MIMET-04627; No of Pages 4

Journal of Microbiological Methods xxx (2015) xxx–xxx

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Journal of Microbiological Methods

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1 Note

2Q1 High-throughput screening assays for antibacterial and antifungal

3Q2 activities of Lactobacillus species

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4Q3 Raffael C. Inglin, Marc J.A. Stevens, Lukas Meile, Christophe Lacroix, Leo Meile ⁎

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5 Laboratory of Food Biotechnology, Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 7, 8092 Zurich, Switzerland

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6 a r t i c l e i n f o a b s t r a c t

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Article history: We describe high-throughput screening techniques to rapidly detect either antimicrobial activity, using an agar- 18
8 Received 27 April 2015 well diffusion assay in microtiter plates, or antifungal activity using an agar-spot assay in 24-well plates. 504 19
9 Received in revised form 28 April 2015 Lactobacillus isolates were screened with minimal laboratory equipment and screening rates of 2000–5000 indi- 20
10

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Accepted 28 April 2015
vidual antimicrobial interactions. 21
11 Available online xxxx
22 © 2015 Published by Elsevier B.V.
12 Keywords:
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14
Antibacterial
Antifungal
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15 Bacteriocin
16
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High-throughput screening
17 Lactobacillus
26
24
23
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27 Fermented food products are part of the daily diet and of high eco- isolates is increased (Maragkoudakis et al., 2009). Recently, an ESI–LC/ 53
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28 nomic importance. The spoilage of such products by microorganisms MS based high-throughput assay to detect novel bacteriocins was de- 54
29 is a major problem in industry (Ross et al., 2002). Lactic acid bacteria veloped but this method requires expensive equipment available in 55
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30 (LAB) like lactobacilli can be isolated from many fermented food prod- only few laboratories (Perez et al., 2014). 56
31 ucts and selected strains exhibit an antibacterial or antifungal activity Here we present a rapid and easy-to-handle screening assay 57
32 (Barbosa et al., 2014; Gaggia et al., 2011; Jiménez et al., 2013). Therefore, for antimicrobial strains. We integrated an agar-well diffusion assay 58
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33 lactobacilli are applied in starter and in protective cultures for (Grinstead and Barefoot, 1992) and an agar-spot assay (Miescher 59
34 fermented products (Delavenne et al., 2013). LAB inhibit growth of Schwenninger and Meile, 2004) into a high-throughput screening 60
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35 spoilage organism via general mechanism such as organic acid produc- assay (HSA). A standardized methodology in multi-well plates using 61
36 tion and environmental pH decrease. In addition, strain-specific inhibi- common laboratory equipment enables rapid screening for antimicrobi- 62
37 tion occurs via production of for example ribosomally encoded al Lactobacillus strains. 63
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38 bacteriocins or low-molecular weight compounds (Castellano et al., Lactobacillus strains (Table 1) were incubated anaerobically in MRS 64
39 2010; Nes et al., 2007). Bacteriocin-related inhibition is associated broth (BioLife, Switzerland) at 30 °C using the oxygen scavenging sys- 65
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40 with blocking of the cell wall biosynthesis or with formation of pores tem AnaeroGen (Thermo Scientific, Switzerland). Growth conditions 66
41 in the cell membrane resulting in leakage of the cell (O'Sullivan et al., of the indicator strains used in this study are listed in Table 2. Lactobacil- 67
42 2002). Low-molecular weight compounds, mainly acids, inhibit via var- lus supernatant for antibacterial assays (HSA-B) was obtained from out- 68
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43 ious mechanisms including pH reduction and inhibition of metabolic re- grown cultures in 1.8-ml 96-deep-well plates (Life Systems Design, 69
44 actions (Batish et al., 1997; Crowley et al., 2013). The application of Switzerland) inoculated from cryo-stocks using a replicator pin. The 70
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45 protective cultures containing natural occurring strains with antifungal plate was centrifuged (6000 ×g, 15 min) and aliquots of the superna- 71
46 or antibacterial activity is a growing trend in food preservation. Strains tant were transferred to a 96-well PCR plate and pasteurized at 75 °C 72
47 with antimicrobial or antifungal activity can be detected by testing for 3 min. The indicator strain media (Table 2) were supplemented 73
48 spoilage-free food isolates in traditional antimicrobial assays. However, with 0.5% agar and 0.1 M K2HPO4. After sterilization, the media were 74
49 screening an antimicrobial activity with such assays, often neglecting tempered to 50 °C and inoculated with 0.5% of an overnight-culture of 75
50 pH effects, is cumbersome (Bao et al., 2010; Zhu et al., 2014). Moreover, the indicator strain. 50 μl of the inoculated agar was transferred to a 76
51 if individual pH adjustment and microfiltration of supernatant are in- 200-μl clear-glass-flat-bottom 96-well microtiter plate (Sigma) using a 77
52 cluded in such assays, labor increases rapidly when the number of multichannel pipette and air-dried for 30 min. 30 μl Lactobacillus super- 78
natant was transferred to each well and air-dried for 15 min. The optical 79
⁎ Corresponding author. density at 600 nm (OD600) was measured (time = t0) using a plate 80
E-mail address: leo.meile@hest.ethz.ch (L. Meile). reader (PowerWave XS) and the plate was subsequently incubated at 81

