Daptomycin-modified magnetic beads integrated with lysostaphin for selective analysis of Staphylococcus

Daptomycin-modified magnetic beads integrated with lysostaphin for selective analysis of Staphylococcus

Journal of Pharmaceutical and Biomedical Analysis 175 (2019) 112785 Contents lists available at ScienceDirect Journal of Pharmaceutical and Biomedic...

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Journal of Pharmaceutical and Biomedical Analysis 175 (2019) 112785

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba

Daptomycin-modified magnetic beads integrated with lysostaphin for selective analysis of Staphylococcus Mengyao Wang, Enci Fan, Yue Wu, Zhifeng Fu ∗ Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Ministry of Education), College of Pharmaceutical Sciences, Southwest University, Chongqing 400716, China

a r t i c l e

i n f o

Article history: Received 21 April 2019 Received in revised form 19 July 2019 Accepted 20 July 2019 Available online 21 July 2019 Keywords: Staphylococcus Lysostaphin Daptomycin Magnetic beads Bioluminescence

a b s t r a c t An antibiotic-affinity method was developed for analyzing Staphylococcus on the basis of the strong binding capability of daptomycin towards Gram-positive bacteria cellular membrane, as well as the selective lytic action of lysostaphin towards Staphylococcus. Daptomycin-modified magnetic beads were adopted to enrich Staphylococcus from sample matrix. Afterwards lysostaphin was adopted to lyse Staphylococcus, which can hydrolyze pentaglycine cross-linkers of peptidoglycan composing the cellular wall of Staphylococcus. The concentration of Staphylococcus was quantified by collecting the bioluminescent signal of the released intracellular adenosine triphosphate of the enriched Staphylococcus. Staphylococcus aureus (S. aureus) was analyzed as a model bacterium to study the feasibility of the proof-of-principle work. For bioluminescent analysis of S. aureus with the developed method, the linear range was 5.0 × 102 –5.0 × 106 colony forming units mL−1 , and the limit of detection was 3.8 × 102 colony forming units mL−1 . The analytical procedure consisting of bacterial enrichment, cell lysis and signal collection can be accomplished within 20 min. Some common Gram-positive bacteria and Gram-negative bacteria all indicated very low interference to the analysis of the target bacterium. It has been successfully used to analyze S. aureus in milk as well as physiological saline injection, indicating its application potential for real samples. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Staphylococcus is one of the leading causes of a variety of dangerous infectious diseases including osteomyelitis, endocarditis, sepsis syndrome and respiratory tract infection, of which Staphylococcus aureus (S. aureus) has been considered as the most threatening [1,2]. The limits of Staphylococcus in food and drug are strictly regulated throughout the world because of its severe threats to human health. To ensure public health, it is urgently needed to develop some rapid, facile and sensitive methods for analyzing Staphylococcus. The traditional approach on the basis of bacterial culture and colony count has been longly adopted as a standard method for Staphylococcus analysis owing to its accuracy and reliability [3]. However, this approach suffers from some drawbacks including time-consuming bacterial culture as well as laborious manipulation [4]. Typically, it takes several days to obtain results, which is unacceptable in emergency situation and point-of-care testing. Alternatively, various molecular biological methods have been widely employed for rapid analysis of bacteria, including PCR [5],

∗ Corresponding author. E-mail address: [email protected] (Z. Fu). https://doi.org/10.1016/j.jpba.2019.112785 0731-7085/© 2019 Elsevier B.V. All rights reserved.

asymmetric PCR [6] as well as real-time PCR [7]. These methods show superior selectivity and sensitivity, but require highly skilled operators and are susceptible to exogeneous contamination. Recently, some molecular recognition elements which can selectively recognize and capture Staphylococcus cells have been adopted to construct biosensors to overcome these problems. The typical recognition elements include antibodies [8,9], aptamers [10,11] and bacteriophages [12,13]. Nevertheless, these common biological elements have been longly limited by the high cost, low stability, as well as unnegligible difference among batches. As a lipopeptide antibiotic, daptomycin exerts its antimicrobial activity towards a very wide spectrum of Gram-positive (G+ ) bacteria [14,15]. With the aid of Ca2+ ion, this antibiotic inserts its fatty chain with high hydrophobicity into the cellular membrane of G+ pathogens, resulting in damage of the cellular membrane without cell lysis [16–18]. This mechanism makes it possible for daptomycin to act as the recognition element to capture G+ bacteria. In our previous work, we have demonstrated the application of the recognition mechanism for the analysis of the total amount of G+ bacteria by using hexadecyl trimethyl ammonium bromide as a non-selective lytic reagent [19]. Unfortunately, as a typical wide-spectrum antibiotic towards most G+ pathogens, obviously


M. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 175 (2019) 112785

Fig. 1. Schematic illustration of Staphylococcus analysis using dapt-MBs.

daptomycin is insufficient to selectively recognize a given bacterium such as Staphylococcus. Lysostaphin is known to be a zinc metalloenzyme firstly isolated from Staphylococcus simulans biovar staphylolyticus in 1964 [20]. It consists of a C-terminal cellular wall targeting domain directing to the peptidoglycan substrate and an N-terminal glycylglycine endopeptidase [21,22]. The glycyl-glycine endopeptidase is responsible for the very strong catalytic activity of lysostaphin for hydrolyzing the pentaglycine cross-linkers of peptidoglycan composing the cellular wall of certain Staphylococcus [23,24]. Especially, lysostaphin possesses highly effective antimicrobial activity against both actively growing and quiescent S. aureus because of the very high proportions of pentaglycine in the cellular wall of this pathogen [25]. Herein a novel antibiotic-affinity strategy was developed to analyze Staphylococcus by using S. aureus as a model. Daptomycinmodified magnetic beads (dapt-MBs) were adopted for enrichment and separation of Staphylococcus from sample matrix. After extracting intracellular adenosine triphosphate (ATP) from the enriched bacteria utilizing the selective lytic action of lysostaphin, S. aureus was analyzed by collecting the bioluminescent (BL) signal of ATP. This work provides a facile, rapid, selective and sensitive approach for analyzing Staphylococcus to meet the demand of public health. 2. Experimental methods 2.1. Fluorescent micrography of fluorescein isothiocyanate (FITC)-stained S. aureus One milliliter of bacterial suspension (1.0 × 108 colony forming units (CFU) mL−1 ) was added with 100 ␮L of FITC-conjugated daptomycin. After 10-min reaction at room temperature (RT), the FITC-stained bacteria were centrifugally washed, and dispersed in 1.0 mL of Tris-HCl buffer. Afterwards 10 ␮L of FITC-stained S. aureus suspension was used to prepare a microscopic specimen on a glass slide. The stained bacterial specimen was observed in the green channel of the fluorescence microscope with an amplification of 1000.

2.2. BL analysis of S. aureus Two milliliters of sample suspension was added with 7.5 ␮L of dapt-MBs suspension, followed by 10-min incubation under constant rotation at RT. The S. aureus-loaded dapt-MBs were then washed thoroughly using Tris-HCl buffer. One hundred microliters of 5.0 ␮g mL−1 lysostaphin solution was used to lyse the enriched bacteria for 2 min to extract the intracellular ATP. Finally, 50 ␮L of lysate was pipetted into a microplate, and added with 50 ␮L of BL reagent to produce signal for the analysis of the target bacteria. 3. Results and discussion 3.1. Interpretation of analysis principle As illustrated in Fig. 1, dapt-MBs were used to enrich Staphylococcus based on the strong binding capability of daptomycin towards G+ bacteria. In this process daptomycin inserted the hydrophobic fatty chain into the cellular membrane of the target bacteria with the aid of Ca2+ ion. After magnetic isolation of Staphylococcus from sample matrix, lysostaphin was adopted to selectively lyse the target bacteria by hydrolyzing the pentaglycine cross-linkers to release intracellular ATP. Afterwards the bacterial concentration was quantified by detecting the BL emission of ATP ˜ −18 based on the fact that ATP level usually keeps rather constant (10 mol cell-1 ) in viable bacterial cells [26]. Due to the usage of the bacterial lyase, this approach shows obvious advantage of much higher selectivity for a given bacterium over other reported magnetic isolation methods [19,27]. 3.2. Characterization of binding capability of daptomycin towards Staphylococcus To study the feasibility of adopting daptomycin as a bacterial recognition element for Staphylococcus, FITC-conjugated daptomycin was used to stain S. aureus. The stained S. aureus cells were imaged in the green fluorescence channel of the microscope. As indicated in Fig. 2, the cellular surface of S. aureus emitted very

M. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 175 (2019) 112785


Fig. 2. Fluorescent micrographs of S. aureus treated using FITC-conjugated daptomycin. (A) Image in bright field, (B) image in green fluorescence channel.

