Isolation, Detection and Inactivation of a Myoviridae Bacteriophage Infecting Bacillus Amyloliquefaciens FB11

Ann Microbiol (2015) 65:1841–1846 DOI 10.1007/s13213-014-1022-9

ORIGINAL ARTICLE

Isolation, detection and inactivation of a Myoviridae bacteriophage infecting Bacillus amyloliquefaciens FB11

Chetsadaporn Pitaksutheepong & Mattika Abhisingha & Jureeporn Dumnin & Wonnop Visessanguan

Received: 19 August 2014 /Accepted: 9 December 2014 /Published online: 4 January 2015 # Springer-Verlag Berlin Heidelberg and the University of Milan 2015

Abstract Bacillus amyloliquefaciens is commonly used as a fermentation. These fermentation industries consider phage starter culture for fermentation of soybeans and soybean attacks a major threat. For instance, production of meals. Like other starter cultures, B. amyloliquefaciens fermented soybean products, like natto (Japan) and FB11 faces threats from phage infection. Frequent phage chungkookjang (Korea), has been disrupted by phage attacks during large-scale cell culture cause serious yield attacks (Nagai and Yamasaki 2009; Umene et al. 2009; losses. To alleviate this problem, phage detection, identifica- Kim et al. 2011), with a number of phages belonging to tion and inactivation are required. The isolation and prelim- the Caudovirales order (Myoviridae and Siphoviridae fam- inary characterization of a phage designated BA01 revealed ilies) being isolated from contaminated nattos and that it is a Myoviridae phage, with some regions of its ge- chungkookjang. Subsequently, extensive studies on the nome sharing sequence similarity with the genome of Bacil- characterization and inactivation of these phages [particu- lus subtilis phage SPO1. BA01 showed strong lytic activity larly those attacking B. subtilis (natto)] have been essential against B. amyloliquefaciens, B. circulans and B. pumilus.A in managing phage decontamination in natto production PCR-based method was developed to detect BA01 in large- factories (Nagai 2012). scale cell culture of B. amyloliquefaciens FB11,whereapair Unfortunately, although B. amyloliquefaciens is used of degenerate primers could detect BA01 with a limit of widely as a starter culture for fermentation of soybeans or detection of 104 PFU/mL. Inactivation of BA01 was soybean meals (Meerak et al. 2007; Seo et al. 2013), there is achieved by either thermal treatment at 70 °C for 5 min or little information on phages of B. amyloliquefaciens. De- treatment with peracetic acid-based disinfectant (0.3 % v/v) cades ago, there was a report on induction of a temperate, for 5 min. or lysogenic, phage named H2 from B. amyloliquefaciens strain H (Zahler et al. 1987). The morphology suggested that it belongs to the Caudovirales order, Siphoviridae family Keywords Bacteriophage . Bacillus amyloliquefaciens . while its genome shares sequence similarity with Group III Caudovirales . Myoviridae . Isolation . Inactivation B. subtilis temperate phages. The most recent report on a B. amyloliquefaciens phage was by Muyombwe et al. Introduction (1999), where a phage was isolated from sewage during an attempt to isolate an endolysin gene. There were no details Fermented soybean meal is commonly used as a protein on phage morphology, classification or host range. source in swine feed, with Bacillus subtilis and Bacillus Despite the relative lack of information, phage infection remains a problem, particularly for a strain used lo- amyloliquefaciens generally being used as starter cultures for cally in Thailand, B. amyloliquefaciens FB11. To control C. Pitaksutheepong (*) : M. Abhisingha : J. Dumnin : phage infections, the phage was isolated and classified W. Visessanguan morphologically into the Caudovirales order, Myoviridae Food Biotechnology Research Unit, National Center for Genetic family. A PCR-based method was then developed for the Engineering and Biotechnology, 113 Thailand Science Park, early detection of phage attack. Finally, chemical and Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand thermal treatments were examined for fast and efficient e-mail: [email protected] ways to inactivate this phage. 1842 Ann Microbiol (2015) 65:1841–1846

