Biosafety assessment of Bifidobacterium animalis subsp. lactis AD011 used for human consumption as a probiotic microorganism

Journal Pre-proof Biosafety assessment of Bifidobacterium animalis subsp. lactis AD011 used for human consumption as a probiotic microorganism

Seockmo Ku, Suyoung Yang, Hyun Ha Lee, Deokyeong Choe, Tony V. Johnston, Geun Eog Ji, Myeong Soo Park PII:

S0956-7135(19)30574-2

DOI:

https://doi.org/10.1016/j.foodcont.2019.106985

Reference:

JFCO 106985

To appear in:

Food Control

Received Date:

30 August 2019

Accepted Date:

06 November 2019

Please cite this article as: Seockmo Ku, Suyoung Yang, Hyun Ha Lee, Deokyeong Choe, Tony V. Johnston, Geun Eog Ji, Myeong Soo Park, Biosafety assessment of Bifidobacterium animalis subsp. lactis AD011 used for human consumption as a probiotic microorganism, Food Control (2019), https://doi.org/10.1016/j.foodcont.2019.106985

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

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Biosafety assessment of Bifidobacterium animalis subsp. lactis AD011 used for human consumption as a probiotic microorganism

Seockmo Kua, Suyoung Yangb, Hyun Ha Leeb, Deokyeong Choea, Tony V. Johnstona, Geun Eog Jib,c, Myeong Soo Parkb,*

a

Fermentation Science Program, School of Agriculture, College of Basic and Applied Sciences,

Middle Tennessee State University, Murfreesboro, TN 37132, USA b

Research Center, BIFIDO Co., Ltd., Hongcheon 25117, Korea

c

Department of Food and Nutrition, Research Institute of Human Ecology, Seoul National

University, Seoul 08826, Korea

E-mail address: S. Ku ([email protected]); S. Yang ([email protected]); H. Lee ([email protected]);

D.

Choe

([email protected]);

T.

V.

Johnston

([email protected]); G. E. Ji ([email protected]); M. S. Park ([email protected])

*

Corresponding Author: Dr. Myeong Soo Park, CTO, BIFIDO Co., Ltd.,

TEL: +82 33 435 4962; FAX: +82 33 435 4963; Email address: [email protected]

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ABSTRACT In this work, the previously identified Bifidobacterium strain, Bifidobacterium animalis subsp. lactis AD011 (obtained from an infant fecal sample), intended as a potential probiotic microorganism, is assessed for its safety using multiple in vitro virulence assays (ammonia and biogenic amine synthesis, hemolytic and mucin degradation activities, antimicrobial susceptibility and conjugal transferability of antibiotic resistance genes to other microorganisms) and comparative genomic analysis. The genome data of B. lactis AD011 was compared with genome sequences of two commercially available probiotic microorganisms (Bifidobacterium lactis BB12 and Bifidobacterium lactis Bl-04) which are designated as generally recognized as safe by the U.S. Food and Drug Administration. The results of these experiments showed no significant potential vulnerabilities and no side effects. B. lactis AD011 showed higher resistance to tetracycline than the European Food Safety Authority cut-off. However, the measured susceptibility is similar to or lower than that of other previously certified GRAS Bifidobacterium strains. Tetracycline resistance of B. lactis AD011 was not conveyed via conjugation with L. fermentum AGBG1, which is susceptible to tetracycline. Complete DNA sequencing of B. lactis AD011 showed the absence of transferable drug resistance plasmids. The three B. lactis strains (AD011, BB12 and Bl-14) compared in this study were found to share very close genomic sequence homology (>99.8 %). Therefore, based on this study, B. lactis AD011 appears to be a safe, bioactive, bifidobacterial food ingredient, starter culture, and/or probiotic microorganism for human health.

Keywords: Bifidobacterium lactis AD011, biosafety, probiotic microorganism, virulence assays, GRAS

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1. Introduction A variety of lactic acid bacteria, including Carnobacterium, Enterococcus, Lactobacillus, Lactococcus,

