Journal of Microbiological Methods 106 (2014) 101–103
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Note
An improved method for rapid generation and screening of Bacillus thuringiensis phage-resistant mutants Annika Gillis ⁎, Jacques Mahillon Laboratory of Food and Environmental Microbiology, Université catholique de Louvain, Croix du Sud, Louvain-la-Neuve, Belgium
a r t i c l e
i n f o
Article history: Received 28 July 2014 Received in revised form 21 August 2014 Accepted 21 August 2014 Available online 29 August 2014
a b s t r a c t A simple method to isolate, screen and select phage-resistant mutants of Bacillus thuringiensis was developed. The traditional double-layer agar method was improved by a combination of the spotting assay using a lytic phage, to generate the bacterial-resistant mutants, with an inverted spotting assay (ISA), to rapidly screen the candidateresistant mutants. © 2014 Elsevier B.V. All rights reserved.
Keywords: Bacillus thuringiensis Phage Phage-resistant mutants Tectivirus
Bacillus thuringiensis is an insecticidal bacterium that has been used worldwide as an ecofriendly biopesticide over the last 70 years. The bioinsecticide formulations of B. thuringiensis are traditionally produced by submerged fermentations, but solid-state fermentations can also be applied (Devi et al., 2005). These fermentations are exposed to contamination by bacteriophages (phages) that can slow or completely stop B. thuringiensis fermentations, decreasing the production and resulting in significant economic losses. Recently, it has been suggested that between 15 and 30% (sometimes up to 50–80%) of the total fermentation batches of B. thuringiensis, can be damaged by phages, particularly by the virulent ones (Liao et al., 2008). Nevertheless, bacteria and phages are involved in continuous cycles of co-evolution, in which emerging phage-resistant bacteria help to preserve the host lineages, whereas phages might continue to overcome these resistant bacteria (Labrie et al., 2010). Thus, the study of phage-resistant bacteria and their remarkable array of resistance mechanisms can provide important clues to tackle the problem of phage(s) contamination in B. thuringiensis fermentations. Nowadays, many phages are known to be associated with B. thuringiensis, either as virulent or temperate phages (Gillis and Mahillon, 2014a). However, phage-resistance in B. thuringiensis and the mechanisms involved are still poorly understood. Recently it was shown that two of the most important B. thuringiensis bioinsecticide serovars (i.e., kurstaki and israelensis) are infected by tectiviruses GIL01- and GIL16-like, respectively (Gillis and Mahillon, 2014b). Sporadically some resistant phenotypes and distinct adaptation ⁎ Corresponding author at: Laboratory of Food and Environmental Microbiology, Université catholique de Louvain, Croix du Sud 2, Box L7.05.12, B-1348 Louvain-la-Neuve, Belgium. Tel.: +32 10 478598; fax: +32 10 473440. E-mail address: [email protected] (A. Gillis).
http://dx.doi.org/10.1016/j.mimet.2014.08.012 0167-7012/© 2014 Elsevier B.V. All rights reserved.