Materials and methods Table 1 Host range of phage BA01 Bacterial strain Inhibition zone Bacterial strains (mm)a/degree of lysisb Bacillus amyloliquefaciens FB11 was used as the host for Bacillus amyloliquefaciens BCC 18049 14.0/++ phage isolation. Indicator strains used for determination of phage host range were obtained from the BIOTEC Culture B. amyloliquefaciens FB11 9.3/++ − Collection (BCC), National Center for Genetic Engineering Bacillus cereus BCC 27609 and Biotechnology, Thailand and from the Japan Collection Bacillus circulans JCM 2504 9.2/++ − of Microorganisms (JCM). Bacteria were grown in Luria Bacillus licheniformis ATCC 53757 − Bertani (LB) medium at 37 °C for 16–18 h. Bacillus megaterium BCC 18046 Bacillus pumilus BCC 6493 9.2/+ Bacillus subtilis subsp. subtilis JCM 1465 − Isolation of bacteriophage B. subtilis BCC 6327 − Bacillus thuringiensis subsp. aizawai − A culture of B. amyloliquefaciens FB11 naturally infected by BCC 14313 bacteriophages was collected from a local factory and centri- a Diameter of inhibition zone is the mean of two determinations fuged. The supernatant was filtered through a 0.2-μmmem- b − No inhibition zone, + turbid inhibition zone, ++ clear inhibition zone brane. A 200 μl aliquot of the filtrate was used to perform double-layer-agar plating (Adams 1959). Single plaques were Partial characterization of the phage genome picked and placed in 10 mL LB medium containing 100 μL B. amyloliquefaciens FB11. The culture was incubated for Three pairs of degenerate primers named BA44F/BA44R (5′- complete lysis at 37 °C and centrifuged at 10,000 g for CTTSAAGMRTG GKKTGARYTRGAAGG-3′/5′-CCYG μ 10 min. The supernatant was filtered through a 0.2- mmem- CYTCMYKWARYGCSATKATRGT-3′), BA31F/BA31R brane and the filtrate was used for double-layer-agar plating as (5′- AAGGACATTAAAGGRTATGAG-3′/5′- CTTT above. To obtain the pure phage strain, single-plaque isolation YKRGWACCCC ATCTYAAATT-3′), and BA19F/BA19R was repeated twice. (5′- GGHTAYTCHKBWTTYTAYRTNGG-3′/5′-TCNS CRTANCC YTCWAWRTCWATWCC-3′)wereusedfor PCR amplification of phage BA01genomic DNA. The Large scale preparation of bacteriophage and DNA extraction primers were designed based on conserved regions of coding sequences of Bacillus phage SPO1 and other Bacillus phages. Phage multiplication and DNA extraction were performed ac- Primers BA44F and BA44R were designed based on three cording to Binetti et al. (2005). transcriptional regulation proteins (gp44, gp51 and gp25.1) of Bacillus phage SPO1 (accession number YP_002300288, Host range determination YP_002300295 and YP_002300396, respectively). Primers BA19F and BA19R were based on the conserved regions of The indicator strains used in this study are listed in Table 1. gp19.1 (Bacillus phage SPO1, YP_002300377) and hypothet- The lytic activity of BA01 was tested as follows: 100 μL ical proteins of Bacillus phages SPP1 and SPBc2 (NP_690719 overnight host culture (1×108 CFU/mL) was mixed with and NP_046674). Primer set BA31F/BA31R was designed 0.7 % melted LB agar. The melted agar was poured on top based on gp31.2 of Bacillus phage SPO1 (YP_002300418) of 2 % solidified LB agar to make double-layer agar. After and endoribonuclease of Bacillus phage SP82 (AAA56884). solidification, 10 μL phage suspension (1×109 PFU/mL) The PCR mixture contained 50 ng phage genomic DNA, 1× μ were dropped onto a lawn of each indicator strain. Plates were PCR buffer, 1.25 mM MgCl2,100 M dNTPs (Fermentas, incubated at 37 °C for 16–18 h and inhibition zone diameters Vilnius, Lithuania), 450 nM of each primer (Biodesign, Klong recorded. Luang Pathumthani, Thailand) and 1 U Taq polymerase (Invitrogen, São Paulo, Brazil). PCR amplifications were performed for 30 cycles of 94 °C for 1 min, 45 °C for 30 sec and Electron microscopy 72 °C for 30 sec. PCR products were analyzed on 1.2 % aga- rose gels by ethidium bromide staining before cloning into Phage suspensions were dropped onto carbon-coated copper pDrive (Qiagen, Hilden, Germany). The recombinant plas- grids and stained with 2 % uranyl acetate. Phage morphology mids were individually transformed into E. coli JM109. Plas- was viewed under a JOEL transmission electron microscope mid DNA was extracted using QIAprep® (Qiagen) and sub- (JEM-1220, Japan) at an accelerating voltage of 80 kV. jected to nucleotide sequencing. BLAST analysis was Ann Microbiol (2015) 65:1841–1846 1843 performed using the BLASTN algorithm available at http:// single-plaque isolation. Electron micrographs revealed a www.ncbi.nlm.nih.gov. phage with a head and a contractile tail with a base plate (Fig. 1).Theheadhadadiameterofapproximately118nm Detection of phage BA01 by PCR analysis and the tail was about 72 nm long and 30 nm wide. The baseplate was 54 nm wide. The inner tube was extended AcultureofB. amyloliquefaciens FB11 that was suspected of 70 nm from the tail shaft and base plate. Based on its mor- phage attack was collected and centrifuged. The supernatant phology, BA01 was classified into the Caudovirales order, was filtered through a 0.2-μm membrane. The filtrate was Myoviridae family. Determination of the host range revealed then boiled for 5 min and 1 μL was used for PCR analysis that BA01 showed strong lytic activity against with primer set BA44F/BA44R under the conditions detailed B. amyloliquefaciens, B. circulans and B. pumilus, but was above. unable to infect B. subtilis, B. megaterium, B. cereus, B. licheniformis and B. thuringiensis (Table 1). Determination of the detection limit of phage BA01

