Optimized universal protocol for electroporation of both coagulase-positive and -negative Staphylococci

Journal of Microbiological Methods 146 (2018) 25–32

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Optimized universal protocol for electroporation of both coagulase-positive and -negative Staphylococci

T

Yusuke Sato'oa, Yoshifumi Aibaa, Kotaro Kigaa, Shinya Watanabea, Teppei Sasaharaa, ⁎ Yasuhiko Hayakawab, Longzhu Cuia, a

Division of Bacteriology, Department of Infection and Immunity, Faculty of Medicine, Jichi Medical University, 3311-1, Yakushiji, Shimotsuke-shi, Tochigi 329-0498, Japan b NEPA GENE Co., Ltd., Chiba, Japan

A R T I C L E I N F O

A B S T R A C T

Keywords: Gene transformation Electroporation Coagulase-positive Staphylococcus Coagulase-negative Staphylococcus

Electroporation is a common technique necessary for genomic manipulation of Staphylococci. However, because this technique has too low efficiency to be applied to some Staphylococcal species and strains, especially to coagulase-negative Staphylococcus (CNS) isolates, basic researches on these clinically important Staphylococci are limited. Here we report on the optimization of electroporation parameters and conditions as well as on the generation of a universal protocol that can be efficiently applicable to both CNS and Coagulase-positive Staphylococci (CPS). This protocol could generate transformants of clinical Staphylococcus epidermidis isolate, with an efficiency of up to 1400 CFU/μg of plasmid DNA. Transformants of 12 other clinically important Staphylococcal species, including CNS and CPS, were also generated with this protocol. To our knowledge, this is the first report on successful electroporation in nine these Staphylococcal species.

1. Introduction The genus of Staphylococcus consists of more than forty species (August 2017), including many medically important pathogens, and is classified into two groups, coagulase-positive Staphylococcus (CPS) and coagulase-negative Staphylococcus (CNS), according to their coagulase production (Foster, 1996). S. aureus is the most notorious pathogen among the CPS, causing various diseases from mild-to-serious in humans and animals (Lowy, 1998; Fitzgerald, 2012). Other CPS such as S. hyicus and S. pseudintermedius are important pathogens in the veterinary field and are known as porcine and canine pathogens, respectively (Foster, 2012; Bannoehr and Guardabassi, 2012). S. argenteus, which was re-classified as a different species from S. aureus, was isolated from a food poisoning outbreak in Japan (Suzuki et al., 2017). Conversely, CNS was thought to be less virulent than S. aureus (Foster, 1996), however, many of these species have been proven to cause abscesses, medical device-related infections (biofilm infection), endocarditis, urethritis and bone infection (Becker et al., 2014). Currently, drug resistance in both CPS and CNS is also an increasing problem worldwide (Becker et al., 2014; Stryjewski and Corey, 2014). Nevertheless, knowledge on physiology of these pathogens, their pathogenicity

dynamics, the processes of infection and drug resistance is limited because many methods used for genetic manipulation are often failed to apply to some species or strains, especially to many species of CNS and clinical isolates. Genetic transformation is one of the most important technologies for genetic manipulation. This technique can directly introduce exogenous nucleic acids into living cell(s) or organism(s) to change their original phenotype, and this altered genome can be inherited by the progeny. In bacteria research, this technology is necessary for bacterial genome study, including knockout or knock-in of gene, introduction of point mutation, and complementation or overexpression of gene. There are several genetic transformation techniques available, including CaCl2 induction, conjugation, natural transformation and electroporation. In the CaCl2 induction, CaCl2 can make bacterial cell more permeable for uptake of foreign nucleic acid (Mandel and Higa, 1970). This technique is usually used for transformation of Escherichia coli with plasmid in vector construction. Conjugation can transfer DNA from one cell to another through a pilus bridge made by cell-to-cell direct contact (Cook and Federle, 2014, Huddleston, 2014). Using natural transformation ability (Lorenz and Wackernagel, 1994) for some bacteria, Neisseria for example, is also an alternative way to carry out gene

Abbreviations: BHI, brain heart infusion; CFU, colony forming unit; CNS, coagulase-negative Staphylococcus; CPS, coagulase-positive Staphylococcus; Pp, poring pulse; SA, Staphylococcus aureus; SE, Staphylococcus epidermidis; Tp, transfer pulse; TS, temperature sensitive ⁎ Corresponding author. E-mail address: [email protected] (L. Cui). https://doi.org/10.1016/j.mimet.2018.01.006 Received 7 November 2017; Received in revised form 4 January 2018; Accepted 15 January 2018 0167-7012/ © 2018 Elsevier B.V. All rights reserved.