features can be observed in the B. thuringiensis strains in response to tectiviruses (Gillis and Mahillon, unpubl. results). Therefore, in a first endeavor to study tectiviral-resistance in B. thuringiensis, generation of a collection of phage-resistant mutants is necessary. Many of the methods described to isolate spontaneous phage-resistant mutants are derived from the classical plaque assay with double-layer agar (DLA) (Adams, 1959; Gratia, 1936). This method basically consists in mixing appropriate dilutions of both bacteria and phage in molten agar or agarose (the top overlay), which in turn, is evenly distributed on top of a solidified standard medium (the bottom underlay) (Kropinski et al., 2009). Using the standard DLA-system, in combination with high titers of a lytic phage that will kill almost all the bacterial population, it is possible to recover single colonies that further tests will confirm if they are phage-resistant. However, we found that this procedure is time consuming and not suitable for large screenings, since it relies on the interplay between the titers of phage and bacteria used to isolate the mutants. Moreover, the majority of the bacterial colonies were found not to be fully phage-resistant in complementary tests. In this study, an improved DLA-based method to rapidly generate and screen phageresistant mutants of B. thuringiensis is reported. B. thuringiensis strain HER1410 and a lytic variant of tectivirus GIL01, referred to herein as GIL01cp25, were used to develop the method. Strain HER1410 is a well-known host for phage GIL01 (Gillis and Mahillon, 2014b). GIL01cp25 has been previously described as producing clear plaques (unable to establish the lysogenic cycle) in susceptible hosts (Fornelos et al., 2011). The traditionally DLA-method was improved by a spotting assay as follows: strain HER1410 was grown in 10 ml LB (Lysogeny Broth) medium (5 g/l NaCl, 5 g/l yeast extract and 10 g/l tryptone) at 30 °C and 120 rpm to an optical density at 595 nm (OD595) of ~0.5. Then, 200 μl of the bacterial culture was added to 10 ml molten
102
A. Gillis, J. Mahillon / Journal of Microbiological Methods 106 (2014) 101–103
LB-top agar (0.7% (w/v)), gently mixed and placed onto LB-agar (1.5% (w/v)) square Petri plates (10 × 10 cm). After agar solidification and plate drying at room temperature (~ 20 min), 15 μl drops of different sterile-filtered GIL01cp25 stocks with high titers (10 7–108 PFU/ml), were added on top of the bacterial lawn, allowed to dry for 20 min, and incubated overnight at 30 °C. To guarantee the emergence of distinct spontaneous bacterial-resistant mutants, one independent HER1410 culture per plate was subjected to infection by drops of different GIL01cp25 stocks. After incubation, spontaneous bacterial colonies emerged, and depending on the bacterial culture tested and the phage stock used, the frequencies were different (Fig. 1A–D). Since 15 μl drops of different phage stocks (plus a negative control drop of LB medium) can be easily arranged in a 10 × 10 cm square Petri plate, the isolation of 80 candidate phage-resistant mutants of strain HER1410 was achieved by using 6 square plate's assays. Only one candidate colony per drop, if any, was isolated, streaked on fresh LB-agar plates and subjected to a double-round of single-colony isolation. Depending on the number of phage-resistant mutants required, the drop volume can be reduced down to 5 μl to accommodate a larger number of phage drops per plate, but a reduction in the emergence of phage-resistant colonies was observed. Therefore, the use of 15 μl as a minimal drop volume is strongly recommended. The second part consists in the screening of the candidate phageresistant mutants. For this purpose, the traditional DLA-method was further modified as follows and named ISA (for inverse spotting assay): 500 μl of a sterile-filtered GIL01cp25 stock (107–108 PFU/ml) was placed in 10 ml molten LB-top agar, gently mixed and distributed onto LB-agar square plates. After agar solidification and drying, plates containing the “phage lawn” were inoculated with the candidate bacterial mutants. For this, the purified single colonies were picked with a sterile-toothpick and inoculated on top of the solidified agar containing GIL01 cp25. Each square plate was inoculated with up to 24 colonies, plus strain HER1410 as control. After 24 h incubation at 30 °C, candidate resistant-colonies were evaluated (Fig. 1E). Based on the colony phenotypes, it was possible to discriminate between
Spong-test
A
C