Fig. 3. Scanning electron micrograph of bacterial cells enriched by dapt-MBs.

strong green fluorescence, which confirmed the obvious affinity behavior of daptomycin towards Staphylococcus. Furthermore, S. aureus captured by dapt-MBs was observed using SEM to evaluate the enrichment capability of dapt-MBs (Fig. 3). It can be seen that S. aureus cells were well captured by dapt-MBs. The enrichment efficiency () of dapt-MBs towards S. aureus can be calculated with the following formula (1):  = (1 − C1 /C0 ) × 100%


volume of dapt-MBs suspension and then achieved a maximum while 7.5 ␮L of dapt-MBs was adopted. With the dosage of daptMBs, the number of captured bacteria was around 7.6 × 105 CFU. Further increased volume of the dapt-MBs suspension led to lower signal-to-blank ratio, probably because concentrated MBs hindered the collection of BL signal. Thus 7.5 ␮L of dapt-MBs was adopted in the following experiments. The concentration of lysostaphin was another critical factor affecting analytical performance. As indicated in Fig. S2, the signal-to-blank ratio achieved a plateau value after the concentration of lysostaphin exceeded 5.0 ␮g mL−1 , implying that lysostaphin at 5.0 ␮g mL−1 was enough to lyse the enriched bacterial cells. Hence, 5.0 ␮g mL−1 was adopted as an optimal lysostaphin concentration for bacteria lysis. Furthermore, a pH value of 7.4 was adopted for bacterial lysis and BL detection because a maximal signal-to-blank ratio was achieved at this value (Fig. S3). The effect of lysis time on the analytical performance for S. aureus was also studied in detail. The data in Fig. S4A indicate that uncaptured S. aureus can be lysed thoroughly 10 min after addition of lysostaphin when no dapt-MB was used (experimental procedure described in Supplementary Material). Interestingly, S. aureus enriched by dapt-MBs can be lysed thoroughly by lysostaphin within 2 min (Fig. S4B). It might be ascribed to the fact that the binding behavior of daptomycin towards S. aureus caused damage of the cellular membrane, furtherly led to greatly shortened lysis time. Thus 2 min was adopted to lyse bacterial cells in the further work. 3.4. Selectivity study

Here, C0 and C1 are the bacterial concentrations before and after a standard bacterial sample is enriched using dapt-MBs, respectively. For a S. aureus standard suspension at 5.0 × 105 CFU mL−1 , the dapt-MBs showed a high enrichment efficiency of 76%. The results demonstrated the enrichment capability of daptomycin as well as the feasibility of the magnetic separation-based antibiotic-affinity strategy.

The selectivity of the developed method was evaluated by comparison between the BL signal of S. aureus and those of three G+ and three Gram-negative (G− ) bacteria. All these studied bacteria were at 5.0 × 105 CFU mL−1 in the selectivity evaluation. The interference factors (IFs) of these potential interferents can be calculated with formula (2) indicated as following:

3.3. Optimization of analytical conditions

In this formula, I, S and D are the BL responses caused by these interferents, S. aureus and a dilution buffer, respectively. The results in Fig. 4 show that the IFs of Escherichia coli (E. coli), Salmonella typhimurium (S. typhimurium) and Shigella dysenteriae (S. dysenteriae) were all below 0.5%. The interference of the above G− bacteria was negligible because they cannot bind with G+ bacteria-targeting daptomycin. Meanwhile, the studied G+ bacteria including Micrococcus luteus (M. luteus), Bacillus subtilis (B. subtilis) and Enterococcus faecium (E. faecium) also indicated very minor interference to

To achieve high sensitivity for BL analysis of S. aureus, various analytical conditions were optimized, such as the amount of daptMBs, the concentration of lysostaphin, pH value and lysis time. A standard S. aureus suspension at 5.0 × 105 CFU mL−1 was used in the optimization experiments. Fig. S1 indicates the effect of the volume of dapt-MBs suspension on the signal-to-blank ratio for BL analysis of S. aureus. The signal-to-blank ratio rose firstly with the

IF= (I-D)/(S-D) × 100%



M. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 175 (2019) 112785

Table 1 Analysis of sterilized milk inoculated with S. aureus and cultured for various durations (n = 5). BL method (CFU mL−1 )

Culture duration (h)


4 8 12

(2.3 ± 0.3) × 10 (1.2 ± 0.2) × 107 (1.1 ± 0.1) × 108



Plate count method (CFU mL−1 )

Relative error (%)

(2.6 ± 0.2) × 105 (1.5 ± 0.1) × 107 (1.2 ± 0.1) × 108

11.5 20.0 8.3

All samples were diluted for 100 times prior to bacterial analysis when the BL method was applied.