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2008 was provided by Dr. Keiichi Hiramatsu and Dr. Takashi Sasaki (Juntendo University). Three E. coli strains, DH5α (Takara, Shiga, Japan), Top10 (Invitrogen, Carlsbad, CA) and BL21 (Takara), were used for plasmid propagation (Table 1). DH5α and Top10 were positive for dcm, which encodes DNA cytosine methylase, whereas, BL21 was negative for dcm.

transformation. These bacteria can uptake foreign DNA directly from environment under natural condition. Electroporation is a technique that uses an electrical pulse to create temporary pores in cell membranes through which nucleic acids can pass into cells. This technique is known as most powerful and effective for introducing exogenous DNA into cells, and is widely used for broad range of bacterial species, including Staphylococcus. Recently, there were several reports on the improvement of electroporation efficiency in Staphylococcus and extension of its usage to other Staphylococcal species (Löfblom et al., 2007; Monk et al., 2012; Cui et al., 2015; Grosser and Richardson, 2016.). To date, however, electroporation methods for only 7 Staphylococcal species (S. aureus, S. epidermidis, S. capitis, S. lugdunensis, S. staphylolyticus, S. xylosus and S. carnosus) were reported (Luchansky et al., 1988; Augustin and Götz, 1990; Brückner, 1997; Löfblom et al., 2007; Monk et al., 2012; Heilbronner et al., 2013; Cui et al., 2015; Hisatsune et al., 2016; Grosser and Richardson, 2016.), and varied for different species. In 2016, we had reported an improved electroporation method for S. aureus, involving the use of a novel multi-pulse system of electroporator, the ELEPO21, which significantly increased the transformation efficiency (Hisatsune et al., 2016). Here we report a further improvement of this method through use of a universal electroporation protocol that could be applicable to many Staphylococcal species of both CPS and CNS. With this protocol, we successfully transformed 13 clinically important Staphylococcal species using an Escherichia coli-S. aureus shuttle vector.

2.2. Plasmid vector Plasmid pKFT (5.7 kbp in size), an E. coli-S. aureus shuttle vector (Kato and Sugai, 2011), was used as a model vector in this study (Table 1). This plasmid contains a temperature-sensitive (TS) replication origin and a tetracycline-resistant gene of Staphylococcus. Plasmid extraction from the culture bacteria was performed using the FastGene Plasmid Mini Kit (Nippon Genetics Co., Tokyo, Japan). Plasmid concentration was measured using a UV–Vis microvolume spectrophotometer NanoDrop Lite (Thermo Fisher Scientific, USA).

2.3. Culture media For pKFT propagation, E. coli carrying pKFT was cultured in LuriaBertani broth with supplementation of 100 μg/mL of ampicillin at 37 °C with agitation. The following three culture media were used as basic media for Staphylococcal competent cell preparation: Brain heart infusion broth (BHI), B2 (10 g of casamino acids, 25 g of yeast extract, 1 g of K2HPO4, 5 g of D-glucose and 25 g of NaCl in 1 L, pH 7.5 adjusted with NaOH; Grosser and Richardson, 2016) and B2low (10 g of Tryptone, 25 g of yeast extract, 1 g of K2HPO4, 5 g of D-glucose and 10 g of NaCl in 1 L, pH 7.5 adjusted with NaOH). The BHI, BHI containing 10% sucrose (w/vol) and B2 (10 g of Tryptone, 25 g of yeast extract, 1 g of K2HPO4, 5 g of D-glucose and 25 g of NaCl in 1 L, pH 7.5 adjusted with NaOH) were also used as recovery media after electric pulse application. Trypticase soy agar containing 5 μg/ml tetracycline was used for colony selection. All media and chemicals were purchased from Becton Dickinson (Sparks, MD), Wako Pure Chemical Industries (Osaka, Japan) and Kanto Chemical (Tokyo, Japan).

2. Materials and methods 2.1. Bacterial strains A total of 13 strains representing nine CNS and four CPS (details provided in Table 1) were used in this study. Among these, Jichi Medical University Bacterial Collection (JMUB) strains were isolated at the Jichi Medical University Hospital from June to December 2015. All JMUB strains were identified by using Bruker Biotyper MALDI-TOF MS system (Bruker Daltonik, MA, USA) and 16S rRNA sequencing. For 16S sequencing, previous reported primers were used (Lane, 1991). Staphylococcus hyicus S-3B isolated from a pig slaughterhouse in Japan in Table 1 Bacteria strains and vector in this study. Species

Strains

CNS or CPSa

Reference

CFU/μg DNAb

Staphylococcus epidermidis Staphylococcus capitis Staphylococcus caprae Staphylococcus hominis Staphylococcus warneri Staphylococcus simulans Staphylococcus haemolyticus Staphylococcus lugdunensis Staphylococcus saprophyticus Staphylococcus hyicus Staphylococcus pseudintermedius Staphylococcus argenteus Staphylococcus aureus Escherichia coli Escherichia coli Escherichia coli

JMUB20 S. capitis 60 JMUB877 JMUB705 JMUB272 JMUB1090 JMUB375 JMUB230 JMUB62 S-3B NVAU06002 Tokyo13069 RN4220 DH5α Top10 BL21

CNS CNS CNS CNS CNS CNS CNS CNS CNS CPS CPS CPS CPS – – –

In this study Cui et al., 2013. In this study In this study In this study In this study In this study In this study In this study In this studyc Sasaki et al., 2007. Suzuki et al., 2017. Peng et al., 1988. Takara Invitrogen Takara

1.38 1.94 5.21 2.09 3.98 2.40 1.54 2.31 4.70 2.06 1.33 2.76 1.80 – – –

± ± ± ± ± ± ± ± ± ± ± ± ±

0.09 × 103 0.21 × 105 0.44 × 102 1.27 × 102 0.76 × 103 0.49 0.21 × 103 0.46 × 102 4.31 × 102 0.47 × 104 0.23 × 104 0.19 × 105 0.31 × 106

Reference Plasmid pKFT

Kato and Sugai, 2011.