fully-resistant mutants and others with some levels of resistance to GIL01cp25 (medium and low, evidenced by the presence of lysis/clearing zones in the colony). The non-resistant colonies were also easily identified (Fig. 1E). Consequently, 40 fully-resistant mutants were selected by this assay and retained for subsequent studies associated with phageresistance in B. thuringiensis. To confirm the bona fide phage-resistance of the 40 selected mutants, rather than an immunity stage due to the presence of the phage, bacterial mutants were screened by PCR using previously described sets of primers targeting different regions of phage GIL01 (Gillis and Mahillon, 2014b; Jalasvuori et al., 2013). No amplification bands were observed for the 40 fully-resistant mutants (data not shown). DNA from phage GIL01cp25 was used as positive control. Also, to confirm that the fully-resistant mutants are derivatives of the parental strain HER1410, random amplified polymorphic DNA (RAPD)-PCR was performed as described by Hu et al. (2004). The RAPD fingerprinting of the fully-resistant mutants displayed similar band profiles than those observed for the parental strain HER1410 (data not shown). To verify the correlation between the resistant colony phenotypes (no lysis/clearing when growing on phage-containing media) and phage resistance, the selected bacterial mutants were exposed to GIL01cp25 in a traditional plaque forming assay as described elsewhere (Gillis and Mahillon, 2014b). No PFUs were observed after incubation (up to 48 h at 30 °C; data not shown). These results confirmed the resistance of the isolated bacteria and showed a direct correlation between the observed colony phenotypes and phage resistance. The same ISA was also used to evaluate the specificity of the bacterial resistance. Another lytic phage, named Vp4, was isolated for this purpose from environmental samples throughout an enrichment process with B. thuringiensis strain GBJ002 as described elsewhere (Van Twest and Kropinski, 2009). Strain GBJ002 is also a known host for phage GIL01 (Gillis and Mahillon, 2014b). ISA-square plates containing a top overlay with either GIL01cp25 or Vp4 phages were inoculated as previously described and incubated 24 h at 30 °C. Fig. 2 depicts the results obtained. The selected mutants were fully-resistant to GIL01cp25, but
Selecon and isolaon of emerging colonies B
Bacterial phage-resistance evaluaon by ISA E
1
2
3
4
5
6
7
8b
9
10
11
12
13
14
c-
15
16
17c
18
19
20b
21a
23
24
D
22
Fig. 1. Isolation and evaluation of phage-resistant mutants of B. thuringiensis HER1410. The flowchart for the whole procedure is shown on top. (A–D) Spotting tests displaying bacterial phage-resistant colonies in lysis zones generated by drops of lytic phage GIL01cp25 after overnight incubation at 30 °C. To illustrate the importance of using independent bacterial cultures and different phage stocks, four different drops are shown. (A, B) Same culture batch of HER1410, but different phage stocks gave rise to few resistant colonies in (A) but none in (B). (C, D) Independent cultures of HER1410 and the same GIL01cp25 stock produced the emergence of resistant colonies at different frequencies. (E) Inverted spotting assay (ISA). Twenty-four isolated colonies and control strain HER1410 (negative control, c-) were grown on top of LB-agar containing phage GIL01cp25. After 24 h incubation at 30 °C, the colony phenotypes showed different levels of resistance to GIL01cp25. Numbers alone indicate fully-resistant colonies, whereas lower-case letters (a) and (b) denote medium and low levels of resistance, respectively (evidenced by lysis/clearing zones in the colony). (c) Highlights a very susceptible mutant since it did not grow in the presence of the lytic phage. Similar observations were made for 80 candidate-resistant mutants, leading to the selection of 40 of those that displayed fully-phage-resistant phenotypes.
A. Gillis, J. Mahillon / Journal of Microbiological Methods 106 (2014) 101–103
103
Phages GIL01cp25
Vp4
R12
R11
R18
R06
R40
R12
R11
R18
R06
R40
R25
R10
R22
R37
R19
R25
R10
R22
R37
R19
R09
R15
R20
R24
R31
R09
R15
R20
R24
R31
R02
R26
R04
R35
R28
R02
R26
R04
R35
R28
R33
HER1410
GBJ002
R05
R39
R33
HER1410
GBJ002
R05
R39
Fig. 2. Resistance-specificity evaluation by ISA of B. thuringiensis HER1410 phage-resistant mutants. Twenty-three isolated phage-resistant mutants (R#) were inoculated on top of LB-agar containing lytic phages GIL01cp25 and Vp4. Phage resistance/susceptibility was evaluated after incubation for 24 h at 30 °C. All the B. thuringiensis mutants tested were fully-resistant to phage GIL01cp25 (left plate), but susceptible to phage Vp4 as it is evidenced by lysis of the bacterial colonies (right plate). HER1410 and GBJ002 are B. thuringiensis strains susceptible to both phages (see text for more details) and were used as controls.