http://dx.doi.org/10.1016/j.mimet.2015.04.011
0167-7012/© 2015 Published by Elsevier B.V.

Please cite this article as: Inglin, R.C., et al., High-throughput screening assays for antibacterial and antifungal activities of Lactobacillus species, J.
Microbiol. Methods (2015), http://dx.doi.org/10.1016/j.mimet.2015.04.011
 2 R.C. Inglin et al. / Journal of Microbiological Methods xxx (2015) xxx–xxx

t1:1 Table 1
t1:2 Lactobacillus strains used for the antimicrobial assay (HSA).

t1:3 Identification code Species Number of isolatesa Antimicrobial activityb Antifungal activityc

t1:4 ace L. acetotolerans 1 0 0

t1:5 aci L. acidipiscis 1 0 0
t1:6 aco L. acidophilus 7 0 4
t1:7 ani L. animalis 1 0 0
t1:8 bre L. brevis 12 2 5
t1:9 buc L. buchneri 6 0 1
t1:10 cas L. casei 23 2 7
t1:11 cet L. ceti 1 0 0
t1:12 cor L. coryniformis 1 0 1
t1:13 cris L. crispatus 1 1 1
t1:14 cru L. crustorum 1 0 1
t1:15 cur L. curvatus 26 2 6
t1:16 del L. delbrueckii 49 11 11

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t1:17 fab L. fabifermentans 1 0 0
t1:18 far L. farciminis 1 0 0

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t1:19 fer L. fermentum 58 6 15
t1:20 dru L. fructivorans 4 0 1
t1:21 gas L. gasseri 1 0 0
t1:22 har L. harbinensis 1 0 0

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t1:23 hel L. helveticus 12 2 3
t1:24 hom L. hominis 1 0 0
t1:25 joh L. johnsonii 2 0 1

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t1:26 lac L. lactis 1 0 0
t1:27 lin L. lindneri 3 1 2
t1:28 mal L. mali 1 0 0

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t1:29 mur L. murinus 1 1 0
t1:30 ota L. otakiensis 1 0 0
t1:31 prc L. paracasei 18 3 3
t1:32 prp L. paraplantarum 8 D 0 2
t1:33 pen L. pentosus 3 2 1
t1:34 pla L. plantarum 67 9 31
t1:35 pon L. pontis 1 0 0
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t1:36 reu L. reuteri 6 1 0
t1:37 rha L. rhamnosus 21 2 6
t1:38 sak L. sakei 25 4 10
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t1:39 skc L. sakei-curvatusd 35 2 3

t1:40 sal L. salivarius 1 0 1
t1:41 san L. sanfranciscensis 1 0 1
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t1:42 ult L. ultunensis 1 0 0

t1:43 zea L. zeae 1 0 0
t1:44 spp. L. spp.e 98 14 37
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t1:45 Total 504 65 154