a

CNS: coagulase-negative Staphylococcus. CPS: coagulase-positive Staphylococcus. Transformation efficiencies (colony forming unit/μg plasmid DNA) of this study. Three (all strains except for S. simulans and S. saprophyticus), five (S. simulans) or ten (S. saprophyticus) independent trials were performed. Average and standard error (SE) are shown. Competent cell preparation and electroporation were performed under the optimized condition (see Supplementary information). c Kindly provided by Dr. Keiichi Hiramatsu and Dr. Takashi Sasaki (Juntendo University). b

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Transformation efficiency (CFU/µg DNA)

A

Fig. 1. Gene transformation of S. aureus and S. epidermidis using our previous method. DNA preparation, competent cell preparation and electroporation were carried out as described previously (Hisatsune et al., 2016). RN4220: S. aureus (SA). JMUB20: S. epidermidis (SE). ELEPO21 was used for all experiments. A. Transformation efficiencies. Vertical line: colony forming unit (CFU)/μg plasmid DNA. Horizontal line: strains. Three independent trials were performed (n = 3). Average and standard error (SE) are shown. B. Representative plate images. 500 μl bacterial solution with 100-folded dilution (RN4220) and without dilution (JMUB20) was inoculated on each plate.

B

100,000

10,000 1,000

100 10

1 RN4220 (SA)

JMUB20 (SE)

Strain

RN4220 (SA)

JMUB20 (SE) Strain

0.2 g, KH2PO4 in 1 L purified water, pH 7.4) and spread on the plate. The plates were incubated at an appropriate temperature (changeable depends on vectors used, e.g. 30 °C for pKFT) for up to 72 h.

2.4. Electroporator Two types of electroporator devices, multiple pulse wave type electroporator ELEPO21 (NEPA GENE, Chiba, Japan) and single exponential pulse wave type electroporator MicroPulser (Bio-Rad, Hercules, CA, USA), were used and their transformation efficiencies were compared. EC-001S Nepa Electroporation Cuvettes 1 mm gap (NEPA GENE) was used as a standard electro cuvette.

2.7. Calculation of transformation efficiency The colonies growing on plates were counted and the efficiency of each condition was calculated. Representative colonies were selected and successful transformation was confirmed using PCR, as described previously (Hisatsune et al., 2016).

2.5. Electrical resistance measurement A portion of 20 μl of washed cell suspensions was transferred into electro-cuvette at room temperature and its electrical resistance was measured with ELEPO21.

2.8. Statistical analysis

2.6. Competent cell preparation and electroporation

3. Results

Using S. epidermidis and plasmid pKFT as a model system, optimal conditions of culture medium, washing buffer, plasmid origin and electroporator parameters, and time and temperature for the ideal recovery of transformants were examined with reference to the relevant literatures (Luchansky et al., 1988; Augustin and Götz, 1990; Schenk and Laddaga, 1992; Löfblom et al., 2007; Monk et al., 2012; Cui et al., 2015; Grosser and Richardson, 2016.). Competent cell preparation and electroporation for Staphylococci were principally carried out according to our previous method (Hisatsune et al., 2016). The detailed protocol and material were described in supplemental material (Supplemental information). Briefly, overnight preculture of Staphylococcus in B2low broth was transferred into pre-warmed fresh B2low broth and cultured at 37 °C for 1 h. Cultured cell suspension was transferred into new centrifuge tubes and centrifuged under condition of 6000 ×g at 4 °C for 10 min. Supernatants were discarded and cell pellets were washed with cold 1.5 M NaCl solution. The pellets were then washed with cold 10% glycerol solution in 1.5 ml tube. This wash was repeated until electrical resistance of the cell suspension exceeded 6 kΩ. Finally, the cell suspension was aliquot into micro-tubes and stored at −80 °C until use. Before electroporation, the competent cells were thawed on ice and subsequently incubated at 25 °C for 5 min. The tube was centrifuge under condition of 15,000 ×g at room temperature for 1 min. The supernatant was discarded and the pellet was re-suspended with electroporation buffer. And then, bacteria suspension was mixed with plasmid DNA extracted from B strain, and incubated at 25 °C for 10 min. The mixture was transformed into 1 mm cuvette and subjected to electroporation with following parameters: For Pp, 1800 V, 2.5 ms, 50 ms, 1 time, +; and for Tp, 100 V, 99 ms, 50 ms, 5 times, +/−. The transformants were recovered by incubation with B2 broth at 30 °C for 5 h, then the cell suspensions were spread on TSA with antibiotic. If needed, the bacterial solution was diluted with autoclaved Dulbecco's phosphate-buffered saline (8 g, NaCl; 2.9 g, Na2HPO4·12H2O; 0.2 g, KCl;

3.1. Transformation efficiencies of Staphylococcus with the previous electroporation protocol

F test and Welch's t-test were used for statistical analysis of data.