were completely susceptible to Vp4, indicating that the displayed resistance is specific to tectivirus GIL01cp25. The selected mutants were also exposed to the wild type (temperate) phage GIL01 by means of a traditional DLA-assay (Gillis and Mahillon, 2014b). No PFUs were observed after incubation (up to 48 h at 30 °C; data not shown). From the bacterial lawns, bacterial were recovered, streaked onto LB-agar plates and screened again by PCR. No positive amplification bands were obtained (data not shown), indicating that the selected bacteria are also resistant to the wild type phage GIL01. In conclusion, this work showed that the combination of a spotting assay with the ISA is a simple, efficient and practical approach for isolating, screening and selecting bacterial mutants that are resistant to lytic phages. Furthermore, the fact that the mutants isolated in this work were also resistant to the temperate phage GIL01 indicates that this method can be used to study resistance to temperate phages, provided that a lytic variant of the phage of interest is available to initially generate the bacterial mutants. Ultimately, this method can be applicable to generate phage-resistant mutants of B. thuringiensis closely-related species, such as Bacillus anthracis or Bacillus cereus, and with minor variations, to most bacterial species that can grow in semi-solid media. Acknowledgments This work was supported by the Foundation for Training in Industrial and Agricultural Research (FRIA), grant to A.G., the National Fund for Scientific Research (FNRS), the Université catholique de Louvain
and the Research Department of the Communauté Française de Belgique (Concerted Research Action) (grants to J.M.). References Adams, M.H., 1959. Bacteriophages. Interscience Publishers, New York, pp. 1–592. Devi, P.S.V., Ravinder, T., Jaidev, C., 2005. Cost-effective production of Bacillus thuringiensis by solid-state fermentation. J. Invertebr. Pathol. 88, 163–168. Fornelos, N., Bamford, J.K.H., Mahillon, J., 2011. Phage-borne factors and host LexA regulate the lytic switch in phage GIL01. J. Bacteriol. 193, 6008–6019. Gillis, A., Mahillon, J., 2014a. Phages preying on Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis: past, present and future. Viruses 6, 2623–2672. Gillis, A., Mahillon, J., 2014b. Prevalence, genetic diversity and host range of tectiviruses among members of the Bacillus cereus group. Appl. Environ. Microbiol. 80, 4138–4152. Gratia, A., 1936. Des relations numeriques entre bactéries lysogenes et particules de bactériophage. Ann. Inst. Pasteur 57, 652–676. Hu, X., Hansen, B.M., Eilenberg, J., Hendriksen, N.B., Smidt, L., Yuan, Z., Jensen, G.B., 2004. Conjugative transfer, stability and expression of a plasmid encoding a cry1Ac gene in Bacillus cereus group strains. FEMS Microbiol. Lett. 231, 45–52. Jalasvuori, M., Palmu, S., Gillis, A., Kokko, H., Mahillon, J., Bamford, J.K.H., Fornelos, N., 2013. Identification of five novel tectiviruses in Bacillus strains: analysis of a highly variable region generating genetic diversity. Res. Microbiol. 164, 118–126. Kropinski, A., Mazzocco, A., Waddell, T., Lingohr, E., Johnson, R., 2009. Enumeration of bacteriophages by double agar overlay plaque assay. In: Clokie, M.J., Kropinski, A. (Eds.), Bacteriophages. vol. 501. Humana Press, pp. 69–76. Labrie, S.J., Samson, J.E., Moineau, S., 2010. Bacteriophage resistance mechanisms. Nat. Rev. Microbiol. 8, 317–327. Liao, W., Song, S., Sun, F., Jia, Y., Zeng, W., Pang, Y., 2008. Isolation, characterization and genome sequencing of phage MZTP02 from Bacillus thuringiensis MZ1. Arch. Virol. 153, 1855–1865. Van Twest, R., Kropinski, A., 2009. Bacteriophage enrichment from water and soil. In: Clokie, M.J., Kropinski, A. (Eds.), Bacteriophages. vol. 501. Humana Press, pp. 15–21.
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Note
An improved method for rapid generation and screening of Bacillus thuringiensis phage-resistant mutants Annika Gillis ⁎, Jacques Mahillon Laboratory of Food and Environmental Microbiology, Université catholique de Louvain, Croix du Sud, Louvain-la-Neuve, Belgium
a r t i c l e
i n f o
Article history: Received 28 July 2014 Received in revised form 21 August 2014 Accepted 21 August 2014 Available online 29 August 2014
a b s t r a c t A simple method to isolate, screen and select phage-resistant mutants of Bacillus thuringiensis was developed. The traditional double-layer agar method was improved by a combination of the spotting assay using a lytic phage, to generate the bacterial-resistant mutants, with an inverted spotting assay (ISA), to rapidly screen the candidateresistant mutants. © 2014 Elsevier B.V. All rights reserved.
