plate-based method
Sample	Number of samples	Positive result^a	Negative result^b	Positive result^a	Negative result^b
Retail milk powder sample	123	0	123	0	123
Retail UHT milk sample	75	0	75	0	75
Retail pasteurized milk sample	25	0	25	0	25
Raw milk sample	66	21^c	45	21	45

A positive result means that there was an exact concentration of S. aureus in the sample higher than the LOD of the method.

A negative result means that the concentration of S. aureus is lower than the LOD of the method.

For each of 21 S. aureus-positive raw milk samples, both the FCM-based method and plate-based method yielded similar results of S. aureus counts (Fig. S3).

LOD, limit of detection; UHT, ultra-high-temperature-treated.

Discussion

PI is a red fluorescent nucleic acid stain that only penetrates cells with damaged membranes. FCM worked well in quantification of viable S. aureus cells in combination with FITC-labelled antibody and PI. However, FCM cannot detect bacterial cells when the concentrations are <10² cells/mL or cells/g, and many studies have added a pre-enrichment procedure to carry out an acceptable LOD (McClelland and Pinder, 1994; Williams et al., 2017). The current study corroborated that a pre-enrichment procedure before FCM analysis was necessary for the detection of S. aureus. Ding et al. (2017) used a nonselective medium, BHI broth, to reduce the enrichment time. On the contrary, we used a selective medium, SCB, for pre-enrichment because SCB could suppress the proliferation of nonrelated bacteria. Since the proliferation rate of S. aureus is influenced by various external conditions, such as temperature and nutrient medium (Baliavichene, 1981), as well as by its physiological status (Fakruddin et al., 2013; Dan Córdoba et al., 2020), the pre-enrichment procedure in this study was optimized to reduce the influence of external conditions on reproduction. S. aureus cells each have a different physiological status, which can lead to different proliferation rates. When high numbers of bacterial cells are incubated, postenrichment cell counts are more predictable (Williams et al., 2017), which was also confirmed in this study.

We tested different pre-enrichment times for optimization, using the coefficient “K,” which is based on the Eq. (1) It was found that ∼10¹ cells/mL of S. aureus in milk and 10¹ cells/g of S. aureus in milk powder could be analyzed by FCM after 5 h of pre-enrichment (Fig. 2). Considering the detection limit of 10–100 CFU/mL or CFU/g in milk and dairy products prescribed by the European Commission regulations (EC, 2007), the pre-enrichment time in this study was determined to be 5 h. Moreover, this method can even detect a single S. aureus cell when a suitable pre-enrichment time is used.

To analyze the correlation between initial and postenrichment bacterial cell numbers, four regression equations were established using four groups of samples (Table 2). There was no significant difference among the four regression equations, which indicated that S. aureus cells proliferate at similar rates in milk and milk powder, and different strains of S. aureus have similar reproduction rates in SCB. Consequently, a fitting regression Eq. (2) was established by combining the data from the four sample groups, which could be applied for calculating the initial number of S. aureus cells in milk and milk powder (Fig. 3). Although utilization of two strains might not be conservative enough to account for potential discrepancies between growth dynamics of other S. aureus strains, this study could be a preliminary methodology study that provide a proof of concept for the application of other strains in this method.

The results (Table 3) demonstrated that the LOD of the established method was 7.50 cells/mL in milk and 8.30 cells/mL in milk powder, and the TTR was nearly 6 h in total, including 5 h of pre-enrichment, 40 min of isolation, and 20 min of staining and analysis. The TTR of the newly established method is much shorter compared with the plate-based method (FDA, 2016). Table 5 shows the reported technologies, including immunoassay methods, PCR-based assays, biosensors, and FCM, for the detection of S. aureus. Compared with the reported technologies, the developed FCM-based method possesses a better LOD and a lower TTR, for quantitative detection of viable S. aureus cells in low numbers.

Table 5.

Comparison of the Characteristics of Different Technologies Mentioned in the Study

Technology	Specific detection	LOD	TTR	Detection of viable cells	References
Immunoassay method	Yes	10 CFU/mL	/	No	Alamer et al. (2018)
Immunoassay method	Yes	2.47 × 10³ CFU/mL8.86 × 10³ CFU/mL	90 min	No	Yan et al. (2017)
PCR	Yes	400 pg/μL	25 h	Yes	Liu et al. (2019a)
PCR	Yes	1 cells per 25 mL	18 h	No	Moezi et al. (2019)
Biosensor	Yes	10⁷ cells in 2 μL	<1 h	No	Ghali et al. (2016)
Biosensor	Yes	5 cells/mL	4 h	No	Zheng et al. (2019)
FCM	No	33 CFU/mL	2 h	No	Meng et al. (2017)
FCM	Yes	200 cells/mL	2 h	No	Shan et al. (2019)
FCM	Yes	7.50 cells/mL, 8.30 cells/g	6 h	Yes	This study

The symbol “/” means “not mentioned.”

TTR, time-to-results.

Thus, this established method could be used for rapidly monitoring the product quality, as well as reducing the time between production date and on-shelf date of dairy products, especially the pasteurized milk with a short shelf life. At present, although the FCM method may be only available with big industries or developed countries, and may not be feasible in remote areas with the underdeveloped testing facility as it requires sophisticated equipment with relatively high cost, it offers great potential for rapid and sensitive detection of S. aureus. We believe that with further development of FCM, and more utilization of the instruments and reagents, the cost would be reduced.

Conclusion

We developed a faster FCM-based method for the detection of viable S. aureus cells in milk and milk powder. Using a 5-h pre-enrichment period, this method could detect low numbers of S. aureus cells within 6 h. The LOD of the established method was 7.50 cells/mL in milk and 8.30 cells/mL in milk powder. This study provided proof of concept for the application of this assay to other pathogenic microorganisms after rational specific antibody screening and pre-enrichment optimization. With further improvement, it is believed that this method can provide an effective platform for the rapid monitoring of the microbiological quality of milk and its products.

Footnotes

Acknowledgment

The authors thank Elsevier Language Editing Services for providing language help.

Disclosure Statement

No conflict of interest, financial or other, exists.

Funding Information

This study was supported by grant 2017YFF0204602 from the National Key Research and Development Program of China, grant 2019MK114 from the Science and Technology Program of the State Administration for Market Regulation, grant AKY1958 and grant AKY1818 from the National Institute of Metrology, P. R. China, grant 2018KJ34 from Nanjing Customs Project, and grant JC2018094 from Nantong Customs Project.

Supplementary Material

Supplementary Figure S1

Supplementary Figure S2

Supplementary Figure S3

Supplementary Table S1

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