We first tested our previous protocol (Hisatsune et al., 2016) for the transformation of S. aureus and S. epidermidis with pKFT (propagated in DH5α) by using the electroporator ELEPO21. As shown in Fig. 1, the transformants of S. aureus was succeeded, while those of S. epidermidis could not be obtained despite three independent tries (independent competent cell preparation and independent electroporation). Electroporation of other CNS, including S. simulans and S. lugdunensis, also failed despite three attempts with this previous protocol (data not shown); our findings demonstrate that the previous protocol is only applicable to a limited number of Staphylococcal species or strains, especially CNS. 3.2. Optimization of electroporation conditions To facilitate electroporation of various Staphylococcal species with simple and convenient method, we tried to develop a universal protocol that could be applied to as many Staphylococcal species as possible. We started by modifying our previous protocol using S. epidermidis JMUB20 because S. epidermidis is one of the most major pathogens in CNS, whose electroporation is widely recognized as a big hurdle to get over. Then, the modified protocol was tested in other Staphylococcal species. The resultant protocol is attached as Supplemental information, and the major modifications in the new protocol are summarized in Table 2. 3.2.1. Plasmid origin The plasmid pKFT was propagated via the three commercially available E. coli strains and their transformation efficiency in JMUB20 was tested. Using pKFT from DH5α and Top10, only < 1.5 CFU/μg DNA transformants of JMUB20 were obtained (Fig. 2A). By contrast, 27

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Table 2 Major difference between present and previous protocols. Item description

Present protocol

Previous protocol (Hisatsune et al., 2016)

E. coli type for plasmid propagation Media for competent cell preparation 1st wash buffer for competent cell preparation Incubation after mixing cells and DNA Total electric energy Recovery (media, temp., time)

BL21 (B strain) B2low 1.5 M NaCl 25 °C, 10 min High (Pp:18 kV/cm, 1.0–3.0 J) B2, 30 °C, 5 h

DH5α (K12 strain) BHI Pure water No incubation Low (Pp: 16 kV/cm, 0.3–1.5 J) BHI + 10% sucrose, RT, 1–1.5 h

been used in our previous protocol with satisfied outcomes (Hisatsune et al., 2016). 2) B2 contains high concentration of NaCl and yeast extract, which had been reported to improve the transformation efficiency (Schenk and Laddaga, 1992). 3) B2low containing low concentrations of NaCl compared to B2 was used for testing NaCl influence on the transformation efficiency and arching frequency. Bacterial cells of logphase cultures in these media were subjected to competent cell preparation, which was similar to our previous study (Hisatsune et al., 2016). As shown in Fig. 2B, the transformation efficiency of JMUB20 prepared with B2low was nearly two times higher than that of prepared with both BHI and B2. The electrical resistances of the competent cells prepared with the BHI, B2 and B2low media were very similar, with values of 6.6–8.8,

consistent with our previous observation in S. aureus (Sato'o et al., 2017), the same plasmid from BL21 increased the transformation efficiency by > 2 log10 folds by the new method. Therefore, E. coli B derivative BL21 strain was used for pKFT propagation in all experiments thereafter.

3.2.2. Medium Bacterial culture medium is an important factor that influences the efficiency of electroporation (Schenk and Laddaga, 1992.). Therefore, three bacterial culture media, BHI, B2 and B2low, for preparing competent cell of JMUB20 were compared in terms of transformation efficiency and incidence of arcing (sparking) during electroporation. The reasons for selection of these three media are as follows: 1) BHI had ＊

A Transformation efficiency (%)

＊

DH5α

Top10 E. coli strain

C

＊

BHI

BL21

D Transformation efficiency (%)

＊

B

B2 Medium

B2low

H2O

NaCl Wash buffer

E

400

＊＊

F ＊

NS

＊

＊

NS

300

200

＊

100

0

0

10

30 10 Time (min)

30

60

on ice RT Cuvette temperature

0

1

3 5 Time (h)

7

24

Fig. 2. Effects of the different materials and methods on the transformation efficiency. Three independent trials were performed (n = 3). Average and SE are shown. A. E. coli strains and types. K12: Top10 and DH5α; B: BL21. B. Culture media for competent cell preparation. BHI: BHI broth; B2: B2 broth (containing 2.5% NaCl); B2low: B2low broth (containing 1.0% NaCl). Regarding B2, we adopted the result without arching during electroporation. C. First wash buffer for competent cell preparation. H2O: cold purified water; NaCl: cold 1.5 M NaCl solution. D. Incubation conditions and periods before electroporation. Black bars, no incubation or 25 °C incubation after mixing cells and DNA. White bars, on ice incubation after mixing cells and DNA. E. Electro-cuvette temperature. RT, room temperature cuvette (preset temperature is 22–28 °C in our laboratory); on ice, on ice pre-chilled cuvette. F. Recovery time after electroporation. In this figure, the values of transformation efficiencies for all tests are presented as the mean percentage relevant to the respective control (set as 100%) and the results are shown, with values of DH5α, BHI, H2O, no incubation, on ice and 1 h considered as 100% in A, B, C, D, E and F respectively. ELEPO21 was used for all experiments and parameters are as follows: Pp parameter, 1800 V for 2.5 ms with a 50 ms interval (one pulse in + direction); Tp parameter, 100 V for 99 ms with a 50 ms interval (five pulses in ± direction). *, p < 0.05; **, p < 0.01; NS, no significance.