a
t1:46 From each species.
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b
t1:47 With antibacterial activity.
c
t1:48 With antifungal activity.
d
t1:49 The species L. sakei-curvatus contains isolates that belong to either one of these species.
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e
t1:50 Species L. spp. contains all Lactobacillus with no further species classification.
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82 conditions preferable for the indicator strain. The OD600 was deter- plate and overlaid with indicator strain inoculated soft-agar (Miescher 104
83 mined after 24 h and 48 h and growth in wells with OD values below Schwenninger and Meile, 2004). 105
84 a threshold of 1.5 × OD600 at t0 was classified as inhibited (Fig. 1a). We screened 504 Lactobacillus strains from 39 different species 106
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85 The antifungal screening assay (HSA-F) was performed in a 24-well mainly isolated from food-products for their activity against potential 107
86 cell-culture plate (Sigma) containing 300 μl of 1.5% MRS agar and 0.1 M spoilage organisms (Table 2). The pH in inhibition zones was checked 108
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87 K2HPO4. Lactobacillus strains were spotted at the center of a well with and ranged between 6.5–6.8, showing that low-pH inhibitory effect 109
88 0.75 μl of an outgrown culture and incubated for 48 h. Thereafter can be excluded. 110
89 wells were overlaid with 100 μl of 0.5% YM soft-agar supplemented The HSA-B revealed 65 strains active against all Enterococcus (n = 111
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90 with 0.1 M K2HPO4 and inoculated either with 103–104 fungal spores/ 14) and Listeria species (n = 13). Among these 65 there was 112
91 ml or with 1% of a yeast overnight-culture. The plates were incubated Lactobacillus plantarum 12 BH, previously described to inhibit these spe- 113
Q5
92 for 24−48 h at conditions preferable for the indicator strain. The inhibi- cies and used as positive control (Wullschleger, 2009). The validation of 114
93 tion areas were visually recorded daily (Fig. 1b). Both assays were per- these 65 strains using an agar-well diffusion assay (Grinstead and 115
94 formed in a sterile bench to avoid contamination when handling a high Barefoot, 1992) revealed no false-positive and false-negative strains. 116
95 amount of plates simultaneously. Initially, all experiments were per- Further, 24 of the 65 inhibiting strains exhibited a protease-sensitive ac- 117
96 formed as triplicates. Due to high reproducibility the following experi- tivity. No qualitative differences were detected between broth- and 118
97 ments were performed as single screening and only the positives were soft-agar approaches. However, the HSA-B soft-agar assay was easier 119
98 then confirmed by an agar-well diffusion assay. Antimicrobial activity to handle, and less water condensed in the wells. 120
99 was tested in an agar-well diffusion assay with and without protease Aspergillus tamarii, Kluyveromyces marxianus, Candida krusei and 121
100 XIV (Sigma, Switzerland) digestion to assess proteinaceous characteris- Rhodotorula mucilaginosa strains were tested in the HSA-F. The HSA-F 122
101 tics of inhibitory compounds. The HSA-B was also performed in broth revealed 154 strains, including the antifungal strain L. plantarum DSM 123
102 instead of soft agar for comparison. The HSA-F was compared to the 20205 as positive control (Miescher, 1999), that inhibited one of the 124
103 modified agar-spot assay where colonies were poured onto an agar four tested indicator strains with R. mucilaginosa being most frequently 125

Please cite this article as: Inglin, R.C., et al., High-throughput screening assays for antibacterial and antifungal activities of Lactobacillus species, J.
Microbiol. Methods (2015), http://dx.doi.org/10.1016/j.mimet.2015.04.011
 R.C. Inglin et al. / Journal of Microbiological Methods xxx (2015) xxx–xxx 3

t2:1 Table 2
t2:2 Indicator strains, their culture conditions and their inhibition by lactobacilli.

t2:3 Indicator strains Cultivation conditionsa Inhibitory lactobacilli

t2:4 Genus Species Strain Identification codeb

t2:5 Enterococcus avium DSM 20679T BHI/37 °C/aerobic bre, cas, del, prc, pen, pla, sak, skc, spp
t2:6 casseliflavus DSM 20680T cas, cur, del, fer, hel, pla, rha, skc, spp
t2:7 cecorum DSM 20682T prc, pen, sak, spp
t2:8 durans DSM 20633T bre, cas, del, prc, pla, rha, sak, spp
t2:9 faecalis DSM 20478Tc cas, del, fer, prc, pla, spp
t2:10 DSM 2570 cas, del, mur, prc, pla, sak, spp
t2:11 DSM 2981 cas, del, mur, prc, pla, sak, spp
t2:12 faecium DSM 20477Tc cas, del, fer, prc, pla, spp
t2:13 SL1.1 del, mur, prc, pla, spp
t2:14 SL.10.9 del, mur
t2:15 gallinarum DSM 20628T cas, cur, del, fer, hel, prc, pen, pla, sak, spp

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t2:16 hirae DSM 20160T bre, del, mur, prc, pla, skc, spp
t2:17 saccharolyticus DSM 20726T bre, cas, cri, cur, fer, hel, lin, pla, rha, sak, skc, spp
t2:18 sulfureus DSM 6905T bre, cas, cur, del, hel, prc, pen, pla, reu, spp