Leuconostoc,

Bifidobacterium,

Oenococcus,

Pediococcus,

Streptococcus,

Tetragenococcus, Vagococcus and Weissella spp., have been utilized to produce fermented foods over the centuries (Fessard & Remize, 2017). Many research groups have reported in vivo and in vitro health benefits of some species/strains of lactic acid bacteria (LAB) and currently categorize some microorganisms capable of imparting functionality to their hosts as probiotics (Kerry et al., 2018). The term, “probiotics” has been defined in various ways by multiple groups and researchers. In 2001, United Nations and World Health Organization Expert Panel defined probiotics as “live microorganisms which when administered in adequate amounts confer a health benefit on the host” (FAO/WHO, 2001). Recently, the National Institute of Health (NIH) defined “probiotics” more simply as, “live microorganisms that are intended to have health benefits” (NIH, 2016). As both of these definitions imply, bioactivity and health functionality rather than taxonomy are the critical characteristics (O’Toole, Marchesi, & Hill, 2017). Therefore, phylogenetically, “probiotic” microorganisms are not limited by species, genus or family. In addition, the term does not indicate whether or not strains have undergone genetic engineering. Biofunctional benefits from probiotic microorganisms may include (i) reduction of the risk of colon cancer, (ii) balancing composition of naturally occurring intestinal microbiota, (iii) avoiding food allergies, (iv) controlling bowl diseases, (v) inhibition of endogenous pathogens, (vi) modulation of the immune system, (vii) lowering serum cholesterol levels, (viii) reduction of intestinal pH and (ix) production of microbial enzymes with potential biocatalytic effects (Jin, Yoon, Johnston, Ku, & Ji, 2018; Kechagia et al., 2013; Ku, 2016; Ku, Park, Ji, & You, 2016). Due

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to growing consumer interest in the connection between food and health and the desire of the food industry to address that interest, the range of application of probiotic bacteria is getting wider, not only in foods in general but also in other areas such as alcoholic beverages, gelling agents, periodontal disease treatment, nutraceuticals, animal feed and cosmetics (Jin et al., 2019; Nagpal et al., 2012). Fundamentally, they are being exploited for new market pioneering by companies offering products with tangible benefits. According to a recently released market analysis report, the global market for probiotics in 2018 was USD 48.38 billion, and the probiotic strains currently most commonly used in the functional food market are Lactobacillus spp. and Bifidobacterium spp. (GVR, 2019). In addition to these two traditionally used LAB genera, other gram positive (e.g. Bacillus, Enterococci and Weissella spp., Elshaghabee, Rokana, Gulhane, Sharma, & Panwar, 2017; Hanchi, Mottawea, Sebei, & Hammami, 2018; Jin et al., 2019; Lee et al., 2012), and gram negative (e.g. Escherichia coli, Nissle, 1917; and Akkermansia muciniphila, Scaldaferri et al., 2016; Zhang, Li, Cheng, Buch, & Zhang, 2019) bacteria and some yeast strains (e.g. Saccharomyces boulardii, Kelesidis & Pothoulakis, 2012) are being introduced to nutraceutical markets. Various researchers and regulatory organizations have recently warned that some LAB and/or probiotic bacteria may cause adverse side effects and/or health risks to their hosts (BgVV, 1999; CORDIS, 2013; EFSA, 2012; FAO/WHO, 2002; FAO/WHO, 2006; Kim et al., 2018; Vankerckhoven et al., 2008; VKM, 2009). Unlike conventional foods or drugs, the exclusive characteristic of probiotic products is that they are likely to be alive when administered to consumers. Due to technical limitations in assessing the safety of probiotics, interdisciplinary assessment approaches, including pathology, genetics, immunology and serology, are required to establish and/or confirm their safety (Sanders et al., 2010). In 2001, the Joint FAO/WHO Expert Consultation in Argentina recognized the need for systematic guidelines for the evaluation of

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probiotics (FAO/WHO, 2006). As a result, minimum requirements for probiotic health claims (e.g. evaluation methods and recommendation criteria) and safety evaluation methods were proposed as FAO Food and Nutrition Paper #85, Probiotics in Food: Health and Nutritional Properties and Guidelines for Evaluation, in 2006 (FAO/WHO, 2006). Following this guidance, appropriate premarketing risk assessments for ensuring consumers’ safety should be performed for all probiotic bacteria based on these (or similar) guidelines or recommendations. Recently, alleviation of the protein-induced food allergy effects of B. animalis subsp. lactis AD011 (AD011) (a non-spore forming, heterofermentative, gram-positive, anaerobe, and a member of the LAB) isolated from infant stool, was evaluated via an in vivo mouse model (Kim et al., 2009; Kim, Choi, & Ji, 2008). In this work, AD011 was evaluated for its safety using in vitro virulence assays (ammonia synthesis, antimicrobial susceptibility, mucin degradation property, transferability of antibiotic resistance gene to other microorganism, hemolytic activity, biogenic amine production and maintenance of genome stability), and comparative genomics based on the recommendations from global probiotics expert consultations.