A series of phage suspensions at titers 101 to 108 PFU/mL was Partial characterization of phage BA01 genome inoculated into the sterile media used for large-scale production of B. amyloliquefaciens FB11; 1 mL of each inoculated Phage BA01 is morphologically similar to phages of the sample was collected, centrifuged, filtered and boiled as men- Spounavirinae subfamily, particularly phage SPO1 of tioned above. Then, 1 μL of each sample was used for PCR B. subtilis (Parker and Eiserling 1983). It was assumed that analysis. BA01 could be related to Bacillus phage SPO1. To test this hypothesis, a preliminary study of the BA01 genome was then Inactivation by chemical treatment conducted. Three pairs of degenerate primers were designed based on six conserved coding regions of Bacillus phage Phage inactivation was tested with commercial disinfectants SPO1 and other Bacillus phages. The three primer pairs were based on either sodium hypochlorite [5.7 % (w/v) active chlo- able to amplify fragments of the corresponding genes from the rine, Clorox, San Rafael, CA] or peracetic acid [5 % (w/w), BA01 genome. PCR amplification using primer pair BA44F/ JohnsonDiversey, Utrecht, the Netherlands). Phage suspen- BA44R yielded an amplicon of 278 bp (Table 2). BLAST sions (107–108 PFU/mL) were mixed with sodium hypochlo- analysis showed that it shared the most similarity with gp44 rite or peracetic acid solutions at concentrations of 0–5 % (v/v) of Bacillus phage SPO1 (86 % similarity). Primers BA19F and 0–0.3 % (v/v), respectively, and incubated at room tem- and BA19R produced two fragments, 418 bp and 748 bp in perature for 5 min. The treated phages were then immediately size, that showed 76 % and 78 % similarity to the gp19.1 of diluted ten-fold with 0.1 M phosphate buffer, pH 7.0. Serial Bacillus phage SPO1. Both fragments share the same 5′-end dilutions of phage suspension were used for determination of sequence. Two amplicons of 421 bp and 593 bp were ampli- phage titers by double-layer-agar plating. fied by primers BA31F/BA31R. The 421-bp fragment showed a maximum similarity with the gp31 of Bacillus Inactivation by thermal treatment

Aliquots of phage suspension (107–108 PFU/ml) were placed into 1.5-mL microtubes and submerged in water baths main- tained at 60 °C, 65 °C or 70 °C. At 5-min intervals, a tube was taken from each water bath and placed on ice for 2–3min. Ten-fold serial dilutions of the phage suspensions in LB medium were then used for determination of phage titers by double-layer-agar plating.