Keywords: Bacillus thuringiensis Phage Phage-resistant mutants Tectivirus
Bacillus thuringiensis is an insecticidal bacterium that has been used worldwide as an ecofriendly biopesticide over the last 70 years. The bioinsecticide formulations of B. thuringiensis are traditionally produced by submerged fermentations, but solid-state fermentations can also be applied (Devi et al., 2005). These fermentations are exposed to contamination by bacteriophages (phages) that can slow or completely stop B. thuringiensis fermentations, decreasing the production and resulting in significant economic losses. Recently, it has been suggested that between 15 and 30% (sometimes up to 50–80%) of the total fermentation batches of B. thuringiensis, can be damaged by phages, particularly by the virulent ones (Liao et al., 2008). Nevertheless, bacteria and phages are involved in continuous cycles of co-evolution, in which emerging phage-resistant bacteria help to preserve the host lineages, whereas phages might continue to overcome these resistant bacteria (Labrie et al., 2010). Thus, the study of phage-resistant bacteria and their remarkable array of resistance mechanisms can provide important clues to tackle the problem of phage(s) contamination in B. thuringiensis fermentations. Nowadays, many phages are known to be associated with B. thuringiensis, either as virulent or temperate phages (Gillis and Mahillon, 2014a). However, phage-resistance in B. thuringiensis and the mechanisms involved are still poorly understood. Recently it was shown that two of the most important B. thuringiensis bioinsecticide serovars (i.e., kurstaki and israelensis) are infected by tectiviruses GIL01- and GIL16-like, respectively (Gillis and Mahillon, 2014b). Sporadically some resistant phenotypes and distinct adaptation ⁎ Corresponding author at: Laboratory of Food and Environmental Microbiology, Université catholique de Louvain, Croix du Sud 2, Box L7.05.12, B-1348 Louvain-la-Neuve, Belgium. Tel.: +32 10 478598; fax: +32 10 473440. E-mail address: [email protected] (A. Gillis).
http://dx.doi.org/10.1016/j.mimet.2014.08.012 0167-7012/© 2014 Elsevier B.V. All rights reserved.
features can be observed in the B. thuringiensis strains in response to tectiviruses (Gillis and Mahillon, unpubl. results). Therefore, in a first endeavor to study tectiviral-resistance in B. thuringiensis, generation of a collection of phage-resistant mutants is necessary. Many of the methods described to isolate spontaneous phage-resistant mutants are derived from the classical plaque assay with double-layer agar (DLA) (Adams, 1959; Gratia, 1936). This method basically consists in mixing appropriate dilutions of both bacteria and phage in molten agar or agarose (the top overlay), which in turn, is evenly distributed on top of a solidified standard medium (the bottom underlay) (Kropinski et al., 2009). Using the standard DLA-system, in combination with high titers of a lytic phage that will kill almost all the bacterial population, it is possible to recover single colonies that further tests will confirm if they are phage-resistant. However, we found that this procedure is time consuming and not suitable for large screenings, since it relies on the interplay between the titers of phage and bacteria used to isolate the mutants. Moreover, the majority of the bacterial colonies were found not to be fully phage-resistant in complementary tests. In this study, an improved DLA-based method to rapidly generate and screen phageresistant mutants of B. thuringiensis is reported. B. thuringiensis strain HER1410 and a lytic variant of tectivirus GIL01, referred to herein as GIL01cp25, were used to develop the method. Strain HER1410 is a well-known host for phage GIL01 (Gillis and Mahillon, 2014b). GIL01cp25 has been previously described as producing clear plaques (unable to establish the lysogenic cycle) in susceptible hosts (Fornelos et al., 2011). The traditionally DLA-method was improved by a spotting assay as follows: strain HER1410 was grown in 10 ml LB (Lysogeny Broth) medium (5 g/l NaCl, 5 g/l yeast extract and 10 g/l tryptone) at 30 °C and 120 rpm to an optical density at 595 nm (OD595) of ~0.5. Then, 200 μl of the bacterial culture was added to 10 ml molten
102
A. Gillis, J. Mahillon / Journal of Microbiological Methods 106 (2014) 101–103