2. Materials and methods 2.1. Microorganisms Bifidobacterium animalis subsp. lactis strains BB-12 (BB-12), Bl-04 (Bl-04) and AD011, Bifidobacterium bifidum BGN4 (BGN4), Bifidobacterium longum BORI (BORI), and Lactobacillus fermentum AGBG1 (AGBG1) were obtained from BIFIDO Co., Ltd (Hongcheon, Korea). Enterococcus faecium KCTC 13225 (KCTC 13225) was obtained from the Korean Collection for Type Cultures. All strains were pre-cultivated twice in MRS (BD BBL™, Franklin Lakes, NJ, USA) broth supplemented by 0.05% L-cysteine-HCl (Sigma, St. Louis, MO, USA) for

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microbial activation prior to the assays. Listeria ivanovii subsp. ivanovii ATCC 19119 (ATCC 19119) was purchased from the American Type Culture Collection and cultivated in Brain Heart Infusion (BHI) (BD BBL™, Franklin Lakes, NJ, USA) medium.

2.2. Ammonia Production Test The evaluation of ammonia production properties of bacterial cells (BGN4, BORI, AD011 and KCTC 13225) was conducted via the protocol described in our previous work (Kim et al., 2018). Specifically, cells were cultivated anaerobically in BHI at 37oC for 5 days. The media supernatants were collected by micro-centrifuge at 10,000 g for 30 min under 4oC (2236R centrifuge; Labogene Aps, Lillerød, Denmark). The pH of each supernatant was adjusted to 7.0 with 1 N NaOH. Two types of chemical cocktails were built to estimate ammonia content using the indophenol blue reaction. Cocktail #1 was made by dissolving 2 g phenol (Sigma, St. Louis, MO, USA) and 0.01 g sodium nitroferricyanide dehydrate (Sigma, St. Louis, MO, USA) in 200 mL of LC-MS grade water. Cocktail #2 was made by dissolving 1 g sodium hydroxide and 0.08 g sodium hypochlorite in 200 mL of LC-MS grade water. One hundred µL of the supernatants and 10 µL of cocktail #1 and #2 aliquots were mixed in 96 well plates (SPL Life Sciences Co., Ltd. Pocheon, Korea) and incubated for an hour at 20°C. Absorbance was measured at 625 nm using a microplate reader (Bio-Rad Laboratories, Philadelphia, PA, USA). All tests were performed in triplicate. As a positive control, KCTC 13225 supernatants were used. As a negative control, sterilized cell-free BHI media was used in this work.

2.3. Biogenic Amines Test

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Biogenic amine production by AD011 was evaluated using our previous protocol (Kim et al., 2018; Kim & Ji, 2015). AD011 was cultivated in two types of media (whole milk and in MRS supplemented with 0.05% L-cysteine-HCl) at 37oC for 15 h. Milk was purchased at a local grocery (Seoul, Korea). After cell cultivation, 5 mL of milk and MRS supernatants were vigorously mixed with 25 mL of 0.1 N HCl. The medium-HCL mixture was microcentrifuged at 10,000 g for 15 min at 4°C (2236R high-speed centrifuge; Labogene Aps, Denmark). The aqueous phase was decanted and the solid phase was reused to extract more components from media samples as described above. After reaction, all particles were removed using a 20–25 μm cut-off flat sheet filter membrane (Whatman Int'l., Ltd., Maidstone, UK, Whatman® Grade 4, Cat#: 28460-019PK) in a Buchner funnel. Subsequently, one mL of permeate was mixed with 0.1 mL of 0.01% (w/v) 1,7-diaminoheptane, 0.5 mL of saturated aqueous sodium carbonate solution (99.0% Samchun Pure Chemical Co., Ltd, Korea), and 1 mL of 1% 5-(dimethylamino)naphthalene-1sulfonyl chloride (dansyl chloride; Sigma, St. Louis, MO, USA) in propanone (Sigma, St. Louis, MO, USA). After vigorously mixing the solutions via vortexer, the samples were incubated for 1 h at 45°C in a light-tight incubator (WBC 1510A; Jeio Tech. Co., Ltd., Korea). 0.5 mL of 10% (w/v) L-proline (Sigma, St. Louis, MO, USA) and 5 mL of HPLC grade ether were added to each sample. All samples were then further incubated at room temperature for about 5 min, followed by dansyl chloride removal. For high-performance liquid chromatography (HPLC) analysis, supernatants were collected and concentrated at 20°C using a Scanvac Speed Vacuum Concentrator (Labogene Aps, Lillerød, Denmark) at 1,500 rpm until completely dry. The sediment powder was collected and mixed with 1 mL of HPLC grade C2H3N (Sigma-Aldrich, St. Louis, MO, USA). The reconstituted sample and standard were filtered through a 0.22 µm syringe filter for HPLC