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t2:19 Escherichia coli DH5α BHI/37 °C/aerobic No inhibition
t2:20 K12 No inhibition
t2:21 Lactobacillus curvatus RI-504 del, mur, prc, pla, sak, skc, spp

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t2:22 Lactococcus lactis MG 1363 MRS/30 °C/anaerobic No inhibition
t2:23 Leuconostoc mesenteroides Y105 BHI/25 °C/aerobic No inhibition
t2:24 Listeria innocua HPB13c BHI/37 °C/aerobic prc, pla, spp

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t2:25 DSM 20649T mur, pla, spp
t2:26 L17 pla
t2:27 L19 cas, del, mur, prc, pla, spp
HPB28c

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t2:28 ivanovii cas, del, fer, hel, prc, pen, pla, sak, skc, spp
t2:29 DSM 20750T cas, del, hel, mur, prc, pen, pla, sak, skc, spp
t2:30 DSM 12491T del, hel, mur, prc, pen, pla, sak, skc, spp
t2:31 monocytogenes ATCC 19114 del, hel, mur, prc, pla, sak, spp
t2:32
t2:33
10403S
H90 2008
D del, mur, prc, pla, sak, spp
del, mur, prc, pla, sak, spp
t2:34 F95 2008 del, mur, prc, pla, sak, spp
t2:35
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Lm15 del, mur, prc, pla, sak, spp
t2:36 Staphylococcus aureus DSM1104 BHI/37 °C/aerobic No inhibition
t2:37 463 No inhibition
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t2:38 DSM 2569 No inhibition

t2:39 Streptococcus mutans DSM 20523T BHI/37 °C/aerobic No inhibition
t2:40 salivarius OMZ513 TSY/37 °C/aerobic No inhibition
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t2:41 ATCC 9759 No inhibition

t2:42 thermophilus DSM 20617T BHI/44 °C/aerobic No inhibition
t2:43 vestibularis CCUG 24686 BHI/37 °C/aerobic No inhibition
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t2:44 DSM 5636T No inhibition

t2:45 Aspergillus tamarii S078c YM/25 °C/aerobic No inhibition
t2:46 Candida krusei 3-69/2c YM/25 °C/aerobic fer, rha
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t2:47 Kluyveromyces marxianus LMEc YM/25 °C/aerobic fer, rha

t2:48 Rhodotorula mucilaginosa LMEc YM/25 °C/aerobic aci, bre, buc, cas, har, cri, cru, cur, del, fer, fru, hel,
joh, lin, prc, prp, pen, pla, rha, sak, skc, sal, san, spp
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a
t2:49 BHI broth (Labo-Life Sàrl, Switzerland); TSY (Jans et al., 2012); YM broth (Becton Dickinson AG, Switzerland).
b
t2:50 Identification code from Table 1 that indicates that isolates from Lactobacillus species were able to inhibit the indicator strain.
c
t2:51
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Indicator strains that were tested as triplicates.

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Fig. 1. Demonstration of the antibacterial (HSA-B) (a) and antifungal (HSA-F) (b) high-throughput screening assays. The HSA-B (a) with 92 Lactobacillus strains and 4 negative control
samples against Lactobacillus curvatus RI-504, a commercial salami starter culture, as an indicator strain shows clear inhibition in wells B5, B6, B8–B12, C1, C2, C5, D3, D5, D6, D8, D11,
E3, E7 and F2; 4 negative control samples for intact growth of RI-504 are gained from supernatants from MRS incubated without lactobacilli in wells A1, A12, H1 and H12. The HSA-F
(b) from 22 Lactobacillus stains against Rhodotorula mucilaginosa LME as an indicator strain. Complete inhibition is detected in well A1. Medium inhibition is detected in the wells B1,
B6, C2, D2 and D3. Weak inhibition is detected in wells B1, C1 and D1; negative controls with no lactobacilli colonies in the wells A5 and A6 are completely grown.

Please cite this article as: Inglin, R.C., et al., High-throughput screening assays for antibacterial and antifungal activities of Lactobacillus species, J.
Microbiol. Methods (2015), http://dx.doi.org/10.1016/j.mimet.2015.04.011
 4 R.C. Inglin et al. / Journal of Microbiological Methods xxx (2015) xxx–xxx

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Please cite this article as: Inglin, R.C., et al., High-throughput screening assays for antibacterial and antifungal activities of Lactobacillus species, J.
Microbiol. Methods (2015), http://dx.doi.org/10.1016/j.mimet.2015.04.011