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electroporation under the previous electric condition, and obtained transformation efficiency of approximately 3.4 × 102 CFU/μg DNA. The efficiency was improved up to 7.5 × 102 CFU/μg DNA under the same Pp parameters (pulse voltage, 1600 V, pulse duration, 2.5 ms with a 50 ms interval; pulse number, one; and pulse direction, +) with modified Tp parameters (pulse voltage, 100 V, pulse duration, 99 ms with a 50 ms interval; pulse number, five; and pulse direction, ± ), suggesting that the conditions of higher voltage, longer pulse length and more pulse times Tp were better for improvement of electroporation efficiency. Therefore, we compared different pulse voltages of ranging from 1200 to 2000 V, then compared the Pp parameters (pulse length, interval, time and direction) under conditions of the fixed Tp parameters. Based on the pre-test results, the Pp parameters of pulse length with a 50 ms interval, pulse time of 1–5, and pulse direction of ± and + were selected to be tested. The results showed that the transformation efficiency was increased by raising the pulse voltage up to 1800 V and 2000 V for ELEPO21 and MicroPulser, respectively (Table 3), and ELEPO21 was more efficient than MicroPulser in all tested voltage ranges. However, electric arcing frequently occurred when the pulse voltage was > 2200 V in both devices (> 50%). For the ELEPO21, the highest transformation rates (1.38 ± 0.09 × 103 CFU/ μg DNA, Table 3) could be obtained by setting the Pp parameters at 1800 V for 2.5 ms with a 50 ms interval (one pulse in + direction) and Tp parameters at 100 V for 99 ms with a 50 ms interval (five pulses in ± direction), and the highest transformation rate for MicroPulser was 4.15 ± 2.05 × 102 CFU/μg of plasmid DNA under the best conditions of 2000 V (Table 3). The maximum efficiency with ELEPO21 was significantly higher than that with MicroPulser (p < 0.01, Welch's t-test).

7.1–9.1 and 6.7–8.5 kΩ (total three tries), respectively, and these electrical resistances were agreed with our criterion, as discussed later. However, electric arcing frequently occurred in cells prepared with B2 (four times in seven trials), whereas no arcing was observed in the cells prepared with BHI and B2low. Because the competent cells prepared by B2low medium had high transformation efficiency without electrical arching, this medium was used for competent cell preparation in the further experiments. 3.2.3. Wash buffer Two wash buffers, hypotonic (purified H2O, 18.2 MΩ) and hypertonic (1.5 M NaCl) solutions, were used for the first washing and their influence on the electroporation were compared. For this comparison, all afterward processes including washing with 10% glycerol buffer were kept same for all subjects. As shown in Fig. 2C, unlike other reported protocols (Löfblom et al., 2007; Monk et al., 2012; Cui et al., 2015; Grosser and Richardson, 2016; Hisatsune et al., 2016), the electroporation efficiency of the cells washed with 1.5 M NaCl solution was approximately 3 times higher than that of cells washed with purified H2O. 3.2.4. Incubation temperature and time Two temperature conditions and three time periods were tested and compared for the pre-incubation of the competent cell/DNA mixture before transfer to the cuvette. As shown in Fig. 2D, transformation efficiency was increased up to 6 times when the incubation of the competent cell/DNA mixture prior to electroporation was performed at 25 °C instead of on ice. Regarding the incubation time prior to electroporation, 10 min showed better results than did the tested time periods of 0, and 30 min.

3.3. Transformation efficiencies of various Staphylococcal species 3.2.5. Cuvette temperature We tested two cuvette temperatures by using a pre-chilled cuvette and a room temperature cuvette. The results showed that the cuvette kept at room temperature prior to electroporation generated 2.5 times more transformants than did the cuvette that was pre-chilled on ice (Fig. 2E).

Finally, we examined the optimized protocol (Supplementary information) for its efficiency and applicability to other Staphylococcal species. A total of 12 Staphylococcal species other than S. epidermidis, including eight CNS and four CPS, were tested and the results are shown in Table 1. We succeeded in generating the plasmid transformants from all tested Staphylococcal species including JMUB230 (S. lugdunensis) and JMUB1090 (S. simulans) from which we had failed to obtain transformant using the previous method, even though the transformation efficiencies were varied from 2.40 to 1.80 × 106 CFU/μg of plasmid DNA.

3.2.6. Recovery conditions The cuvettes containing the competent cell/DNA mixture were subjected to high-voltage electrical pulses of defined magnitude and length. The electroporated cells were then allowed to recover in three different media (B2, BHI and BHI with 10% sucrose) before being spread on drug plates. Results showed that the transformation efficiency was > 8 times higher when the transformants were recovered in B2 medium compared to the other two media (p < 0.05, Welch's ttest). The transformation efficiencies for B2 and BHI were 846.9 ± 45.9% and 64.3% ± 15.6%, respectively, once the value for BHI with 10% sucrose medium was set as 100% (average ± SE, n = 3). Recovery times for greatest success were also tested with B2 medium. As shown in Fig. 2F, the cells immediately spread on the agar medium without incubation for recovery generated a few colonies of transformants, and the number of colonies increased with the duration of incubation up to 7 h, however, there was no statistic difference in the number of colonies between 5- and 7-h incubation periods (p = 0.34). In addition, incubation for 24 h resulted in no transformant colony formation.