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analysis. Unwanted particles in sample solutions and standard solutions were filtered out by 0.22 μm cut-off syringe filter membranes (PALL Corporation, Port Washington, NY, USA) prior to HPLC analysis. HPLC analysis was performed by the National Instrumentation Center for Environmental Management (NICEM) at Seoul National University. The system used for HPLC analysis was the Thermo Dionex Ultimate 3000 HPLC (Thermo Fisher Scientific, St Peters, MO, USA). Cadaverine, histamine, putrescine and tyramine were separated using a VDSpher C-18 column (4.6  250 mm, 5 µm) (VDS Optilab Chromatographie Technik GmbH, Berlin, Germany). Cadaverine (>97.0%, Cat. #33211), histamine (>97.0%, Cat. #H7125), putrescine (>98.5%, Cat. #51799), and tyramine (>99%, Cat. #T90344) were purchased from Sigma-Aldrich (St. Louis, MO, USA) as standard samples. The mobile phase consisted of (A) acetonitrile and (B) HPLC grade water, and separation was achieved using the following gradient: 0–1 min, 60% A; 1–25 min, 100% A; 25–30 min, 60% A. The injection volume was 20 μL, the flow rate was set at 0.8 mL/min, and the column temperature was maintained at 30°C. The UV detection wavelength was 250 nm.

2.4. Hemolytic and mucin degradation activities Evaluation of hemolytic and mucin degradation activities of AD011 was carried out using our previous protocol (Kim et al., 2018). For the evaluation of the hemolytic capacity, AD011 was anaerobically cultured in BL Agar (BD Difco™, Franklin Lakes, NJ, USA) supplemented with 5% sheep blood at 37oC for 2 days. ATCC 19119 was aerobically cultured in BHI media supplemented with 1.5% agar and 5% sheep blood at 37oC for 2 days. After anaerobic cultivation, all plates were checked for the presence of zones around the microbial colonies by holding the plates to a light source (or placement on a colony counter) and viewed through both the back and

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the front. Strains that produced green-hued zones around the microbial colonies were classified as α-hemolytic (incomplete lysis). Strains displaying clean blood lysis zones around the colonies were classified as hemolytic (β-hemolysis) bacteria. If there were any changes in the blood agar around the microbial colonies, the bacteria were classified as γ-hemolytic. For the evaluation of mucin degradation activity of AD011, dextrose-free MRS was formulated as a stock solution according to our previous reports (Kim et al., 2018; Ku, You, & Ji, 2009; Ku, You, Park, & Ji, 2015; Ku, You, Park, & Ji, 2016). 0.5 to 1% (w/v) of dextrose or crude mucin powder was added to dextrose-free MRS as the major carbon sources to produce modified MRS. Commercially available crude mucin powder bound sialic acid 0.5–1.5% (Type III, bound sialic acid 0.5–1.5%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The pH of all broths was adjusted to 6.9 to 7.0 via the addition of 1N NaOH. AD011 was treated to each modified MRS and sample bacteria were anaerobically cultured at 37°C for two days. AD011 cell samples cultured in dextrose-free MRS were used as a control group. Cell growth was assessed by measuring absorbance at 550 nm using a microplate reader (Bio-Rad Laboratories, Philadelphia, PA, USA) at 0, 12, 24, 36, and 48 h. The initial optical density value of the media was subtracted from the final value for each test sample.

2.5. Genetic comparison, stability and virulence gene analysis To conduct genetic comparison analysis between AD011 and two commercial probiotic Bifidobacterium strains (B. lactis BB-12 from Nestle USA [GRAS Notice No. 49], and B. lactis Bl-04 from Danisco USA, Inc. [GRAS Notice No. 445]) (FDA, 2019a–b), whole genome sequence of AD011 was performed by the Chunlab, Inc. (Seoul, Korea) using PacBio Sequel Systems (Pacific Biosciences, CA, USA). The sequences were analyzed using CLgenomics™ program

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(Chunlab, Inc., Seoul, Korea). Orthologous Average Nucleotide Identity (OrthoANI) and TetraNucleotide Analysis (TNA) were carried out to calculate and compare nucleotide identity values. The sequences of the three tested Bifidobacterium lactis strains (AD011, BB-12 and Bl-04) are also available on the taxonomically united database, EZBIOCLOUD (EzBioCloud, 2019). To assess potential pathogenicity of AD011, virulence factor analysis was performed using the VirulenceFinder 2.0 server of the Center for Genomic Epidemiology, as conducted in our previous study (Kim et al., 2018). The genome sequence of AD011 was compared with toxic or pathogenic genes in E. coli (Stx1 and Stx2), Enterococcus (hylA and hylB), Listeria (prfA), and Staphylococcus aureus (aur).