Table 2 Recovery tests of physiological saline injection spiked with S. aureus (n = 5). Sample number

Spiked amount (CFU mL−1 )

Found amount (CFU mL−1 )

RSD (%)

Recovery (%)

1 2 3

5.0 × 10 5.0 × 104 5.0 × 103

4.8 × 105 4.5 × 104 4.4 × 103

8.6 5.4 7.1

96.0 90.0 88.0


analytical procedure consisting of bacterial enrichment, cell lysis and signal collection can be completed within 20 min. This method can also be used to analyze other Staphylococcus bacteria under the same conditions for S. aureus analysis. As a coagulase-negative Staphylococcus, Staphylococcus epidermidis (S. epidermidis) was less susceptible to the lytic action of lysostaphin than S. aureus because of the lower level of glycyl-glycine linkage in the peptidoglycan structure [28]. It has already been reported that greatly increased amount of lysostaphin and longer lysis duration are required for killing S. epidermidis by lysostaphin [29,30]. Because of the lower susceptibility, S. epidermidis cannot be lysed thoroughly by lysostaphin under the same conditions for S. aureus analysis, leading to a lower sensitivity. For the analysis of S. epidermidis, the linear range is 1.0 × 104 – 1.0 × 108 CFU mL−1 , and the regression equation is lgS = 0.65lgC-1.26 (R2 = 0.9978). The limit of detection for S. epidermidis is 7.6 × 103 CFU mL−1 at 3␴. 3.6. Real sample analysis

Fig. 4. BL responses of different bacteria all at 5.0 × 105 CFU mL−1 (n = 5).

S. aureus analysis with IFs all below 1.9%. Our previous work had already demonstrated the binding capability of daptomycin towards many G+ bacteria [19]. In this work, we also tried to use lysostaphin to treat the suspensions of M. luteus, B. subtilis and E. faecium, and then collect the BL signals of released ATP. However, the collected BL signals of the three interfering bacteria were very close to the background. Based on the above facts, although the three G+ interfering bacteria can be enriched by dapt-MBs, they cannot be lysed by lysostaphin since there was no pentaglycine cross-linking structure in their cellular wall. For mixture 1 consisting of all these six interferents, the IF was 0.9%. Moreover, for mixture 2 consisting of S. aureus and the six interferents, the BL signal was only 2.6% higher than that of S. aureus alone. The potential interference from some small-molecular substances including ATP, Na+ , Zn2+ , Fe3+ , lysine, tryptophan, methionine, leucine was investigated by analyzing 5.0 × 105 CFU mL-1 S. aureus suspensions spiked with the substances all at 1.0 ␮M. All of these spiked S. aureus suspensions produced BL signals very close to that of S. aureus alone. The above data indicated that the proposed method possessed ideal selectivity for BL analysis of the target pathogen. 3.5. Analytical performance S. aureus can be analyzed within a wide concentration range of 5.0 × 102 –5.0 × 106 CFU mL−1 , with a regressive equation of lgS = 0.27 + 0.59lgC (R2 = 0.9915). Here C is the bacterial concentration (CFU mL−1 ), and S is the intensity of BL emission (a. u.). The limit of detection for S. aureus is 3.8 × 102 CFU mL−1 at 3␴. The whole

For estimation of the application potential in real sample analysis, sterilized milk was inoculated with the target bacteria and cultured for various durations at RT. Afterwards the S. aureus contaminated milk samples were analyzed using the developed BL method. The results were then compared with those obtained from the traditional plate count method. From the results indicated as Table 1, the two methods imply acceptable consistence with relative difference all below 20.0%. The application reliability was also studied by recovery tests conducted using physiological saline injection solution spiked with S. aureus suspensions. The recovery values indicated in Table 2 are between 88.0% and 96.0%, with RSD values not higher than 8.6%. All these results demonstrate the feasibility for the analysis of contaminated food as well as pharmaceutical samples. 4. Conclusion Overall, a novel BL approach was proposed for the analysis of Staphylococcus utilizing the strong binding capability of daptomycin towards G+ bacteria, as well as the selective lytic action of lysostaphin towards Staphylococcus. The target pathogen was enriched by dapt-MBs and then lysed by lysostaphin. The extracted intracellular ATP was analyzed to quantify Staphylococcus with high sensitivity. Due to the presence of numerous pentaglycine cross-linkers in the cellular wall of Staphylococcus, this method provided excellent sensitivity for S. aureus analysis. S. epidermidis is less susceptible to the lytic action of lysostaphin because of lower level of glycyl-glycine linkage in the cellular wall, leading to lower analytical sensitivity. By adopting antibiotics and lyases acting on other bacteria, this method can also be extended to the analysis of other targets.

M. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 175 (2019) 112785

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