4. Discussion In this study, we developed a new universal electroporation protocol that can be applicable to many Staphylococcal species of both CPS and CNS. In order to extend its application range as much as possible and enhance the transformation efficiency, we tested and modified almost all conditions that were studied in our previous protocol (Table 2). The follows are the main items we focused on in the current study. Firstly, we tested E. coli stains originated from both K12 and B for plasmid propagation. K12 type E. coli strains are positive for dcm and methylate the cytosine of plasmid DNA and this modified plasmid can be easily cleaved in Staphylococcus cells by type IV restriction enzyme (Corvaglia et al., 2010; Xu et al., 2011; Monk and Foster, 2012). However, there were several reports succeeded in high efficient transformation of Staphylococcal cells using plasmid prepared in E. coli K12 derivative strain where the dcm was knocked out (Monk et al., 2012) or the restriction barrier was inhibited by a heat inactivation process (Löfblom et al., 2007; Cui et al., 2015). Conversely, B type E. coli strains are negative for dcm. The non-cytosine-methylated plasmid DNA from B strains can be easily accepted by Staphylococcus due to the absence of cutting by type IV enzyme. In agreement with those points, our data also clearly showed that plasmid prepared in E. coli B derivative BL21 strain had > 2 log10 higher efficiency compared to that from K12

3.2.7. Electrical conditions We previously reported that the multiple pulse wave type electroporator was superior to the single exponential pulse wave type electroporator for the transformation of S. aureus, but the voltage value and electric pulse duration largely influenced on the results (Hisatsune et al., 2016). To determine optimal conditions for high transformant efficiency, we tested different combinations of the Pp and Tp parameters, and the results are summarized in Table 3. We first performed 29

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Table 3 Transformation efficiencies and electrical conditions for the two devices used this study with JMUB20 (SE). Devices

Poring pulse (Pp)

Transfer pulse (Tp)

Efficiency (CFU/μg)

Pulse voltage

Pulse length

Pulse interval

Pulse times

Pulse direction

Pulse voltage

Pulse length

Pulse interval

Pulse times

Pulse direction

ELEPO21

1200 1200 1200 1200 1200 1200 1200 1400 1400 1400 1400 1400 1600 1600 1600 1600 1600 1800 1800 1800 2000 2000

2.5 3.5 5.0 2.5 2.5 2.5 3.5 2.5 3.5 5.0 2.5 2.5 2.5 2.5 3.5 5.0 2.5 2.5 3.5 5.0 1.5 2.5

50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50

1 1 1 2 3 1 1 1 1 1 2 1 1 1 1 1 2 1 1 1 1 1

+ + + + + ± ± + + + + ± + + + + + + + + + +

100 100 100 100 100 100 100 100 100 100 100 100 50 100 100 100 100 100 100 100 100 100

99 99 99 99 99 99 99 99 99 99 99 99 50 99 99 99 99 99 99 99 99 99

50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50

5 5 5 5 5 5 5 5 5 5 5 5 3 5 5 5 5 5 5 5 5 5

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

5.80 ± 3.19 × 10 6.67 ± 1.48 × 10 7.93 ± 0.71 × 10 1.21 ± 0.22 × 102 6.07 ± 1.49 × 10 9.07 ± 0.92 × 10 1.18 ± 0.15 × 102 2.13 ± 0.07 × 10 6.20 ± 2.71 × 10 1.52 ± 0.10 × 102 3.14 ± 1.35 × 102 2.65 ± 0.30 × 102 3.35 ± 0.31 × 102a 7.42 ± 0.14 × 102 8.03 ± 1.67 × 102 4.43 ± 2.02 × 102 5.41 ± 1.47 × 102 1.38 ± 0.09 × 103b 5.64 ± 2.39 × 102 3.47 ± 0.16 × 102 8.67 ± 1.11 × 102 4.33 ± 0.76 × 102

MicroPulser

1200 1400 1600 1800 2000

ND ND ND ND ND

ND ND ND ND ND

ND ND ND ND ND

ND ND ND ND ND

ND ND ND ND ND

ND ND ND ND ND

ND ND ND ND ND

ND ND ND ND ND

ND ND ND ND ND

2.7 ± 1.3 3.00 ± 0.02 × 10 6.11 ± 2.31 × 10 3.60 ± 0.85 × 102 4.15 ± 2.05 × 102

ND, not determined. Three independent trials were performed (n = 3). Average and SE are shown. Bold means the highest value of each device. a The most efficient electric condition in the previous study (Hisatsune et al., 2016). b It is the same result as that in Table 1.

Thirdly, we tested wash buffers. When preparing competent cells, hypotonic buffers or purified water are commonly used for washing cells, such as in our previous study (Hisatsune et al., 2016). However, in this study, hypertonic NaCl solution enhanced the transformation efficiency. This probably was due to the effect of NaCl on biofilm matrix removal. Biofilm matrix can protect bacterial cells from environmental stress, such as hypoxia, salinity, host immune system and antibiotics (Archer et al., 2011.), and the biofilm matrix surrounding cells can be removed by high concentration NaCl solution without having severe damage on the cells (Chiba et al., 2015). We adopted this method to prepare competent cells and confirmed that the removal of biofilm components enhanced the efficiency of the technique. However, this method has side effects such as abundant ions; compared with the conventional methods using hypotonic buffers and water, excessive ions often remain in competent cells when using this method. To completely remove the abundant ions, we measured the electrical resistance after wash with 10% glycerol. We adopted > 6 kΩ in the final solution as a criterion. If this criterion is met, arching during electroporation can be significantly suppressed. The other side effect is the limited application; given that this hypertonic buffer method improves efficiency by removing biofilm matrix material, it may have no effect on or be damaging to strains that make less or do not have a biofilm matrix. Fourthly, we tested temperatures for handling competent cells. We assessed two temperature conditions: on ice and room temperature. Some researchers have reported that on ice electroporation reduces arching during electroporation (Grosser and Richardson, 2016), whereas other researchers, including us, have reported that on-ice (cold) electroporation does not yield good results (Augustin and Götz, 1990; Cui et al., 2015; Hisatsune et al., 2016). The suppressions of arching and improvement of efficiency are contradictory issues. As