2.6. Antimicrobial resistance transferability and antibiotic susceptibility tests Experiments were conducted using our previous protocol (Kim et al., 2018). Donor (AD011) and recipient cell suspensions (AGBG1) were mixed in a ratio of 1:1 (v/v) and centrifuged at 7,000 g for 10 min for pellet harvesting. The collected mixed microbial cell pellets were resuspended in MRS and anaerobically incubated at 37°C for 12 h. The bacterial biomass was collected via microfilter membrane (0.45 µm cut-off). The collected microorganisms were then spread on conventional MRS agar and anaerobically incubated at 37°C for 24 h for activation. Cultured active cells were harvested and plated on MRS agar containing tetracycline, and cultured aerobically for 36 h at 37°C. Bacterial colonies were then counted. Minimum inhibitory concentrations (MICs) of AD011 were evaluated using ISO 10932:2010 protocol (Milk and milk products — Determination of the minimal inhibitory concentration of antibiotics applicable to bifidobacteria and non-enterococcal lactic acid bacteria) with 20 antibiotic stocks (ISO, 2010). The following antimicrobial agents were treated in the

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ranges (1,024 to 0.0032 g/mL) after microfilter sterilization: penicillin G (Sigma, Lot#087M4834V),

carbenicillin

Lot#116M4834V),

ampicillin

Lot#SZBD263XV),

gentamicin

(Sigma, (Sigma,

Lot#116M4834V),

methicillin

(Sigma,

Lot#BCBW1243),

dicloxacillin

(Sigma,

Lot#SLBP3082V),

streptomycin

(Sigma,

(Sigma,

Lot#SLBT8451), kanamycin (Sigma, Lot#066M4019V), neomycin (Sigma, Lot#LRAB3300), cephalothin (Sigma Lot#056M4858V), tetracyclin (Sigma, Lot#126M4769V), polymyxin B (Sigma, Lot#SLBT8451), erythromycin (Sigma, Lot#WXBC4044V), metronidazole (Sigma, Lot#MKBZ3056V), vancomycin (United States Pharmacopeia, Lot#R07250), chloramphenicol (Sigma, Lot#SLBR8869V), rifampicin (Sigma, Lot#MKCC2435), clindamycin (Sigma, Lot#021M1533), mupirocin (Sigma, Lot#106M4733V), and trimethoprim/sulfamethoxazole (Sigma, Lot#097M4017V).

Antimicrobial agents were added into lactic acid bacteria

susceptibility test medium (LSM; Klare et al. 2005), consisting of 90% (v/v) Iso-Sensitest media (Mbcell Iso-Sensitest Broth, Seoul, Korea) and 10% (v/v) MRS (BD Difco™MRS Lactobacilli broth, Franklin Lakes, NJ, USA) for testing.

3. Results and discussion 3.1. Toxic nitrogenous compound (ammonia and biogenic amines) production properties of AD011 Some lactic acid bacteria metabolize the amino acids contained in freshly crushed fruit juice (must) and wine, releasing ammonia and raising the pH to make the environment suitable for their growth during fermentation (Muñoz, Moreno-Arribas, & de las Rivas, 2011). It is known that a large amount of ammonia is produced in the intestinal tract as a metabolite of nitrogenous compounds by naturally occurring intestinal microbiota (Richardson, McKain, & Wallace, 2013).

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The non-ionized form of ammonia is known to show high permeability to the colonic epithelium. Ammonia molecules are carried to the liver and converted to urea for detoxification. High ammonia synthesis from gut microbiota may be noxious to the nerve system, and the active production of such ammonia may possibly be the cause of intestinal degeneracy (Hambly, Rumney, Fletcher, Rijken, & Rowland, 1997). Recently, controlling intestinal microbiota via probiotic supplementation and/or consumption of fermented foods has been proposed as an approach to reducing ammonia in the intestinal tract and treating hyperammonemia. However, it is also known that some Lactobacillus strains have the ability to produce trace amounts of ammonia during their growth (Vince & Burridge, 1980). Biogenic amines (BAs) are known as low molecular mass organic bases with aromatic and heterocyclic structures that are easily detected in proteinaceous foods (e.g. dairy, meat and fish), alcoholic beverages and fermented food products (Suzzi & Gardini, 2003). According to Ekici and Omer (Ekici & Omer, 2018), histamine, putrescine, cadaverine, tyramine, tryptamine, betaphenylethylamine, spermine and spermidine are known to be frequently detected BAs in foods. Various lactic acid bacteria are known to actively convert amino acids to amine-containing compounds, and this BA production is related to lactic acid bacterial defense mechanisms (Capozzi et al., 2012; Spano et al., 2010). According to Barbieri et al. (Barbieri, Montanari, Gardini, & Tabanelli, 2019), enterococci, lactobacilli, streptococci, lactococci, pediococci, and oenococci are considered to be major BA-producing lactic acid bacteria. Leuconostoc and Weissella spp. are categorized as minor BA producers. Costa et al. (Costa et al., 2015) suggested that the level of BA in fermented cow and goat milks could be an inclusion criterion for choosing bacterial starter cultures to produce fermented products. High levels of BAs may cause adverse health effects including headaches, sweating, burning nasal secretions, migraine, neurological disorders, nausea,