LB-top agar (0.7% (w/v)), gently mixed and placed onto LB-agar (1.5% (w/v)) square Petri plates (10 × 10 cm). After agar solidification and plate drying at room temperature (~ 20 min), 15 μl drops of different sterile-filtered GIL01cp25 stocks with high titers (10 7–108 PFU/ml), were added on top of the bacterial lawn, allowed to dry for 20 min, and incubated overnight at 30 °C. To guarantee the emergence of distinct spontaneous bacterial-resistant mutants, one independent HER1410 culture per plate was subjected to infection by drops of different GIL01cp25 stocks. After incubation, spontaneous bacterial colonies emerged, and depending on the bacterial culture tested and the phage stock used, the frequencies were different (Fig. 1A–D). Since 15 μl drops of different phage stocks (plus a negative control drop of LB medium) can be easily arranged in a 10 × 10 cm square Petri plate, the isolation of 80 candidate phage-resistant mutants of strain HER1410 was achieved by using 6 square plate's assays. Only one candidate colony per drop, if any, was isolated, streaked on fresh LB-agar plates and subjected to a double-round of single-colony isolation. Depending on the number of phage-resistant mutants required, the drop volume can be reduced down to 5 μl to accommodate a larger number of phage drops per plate, but a reduction in the emergence of phage-resistant colonies was observed. Therefore, the use of 15 μl as a minimal drop volume is strongly recommended. The second part consists in the screening of the candidate phageresistant mutants. For this purpose, the traditional DLA-method was further modified as follows and named ISA (for inverse spotting assay): 500 μl of a sterile-filtered GIL01cp25 stock (107–108 PFU/ml) was placed in 10 ml molten LB-top agar, gently mixed and distributed onto LB-agar square plates. After agar solidification and drying, plates containing the “phage lawn” were inoculated with the candidate bacterial mutants. For this, the purified single colonies were picked with a sterile-toothpick and inoculated on top of the solidified agar containing GIL01 cp25. Each square plate was inoculated with up to 24 colonies, plus strain HER1410 as control. After 24 h incubation at 30 °C, candidate resistant-colonies were evaluated (Fig. 1E). Based on the colony phenotypes, it was possible to discriminate between
Spong-test
A
C
fully-resistant mutants and others with some levels of resistance to GIL01cp25 (medium and low, evidenced by the presence of lysis/clearing zones in the colony). The non-resistant colonies were also easily identified (Fig. 1E). Consequently, 40 fully-resistant mutants were selected by this assay and retained for subsequent studies associated with phageresistance in B. thuringiensis. To confirm the bona fide phage-resistance of the 40 selected mutants, rather than an immunity stage due to the presence of the phage, bacterial mutants were screened by PCR using previously described sets of primers targeting different regions of phage GIL01 (Gillis and Mahillon, 2014b; Jalasvuori et al., 2013). No amplification bands were observed for the 40 fully-resistant mutants (data not shown). DNA from phage GIL01cp25 was used as positive control. Also, to confirm that the fully-resistant mutants are derivatives of the parental strain HER1410, random amplified polymorphic DNA (RAPD)-PCR was performed as described by Hu et al. (2004). The RAPD fingerprinting of the fully-resistant mutants displayed similar band profiles than those observed for the parental strain HER1410 (data not shown). To verify the correlation between the resistant colony phenotypes (no lysis/clearing when growing on phage-containing media) and phage resistance, the selected bacterial mutants were exposed to GIL01cp25 in a traditional plaque forming assay as described elsewhere (Gillis and Mahillon, 2014b). No PFUs were observed after incubation (up to 48 h at 30 °C; data not shown). These results confirmed the resistance of the isolated bacteria and showed a direct correlation between the observed colony phenotypes and phage resistance. The same ISA was also used to evaluate the specificity of the bacterial resistance. Another lytic phage, named Vp4, was isolated for this purpose from environmental samples throughout an enrichment process with B. thuringiensis strain GBJ002 as described elsewhere (Van Twest and Kropinski, 2009). Strain GBJ002 is also a known host for phage GIL01 (Gillis and Mahillon, 2014b). ISA-square plates containing a top overlay with either GIL01cp25 or Vp4 phages were inoculated as previously described and incubated 24 h at 30 °C. Fig. 2 depicts the results obtained. The selected mutants were fully-resistant to GIL01cp25, but