derivatives DH5α and Top10. Recently, we reported an alternative method to improve transformation efficiency of S. aureus by using congenitally dcm-negative E. coli B type strain for plasmid propagation (Sato'o et al., 2017). This improvement was also confirmed with other Staphylococci species tested in this study. Nevertheless, the possible restriction enzyme cleavage by other than type IV cannot be avoided with this protocol and dcm-negative E. coli mutant. Thus, heat inactivation may be useful in some strains (Supplementary information). Secondly, culture media for preparation of Staphylococcal competent cell were tested. In an early report by Schenk. et al., medium type had a big influence on the competency of Staphylococcus, and concentrations of NaCl and yeast extract were important for success of electroporation (Schenk and Laddaga, 1992). They made a significant improvement of transformation efficiency by raising NaCl concentration of medium from 0.5% (original B medium, Augustin and Götz, 1990) to 2.5% (B2 medium, Schenk and Laddaga, 1992). In this study, however, we observed a frequent occurrence of electrical arching when using competent cells prepared with B2 medium, suggesting that high NaCl concentration in medium causes frequent arching as a result of turning out high electrical energy during the electroporation. Actually, high NaCl concentration in medium may result in the increment of intracellular ion concentration, and in turn, causes ion leakage resulting in increased ion concentration in the medium during the electroporation, which leads to electrical arcing. Alternatively, another possible explanation is that high NaCl concentration may cause fragile bacteria that cannot bear high-energy electroporation. Therefore, in the current study, we tested low-NaCl B2 medium, B2low (1.0% NaCl), and confirmed that this medium had improved efficiency without causing arching, indicating that NaCl concentration is a sensitive and important factor for improving transformation efficiency and repressing arching in Staphylococcal spices. 30

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Supplementary data to this article can be found online at https:// doi.org/10.1016/j.mimet.2018.01.006.

described above, we confirmed that both (less arching and more colony formation) can be achieved with careful preparation and measurement of electrical resistance. Then, we tested electric conditions. Our previous study demonstrated that electric parameter was crucial for the transformation efficiency (Hisatsune et al., 2016). Therefore, optimization of the electric condition was a main issue in the current study. As a result of various tests, the optimum electrical condition was found out as follows: 1800 V for 2.5 ms with a 50 ms interval (one pulse in + direction) for Pp parameter, and 100 V for 99 ms with a 50 ms interval (five pulses in ± direction) for Tp parameter. Compared to the optimum electric parameters established with S. aureus in our previous study (Hisatsune et al., 2016, Table 3), Pp voltage was raised from 1600 V to 1800 V, and the voltage, pulse length and times for Tp were raised from 50 V to 100 V, 50 ms to 99 ms, 3 times to 5 times, respectively. As the results of those changes, the electric energy also rose from 0.3–1.5 J to 1.0–3.0 J (Table 2), which seemed appropriate for enhancing efficiency in other Staphylococcal species than in S. aureus. This is consistent with the previous study (Grosser and Richardson, 2016), where they demonstrated that the optimal electric voltage for S. epidermidis was 2000 V, whereas for S. aureus was 1800 V. Also, multi-pulse type electroporator enhanced the efficiency in another Staphylococcus, similar to our previous study (Hisatsune et al., 2016). Finally, incubation time for transformant recovery was tested. There are two commonly used recovery methods: short incubation (1–2 h), (Augustin and Götz, 1990; Schenk and Laddaga, 1992; Löfblom et al., 2007; Monk et al., 2012; Cui et al., 2015; Hisatsune et al., 2016; Grosser and Richardson, 2016) and long incubation (4 h) (Grosser and Richardson, 2016). In the present protocol, we used higher energy for electroporation and this high energy might have caused greater damage of bacterial cells. Thus, long (5 h) incubation to recover from the damage increased the transformation efficiency, and it is thought that there is bacteria proliferation during recovery that may partially contribute to this increasing. However, indeed too long a recovery period decreased the efficiency. This result may be due to plasmid loss or cell death because of excessive culture duration. Overall, by introducing these changes into our previous protocol, the transformation efficiency was greatly improved in the clinical isolates of S. epidermidis, S. lugdunensis and S. simulans with efficiencies of from 2.4 to 1300 CFU/μg DNA, whereas, no any transformants of those isolates had been obtained with the previous protocol (Table 1 and Fig. 1). Furthermore, even in S. aureus and S. capitis, the efficiencies were improved by approximately three times and approximately 2–6 times, respectively, compared with those of previous studies (Hisatsune et al., 2016; Cui et al., 2015). We also confirmed that this could be applied in at least 8 tested Staphylococcal species other than the aforementioned three species. To date, to our knowledge, protocols for only a few Staphylococcus species have been reported (Luchansky et al., 1988; Augustin and Götz, 1990; Brückner, 1997; Löfblom et al., 2007; Monk et al., 2012; Heilbronner et al., 2013; Cui et al., 2015; Hisatsune et al., 2016; Grosser and Richardson, 2016.), and those were not verified to be applicable to various species and clinical isolates. However, our optimized protocol can be applicable to at least 13 Staphylococcal species which are recognized as the major pathogens in humans and animals. Although fine-tuning and optimization of various parameters, such as the type of media, washing buffer, heat inactivation, DNA concentration, and cell concentration, may be occasionally necessary depending on the type of strain/species, it is considered that the present protocol can be used as a standard and first choice method.