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increased cardiac output, tachycardia, and hypotension. However, the exact level of BAs causing acute side effects in the human body is not yet known (EFSA, 2011). Recently, Barbieri et al. (Barbieri, Montanari, Gardini, & Tabanelli, 2019) summarized the five types of BAs (histamine, tyramine, 2-phenylethylamine, cadaverine and putrescine) produced as a result of amino acid metabolism by various lactic acid bacteria species (Enterococcus, Lactobacillus, Streptococcus, Lactococcus,

Pediococcus,

Oenococcus

spp.).

Some

Bifidobacterium

spp.,

such

as

Bifidobacterium CCDM 94, B. adolescentis CCDM 223, B. animalis ssp. lactis CCDM 239, 240, 241, and 374, B. bifidum CCDM 559, and B. longum CCDM 569, are known to have the ability to synthesize BAs. In our previous study (Kim et al., 2018), we reported that BORI and BGN4 were not capable of producing cadaverine, histamine, or tyramine, but both strains produced 24.23 and 16.58 µg/mL of putrescine in common. Therefore, evaluation of the ammonia and BA production capacities of probiotic bacteria may be regarded as an available option for evaluating their safety. In this study, we searched for ammonia (Table 1) and four biogenic amines (Table 2) in AD011 supernatants, but neither ammonia nor BAs were detected. Because Enterococcus faecium strains are known to actively produce ammonia as their metabolite, KCTC 13225 was used as a positive control in this assay (Table 1). KCTC 13225 produced 109.3 ± 7 μg/mL ammonia. We previously reported that both BORI and BGN4 lack ammonia production properties (Kim et al., 2018). BORI and BGN4, which were used as negative controls, did not produce ammonia, and this data reconfirms their lack of ammonia production properties. We attempted to detect four major BAs (cadaverine, histamine, putrescine and tyramine) from two different media (MRS and whole milk) after 15 h of bacterial cultivation, but none were detected. Because ammonia and/or BAs could be used as a quality index for fermented food products, the lack of ammonia and BA production properties in AD011

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suggests that AD011 is likely to be applicable for the production of functional or fermented foods in the future without significant adverse effects from ammonia and BAs.

3.2. Hemolytic and mucin degradation activities Probiotic microorganisms must be safe in their host's bodies and should not represent a risk to the host's life. Many influential committees and expert groups have suggested that probiotic bacteria should be incapable of causing liquefaction/degradation of red blood cells and/or intestinal periplasmic material, such as gelatin and mucin in the host body. Specifically, according to the FAO/WHO Working Group Guidelines for the Evaluation of Probiotics in Food (FAO/WHO, 2002) and the Final Report of Probiotic Microorganism Cultures in Food from the German Federal Institute for Health Protection of Consumers and Veterinary Medicine (BgVV) Working Group (BgVV, 1999), conducting the study of hemolytic characteristics of probiotics is recommended due to its significance in assuring microbial safety. Among the various probiotics, Lactobacillus spp. are classified as α-hemolytic microorganisms (Goldstein, Tyrrell, & Citron, 2015). However, Gómez et al. (Gómez, Ramiro, Quecan, & de Melo Franco, 2016) reported that some Lactobacillus species and strains (L. sakei MBSa1 bac+, L. curvatus MBSa3 bac+ and L. lactis 368 bac–) exhibit significant β-hemolysis. In our hemolysis testing, ATCC 19119 was used as a positive control and sharply defined clean and/or colorless zones were formed around the colonies on blood agar plates (Fig. 1). Microbial colonies around AD011 showed no transparent and/or clean zones on the BL Agar supplemented with 5% sheep blood, and were thus considered as γ-hemolytic or nonhemolytic. This study therefore indicates that AD011 is not capable of inducing hemolysis. The mucolytic bacteria studied by various groups in the 1980s were human pathogenic bacteria (Levy & Aminoff, 1980; Prizont, 1982; Slomiany et al., 1992; Tailford, Crost, Kavanaugh,