Selecon and isolaon of emerging colonies B
Bacterial phage-resistance evaluaon by ISA E
1
2
3
4
5
6
7
8b
9
10
11
12
13
14
c-
15
16
17c
18
19
20b
21a
23
24
D
22
Fig. 1. Isolation and evaluation of phage-resistant mutants of B. thuringiensis HER1410. The flowchart for the whole procedure is shown on top. (A–D) Spotting tests displaying bacterial phage-resistant colonies in lysis zones generated by drops of lytic phage GIL01cp25 after overnight incubation at 30 °C. To illustrate the importance of using independent bacterial cultures and different phage stocks, four different drops are shown. (A, B) Same culture batch of HER1410, but different phage stocks gave rise to few resistant colonies in (A) but none in (B). (C, D) Independent cultures of HER1410 and the same GIL01cp25 stock produced the emergence of resistant colonies at different frequencies. (E) Inverted spotting assay (ISA). Twenty-four isolated colonies and control strain HER1410 (negative control, c-) were grown on top of LB-agar containing phage GIL01cp25. After 24 h incubation at 30 °C, the colony phenotypes showed different levels of resistance to GIL01cp25. Numbers alone indicate fully-resistant colonies, whereas lower-case letters (a) and (b) denote medium and low levels of resistance, respectively (evidenced by lysis/clearing zones in the colony). (c) Highlights a very susceptible mutant since it did not grow in the presence of the lytic phage. Similar observations were made for 80 candidate-resistant mutants, leading to the selection of 40 of those that displayed fully-phage-resistant phenotypes.
A. Gillis, J. Mahillon / Journal of Microbiological Methods 106 (2014) 101–103
103
Phages GIL01cp25
Vp4
R12
R11
R18
R06
R40
R12
R11
R18
R06
R40
R25
R10
R22
R37
R19
R25
R10
R22
R37
R19
R09
R15
R20
R24
R31
R09
R15
R20
R24
R31
R02
R26
R04
R35
R28
R02
R26
R04
R35
R28
R33
HER1410
GBJ002
R05
R39
R33
HER1410
GBJ002
R05
R39
Fig. 2. Resistance-specificity evaluation by ISA of B. thuringiensis HER1410 phage-resistant mutants. Twenty-three isolated phage-resistant mutants (R#) were inoculated on top of LB-agar containing lytic phages GIL01cp25 and Vp4. Phage resistance/susceptibility was evaluated after incubation for 24 h at 30 °C. All the B. thuringiensis mutants tested were fully-resistant to phage GIL01cp25 (left plate), but susceptible to phage Vp4 as it is evidenced by lysis of the bacterial colonies (right plate). HER1410 and GBJ002 are B. thuringiensis strains susceptible to both phages (see text for more details) and were used as controls.
were completely susceptible to Vp4, indicating that the displayed resistance is specific to tectivirus GIL01cp25. The selected mutants were also exposed to the wild type (temperate) phage GIL01 by means of a traditional DLA-assay (Gillis and Mahillon, 2014b). No PFUs were observed after incubation (up to 48 h at 30 °C; data not shown). From the bacterial lawns, bacterial were recovered, streaked onto LB-agar plates and screened again by PCR. No positive amplification bands were obtained (data not shown), indicating that the selected bacteria are also resistant to the wild type phage GIL01. In conclusion, this work showed that the combination of a spotting assay with the ISA is a simple, efficient and practical approach for isolating, screening and selecting bacterial mutants that are resistant to lytic phages. Furthermore, the fact that the mutants isolated in this work were also resistant to the temperate phage GIL01 indicates that this method can be used to study resistance to temperate phages, provided that a lytic variant of the phage of interest is available to initially generate the bacterial mutants. Ultimately, this method can be applicable to generate phage-resistant mutants of B. thuringiensis closely-related species, such as Bacillus anthracis or Bacillus cereus, and with minor variations, to most bacterial species that can grow in semi-solid media. Acknowledgments This work was supported by the Foundation for Training in Industrial and Agricultural Research (FRIA), grant to A.G., the National Fund for Scientific Research (FNRS), the Université catholique de Louvain
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