Author contributions YS designed this project, performed experiments and wrote manuscript. YA and KK assisted experiments. SW and TS isolated and identified JMUB strains. YH advised electronic and mechanical engineering. LC managed this project and wrote manuscript. Conflict of interest and funding Yasuhiko Hayakawa is president of NEPA GENE Co., Ltd. This research is partially supported by NEPA GENE Co., Ltd., Grant-in-Aid for Scientific Research (JSPS KAKENHI Grant Number 26460536) and Grant-in-Aid for Young Scientists (B) (JSPS KAKENHI Grant Number 17K15691). Acknowledgments We thank Dr. Keiichi Hiramatsu, Dr. Takashi Sasaki (Juntendo University) and Dr. Yasunori Suzuki (Tokyo Metropolitan Institute of Public Health) for kindly providing us with bacterial strains, and Dr. Motoyuki Sugai and Dr. Fuminori Kato (Hirosima University) for kindly providing us with a plasmid vector. References Archer, N.K., Mazaitis, M.J., Costerton, J.W., Leid, J.G., Powers, M.E., Shirtliff, M.E., 2011. Staphylococcus aureus biofilms: properties, regulation, and roles in human disease. Virulence 2, 445–459. Augustin, J., Götz, F., 1990. Transformation of Staphylococcus epidermidis and other staphylococcal species with plasmid DNA by electroporation. FEMS Microbiol. Lett. 54, 203–207. Bannoehr, J., Guardabassi, L., 2012. Staphylococcus pseudintermedius in the dog: taxonomy, diagnostics, ecology, epidemiology and pathogenicity. Vet. Dermatol. 23, 253–266. Becker, K., Heilmann, C., Peters, G., 2014. Coagulase-negative staphylococci. Clin. Microbiol. Rev. 27, 870–926. Brückner, R., 1997. Gene replacement in Staphylococcus carnosus and Staphylococcus xylosus. FEMS Microbiol. Lett. 151, 1–8. Chiba, A., Sugimoto, S., Sato, F., Hori, S., Mizunoe, Y., 2015. A refined technique for extraction of extracellular matrices from bacterial biofilms and its applicability. Microb. Biotechnol. 8, 392–403. Cook, L.C., Federle, M.J., 2014. Peptide pheromone signaling in Streptococcus and Enterococcus. FEMS Microbiol. Rev. 38, 473–492. Corvaglia, A., Francois, P., Hernandez, D., Perron, K., Linder, P., Schrenzel, J., 2010. A type III-like restriction endonuclease functions as a major barrier to horizontal gene transfer in clinical Staphylococcus aureus strains. Proc. Natl. Acad. Sci. U. S. A. 107, 11954–11958. Cui, B., Smooker, P.M., Rouch, D.A., Daley, A.J., Deighton, M.A., 2013. Differences between two clinical Staphylococcus capitis subspecies as revealed by biofilm, antibiotic resistance, and pulsed-field gel electrophoresis profiling. J. Clin. Microbiol. 51, 9–14. Cui, B., Smooker, P.M., Rouch, D.A., Deighton, M.A., 2015. Enhancing DNA electrotransformation efficiency on a clinical Staphylococcus capitis isolate. J. Microbiol. Methods 109, 25–30. Fitzgerald, J.R., 2012. Livestock-associated Staphylococcus aureus: origin, evolution and public health threat. Trends Microbiol. 20, 192–198. Foster, T., 1996. Staphylococcus. In: Baron, S. (Ed.), Medical Microbiology, 4th edition. University of Texas Medical Branch at Galveston, Galveston (TX), pp. 1996 (Chapter 12). Foster, A.P., 2012. Staphylococcal skin disease in livestock. Vet. Dermatol. 23, 342–351. Grosser, M.R., Richardson, A.R., 2016. Method for preparation and electroporation of S. aureus and S. epidermidis. Methods Mol. Biol. 1373, 51–57. Heilbronner, S., Hanses, F., Monk, I.R., Speziale, P., Foster, T.J., 2013. Sortase A promotes virulence in experimental Staphylococcus lugdunensis endocarditis. Microbiology 159, 2141–2152. Hisatsune, J., Sato'o, Y., Yu, L., Kutsuno, S., Hayakawa, Y., Sugai, M., 2016. Efficient transformation of Staphylococcus aureus using multi-pulse electroporation. J. Microbiol. Methods 130, 69–72. Huddleston, J.R., 2014. Horizontal gene transfer in the human gastrointestinal tract: potential spread of antibiotic resistance genes. Infect. Drug. Resist. 7, 167–176. Kato, F., Sugai, M., 2011. A simple method of markerless gene deletion in Staphylococcus aureus. J. Microbiol. Methods 87, 76–81. Lane, D.J., 1991. 16S/23S rRNA sequencing. In: Stackebrandt, E., Goodfellow, M. (Eds.), Nucleic Acid Techniques in Bacterial Systematics. John Wiley and Sons, New York, NY, pp. 115–175.

5. Updated protocol The current protocol is posted at our following website: http:// www.jichi.ac.jp/bacteriology/protocol/. We will keep on updating the latest protocol to our website, if any alterations for improved efficiency are added. 31

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