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& Juge, 2015), and mucolytic capacity has, until recently, been interpreted in relation to the virulence and/or toxicity of microorganisms. Various intestinal pathogens (e.g. Escherichia coli, Vibrio cholera, Campylobacter jejuni) are known to hydrolyze glycoprotein-based mucus gel layers and to possess the ability to metabolize mucus-derived monosaccharides (Sicard, Le Bihan, Vogeleer, Jacques, & Harel, 2017). The Guidelines for the Evaluation of Probiotics in Food by the FAO/WHO Working Group (FAO/WHO, 2002) did not mention the evaluation of mucin degradation activity of probiotics. However, according to a recently summarized review work (Tailford, Crost, Kavanaugh, & Juge, 2015), various Bifidobacterium species and strains (B. bifidum D119, L22, ATCC 35914, A8, 324B, 156B, 85B and DSM 20456, B. longum subsp. infantis VIII-240 and ATCC 15697, and B. breve NCIMB8807 and B. longum subsp. longum NCIMB8809) exhibit significant mucolytic properties. Ruas-Madiedo et al. (Ruas-Madiedo, Gueimonde, Fernández-García, de los Reyes-Gavilán, & Margolles, 2008) reported that when some

Bifidobacterium

strains

possess

two

specific

genes

(engBF

[endo-α-N-

acetylgalactosaminidase] and afcA [1,2-α-l-fucosidase]), they have a strong ability to degrade high-molecular weight porcine mucin. Considering these reports, it is probably prudent to evaluate the mucolytic properties of probiotic strains. Although some groups have interpreted this mucolytic process as part of the normal metabolism of microorganisms, the Steering Committee of the Norwegian Scientific Committee for Food Safety (VKM, 2009) suggested that reduced intestinal mucin production or increased mucin degradation may have pathological consequences in patients. Therefore, the administration of probiotics with mucolytic functionality should be avoided in these patients. They also categorized mucin degradation capability as a possible probiotic microorganisms’ virulence factor. The BgVV also recommended examination of mucin reduction properties in probiotics to prove microbial safety (BgVV, 1999).

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In this work, AD011 growth after incubation in five different modified MRS medias (dextrose-free MRS (control group), dextrose-free MRS with 0.5 and 1% (w/v) of mucin as the only carbon source, MRS with 0.5 and 1% dextrose) was tested by measuring absorbance at 550 nm (Fig. 2). In modified MRS supplemented with 0.5 and 1% (w/v) dextrose, the maximum growth of AD011 was generally observed at 36 h. However, microbial growth was not observed at all when AD011 was cultured in a medium containing mucin instead of dextrose or control media for 48 h. NIH genetic sequence database (Genbank) showed that AD011 was a strain free of engBF and afcA genes. These observations indicate that AD011 has no mucin hydrolysis capability.

3.3. Genetic Comparison of AD011, BB-12, and Bl-04 Analysis of the whole genome sequence is considered to be one of the gold standards to define taxonomy, phenotypic characteristics, and potential virulence (Quainoo et al., 2017). Identifying the genomic differences between two closely related edible probiotic strains (safetytested strains vs. strain to be tested) using complete microbial genome sequences can be used to identify potential probiotic characteristics and establish microbial safety. In this study, the whole genome sequence of AD011 was obtained, and through comparative genomic analysis, the common features and phylogenetic differences among B. lactis strains (AD011, BB-12, and Bl04) was pursued. Table 3 shows genomic information on these three B. lactis strains. B. lactis strains AD011, BB-12, and Bl-04 consist of one circular chromosome with 1,933,695-bp, 1,942,198-bp, and 1,938,709-bp, respectively, and have G+C content of 60.49%, 60.48%, and 60.48%, respectively. None of the strains harbor a plasmid. The relatedness measure (as %) between the genomes of AD011, BB-12 and Bl-04 was analyzed by OrthoANI and TNA. OrthoANI analysis showed that the whole genome of B. lactis AD011 has 99.85% and 99.93%

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similarity to those of BB-12 and Bl-04, respectively. B. lactis strains AD011, BB-12, and Bl-04 show 99.99% homology in genome sequences by tetra-nucleotide analysis (TNA) values. The results imply that these strains share common characteristics and potentially similar physiological function in their hosts. The genome sequence of AD011 has been deposited at GenBank under the accession number CP001213.1 (Kim et al., 2009; Nucleotide GenBank, 2019).

3.4. Analysis of presence of virulence genes The potential pathogenicity of AD011 was assessed by analyzing AD011 genes for those encoding virulence factors found in well-known food poisoning bacteria including Shiga toxin producing E. coli (STEC), Enterococcus, Listeria, and Staphylococcus aureus. Genes encoding for Shiga toxin (stx1 and stx2) from STEC, Zinc metalloproteinase (a.k.a. exoenzyme or protease) (aur) from Staphylococcus aureus, positively regulates expression of listeriolysin and other virulence factors genes (prfA) from Listeria, hyaluronidase (hylA and hylB) from Enterococcus and other virulence genes were not found in AD011.