Results

Isolation and preliminary characterization of phage BA01

Phage BA01 was isolated from an infected culture of B. amyloliquefaciens FB11 and purified by three rounds of Fig. 1 Electron micrograph of phage BA01. Bar 100 nm 1844 Ann Microbiol (2015) 65:1841–1846

Table 2 Sequence analysis of PCR products

Primer Product Identity Gene/phage E-value Accession pair (bp) (%) number

BA44F/ 278 86 % gp44/Bacillus 7e-76 FJ230960 BA44R SPO1 BA19F/ 418 76 % gp19.1/Bacillus 2e-72 FJ230960 BA19R SPO1 748 78 % gp19.1/Bacillus 3e-156 FJ230960 SPO1 BA31F/ 421 83 % gp 31/ Bacillus 2e-105 FJ230960 BA31R SPO1 593 82 % gp34.56/Bacillus 6e-113 FJ230960 SPO1

phage SPO1 (83 % similarity) while the 593 bp fragment showed 82 % similarity with the gp34.56 of Bacillus phage SPO1. Altogether, these results suggested that some parts of the BA01 genome shared high similarity with that of the lytic Bacillus phage SPO1—a finding that may facilitate future characterization of this phage. The three primer pairs were also used to amplify genomic DNA of B. amyloliquefaciens strains that are lysogens of Fig. 2a,b Detection of BA01. a PCR analysis to detect the presence of phages PBSX and phi105 as well as strain FB11. No amplifi- BA01 in a large-scale culture of Bacillus amyloliquefaciens FB11. Lanes: cation products were detected for any strain (data not shown), 1 Culture sample treated with DNase, 2 untreated. DNase treatment was performed to eliminate DNA in the culture. b Limit of detection of BA01 demonstrating that the primers do not have non-specific bind- by PCR analysis. Lanes: 1–8 Culture medium spiked with different titers ing to DNA of the bacterial host and phages PBSX and phi of BA01 (108–101 PFU/mL) was used for PCR analysis. DNA markers 105. In addition, it implies that BA01 is not a prophage of (bp) are indicated on the left B. amyloliquefacines FB11.

the reduction of phage titer was less than 1 log PFU/mL after PCR detection of phage BA01 10 incubation for 30 min (Fig. 3a). However, a significant reduction of 5log was observed when the phages were treated at Due to the heavy losses resulting from of phage infections, 10 65 °C for the same period of time. Increasing the incubation rapid detection is required to monitor phage attack. PCR anal- temperature to 70 °C led to effective inactivation of BA01 ysis would be considered one of the methods of choice. Since after incubation for 5 min, where viruses were undetectable primers BA44F and BA44R were able to amplify a specific by double-layer-agar plating (<10 PFU/mL). genome fragment of BA01, they were chosen for detection of BA01. Naturally infected cultures of B. amyloliquefaciens FB11 were examined for the presence of phage BA01 by Inactivation of phage BA01 by chemical treatments PCR analysis. Primers BA44F and BA44R yielded 278-bp amplicons from all infected samples (Fig. 2a). Sequence anal- Disinfectants based on sodium hypochlorite and peracetic acid ysis showed that the amplicon sequences shared similarity were tested for their effect on inactivation of BA01. Sodium with the gp44 of Bacillus phage SPO1 and were identical to hypochlorite solutions at concentrations 0.1–2.0 % (v/v) had the amplicon from purified genomic DNA of BA01. The limit little effect on phage titers (<1log )(Fig.3b), but significant of detection of PCR analysis was subsequently determined; 10 reductions were observed when higher concentrations were growth medium spiked with BA01 suspensions was assayed used. A 6log reduction was obtained after a treatment with by PCR analysis and the limit of detection was determined to 10 the highest concentration (5 % v/v). However, the solution be 104 PFU/mL (Fig. 2b). was rather corrosive at this concentration and therefore may not be practical for phage inactivation. In the case of peracetic Inactivation of phage BA01 by thermal treatment acid solution, the use of concentrations between 0.025– 0.125 % (v/v) did not effectively inactivate the phage. An

Phage suspensions were incubated at 60 °C, 65 °C and 70 °C. approximate reduction of 1 log10 was observed (Fig. 3c). Treatmentat60°CdidnotinactivateBA01effectively,since BA01 titers showed sharp reductions at higher concentrations Ann Microbiol (2015) 65:1841–1846 1845

Spounavirinae subfamily, Spounalikevirus genus (Stewart et al. 2009). BA01 showed rather strong lytic activity against B. amyloliquefaciens, B. circulans and B. pumilus, but was unable able to infect B. subtilis subsp. subtilis JCM1465 and B. subtilis BCC6327, which were isolated from a Thai fermented soybean product and fermented natto, respectively. While, the inability of phage BA01 to infect B. subtilis may seem surprising because B. amyloliquefaciens and B. subtilis are closely related, this may be explained by the findings of Kubo et al. (2011). In this latter study, spore-forming Bacillus strains, primarily B. subtilis subsp. subtilis and B. amyloliquefaciens, were collected from dried rice straw from many harvest sites in Japan. Interestingly, natto- fermenting B. subtilis subsp. subtilis strains were all susceptible to B. subtilis phage ϕNIT1. However, the non-natto fermenting B. subtilis subsp. subtilis and B. amyloliquefaciens strains were either resistant or susceptible to the phage. This finding suggests that susceptibility of bacteria to a phage might be strain-specific. In addition, SPO1-like phages, particularly those in the Spounalikevirus (Bacillus phage SPO1) and Twortlikevirus genera (Listeria phages A511 and P100) of the Spounavirinae subfamily, have different host ranges despite sharing considerable sequence homology (Klumpp et al. 2010). Due to frequent failures of large-scale B. amyloliquefaciens FB11 cultures, rapid detection and efficient decontamination methods are required. With the aim of solving this problem, PCR analysis using primers BA44F/BA44R allowed rapid detection of BA01, with a 104 PFU/mL limit of detection, in less than 3 h including sample treatment, PCR analysis and gel electrophoresis. Fig. 3a–c Inactivation of BA01 by heat and chemical treatments. a Phage suspensions were treated at 60 °C, 65 °C and 70 °C for 0– Inactivation of phage BA01 was achieved by thermal and 30 min. b, c Disinfectants based on sodium hypochlorite (b)and chemical treatments. BA01 was completely inactivated after peracetic acid (c) were used to inactivate BA01. The concentrations of incubation at 70 °C for 5 min, suggesting that the phage was sodium hypochlorite and peracetic acid solutions were assayed between not resistant to high heat and that thermal treatment may be 0–5 % (v/v) and 0–0.3 % (v/v), respectively. The values in each graph are the means of three determinations useful for bringing phage infections under control. For chemical treatments, the use of a disinfectant based on 5 % (w/w) and, at the concentration of 0.3 % (v/v), BA01 was undetect- peracetic acid was capable of inactivating BA01, agreeing able by double-layer-agar plating (<10 PFU/mL). with reports of fast and effective inactivation of several phages like Lactobacillus, Lactococcus and Streptococcus phages by peracetic acid (Binetti and Reinheimer 2000; Suárez and Reinheimer 2002; Quiberoni et al. 2003; Capra et al. 2004; Discussion Ebrecht et al. 2010;Mercantietal.2012). However, inactivation of BA01 using sodium hypochlorite-based solutions was Phage BA01 was isolated from an infected culture of less effective, possibly because the disinfectant used in this B. amyloliquefaciens FB11. The phage was classified mor- study contained low available free chlorine [5.7 % (w/v)]. The phologically into the Caudovirales order, Myoviridae family. use of hypochlorite solution with 14 % available free chlorine Its structure is similar to lytic phages of the Spounavirinae was previously reported to be effective for inactivation of subfamily, namely Bacillus phage SPO1 and, Listeria phages Lactoccoccus phages (Dilek Avsaroglu et al. 2007). A511 and P100 (Klumpp et al. 2008, 2010). Partial character- In conclusion, phage infections of B. amyloliquefaciens ization of the BA01 genome revealed that some parts of its FB11 can be alleviated or controlled by the combined genome shared high (>86 %) similarity to the genome of methods of rapid detection and inactivation by thermal and Bacillus phage SPO1, which is the type member of the heat treatments. PCR-based detection facilitates early 1846 Ann Microbiol (2015) 65:1841–1846 detection of BA01 during the fermentation process, while the to Natto (fermented soybean) production. Appl Environ Microbiol – ability to inactivate phages by the use of 0.3 % (v/v) peracetic 77:6463 6469 Meerak J, Lida H, Watanabe Y, Miyashita M, Sato H, Nakagawa Y, acid or thermal treatment at 70 °C for 5 min enables effective Tahara Y (2007) Phylogeny of gamma-polyglutamic acid-pro- elimination of phages from the fermentation process. ducing Bacillus strains isolated from fermented soybean foods manufactured in Asian countries. J Gen Appl Microbiol 53: Acknowledgments This work was supported by the National Center 315–323 for Genetic Engineering and Biotechnology (BIOTEC), Thailand (grant Mercanti DJ, Guglielmotti DM, Patrignani F, Reinheimer JA, Quiberoni number P-11-00852). We thank Dr. Samaporn Teeravachyan for proof- A (2012) Resistance of two temperate Lactobacillus paracasei bac- reading the manuscript. teriophages to high pressure homogenization, thermal treatments and chemical biocides of industrial application. 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