3.5. Analysis of Antibiotic Susceptibility and Transferability of Antibiotic Resistance to AD011 The natural expression of resistance to certain antibiotics by microorganisms is referred to as intrinsic or natural resistance. Microorganisms can also acquire antibiotic resistance by taking up DNA that confers resistance from other microorganisms or by genetic mutation; this is referred to as acquired resistance (EFSA, 2012). The emergence of drug-resistant superbugs (microorganisms resistant to a wide range of antibiotics) is attributed to widespread antibiotic overuse, and is now regarded as a major public health issue (CDC, 2018; European Commission, 2019). The use of probiotic bacteria as “living medicine” is therefore being investigated as a means

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of addressing antibiotic overtreatment in both human and animals. To ensure that probiotic bacteria do not inadvertently contribute to overall antibiotic resistance in the microbiome, verification of the inability to transmit or accept antibiotic genes is a necessary step to demonstrating the biosafety of potential probiotic microorganisms. Despite attempts to alleviate pressure on antibiotics overuse to humans and livestock via probiotic treatments, some probiotic strains have been considered to reserve antibiotic resistance genes in their plasmid and have the potential to transmit the antibiotic resistance genes to other bacteria through horizontal gene transfer (between individuals) (a.k.a. lateral gene transfer) with mobile genetic elements (Imperial & Ibana, 2016). Therefore, addressing the antibiotic resistance and transferability of commercial probiotics are crucial in terms of the biosafety assessment of bacteria used for human consumption. The FAO/WHO Working Group recently recognized the importance of ensuring the safety of antibiotic resistance and understanding the characteristics of probiotic strains with respect to their ability to acquire such abilities (FAO/WHO, 2002). In addition, the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) clearly recommended, “all bacterial products intended for use as feed additives must be examined to establish the susceptibility of the component strain(s) to a relevant range of antimicrobials of human and veterinary importance” in their Guidance on the Assessment of Bacterial Susceptibility to Antimicrobials of Human and Veterinary Importance (EFSA, 2012). One of the objectives of the Assessment and Critical Evaluation of Antibiotic Resistance Transferability in the Food Chain (ACE-ART) consortium, was to assess whether antibiotic resistance genes in certain LAB could be transferred to their partner cells (CORDIS, 2013). In general, the MIC values of AD011 were similar to or significantly lower than the established cut-off values suggested by FEEDAP (EFSA, 2012) (Table 4). Specifically, the MIC

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values of AD011 for ampicillin, streptomycin, erythromycin, vancomycin, chloramphenicol, and clindamycin were 0.5, 128, 0.063, <0.25, 2, and <0.032, respectively. The exceptions were gentamicin and tetracycline, whose MIC values of were slightly higher than those established by FEEDAP (AD011 vs. EFSA cut-off: gentamycin, 256 vs. 64; tetracycline, 16 vs. 8). According

to

recent

reports,

Bifidobacterium

spp.

carrying

aminoglycoside

phosphotransferases (APH)-related genes tend to be resistant to gentamicin (Duranti et al., 2017; Fouhy et al., 2013; Toth et al., 2010). A wide variety of Bifidobacteria species (e.g. B. bifidum, B. breve, B. animalis and B. adolescentis) have shown significant gentamicin resistance (Duranti et al., 2017; Moubareck et al., 2005). A putative gentamicin resistant gene (bla:BLA_0835; APH precursor) is included in the genome sequence of AD011 held in the Kyoto Encyclopedia of Genes and Genomes (KEGG, 2019). Accordingly, our data confirms earlier findings. However, it is noteworthy that the AD011 MIC value for gentamicin was equal to the cut-off value of B. animalis subsp. lactis strain Bi-07 (256 μg/mL), which has received a GRAS no question letter from the US FDA (FDA, 2019b). The MIC value of B. lactis AD011 for tetracycline was comparable to those of other commercially available GRAS-designated strains such as B. lactis BB-12 (GRN # 49) (FDA, 2019a), HN019, Bl-04, B420, Bi-07 (GRN 445) (FDA, 2019b), and Bf-6 (GRN # 377) (FDA, 2019c) strains, and B. breve M-16V (GRNs 453–455) (FDA, 2019d–f), which have received the FDA’s no question letters for use as ingredients in infant formulas and/or selected conventional foods. As shown in Table 4, most Bifidobacterium spp. have shown resistance to tetracycline. Tetracycline resistance in B. animalis subsp. lactis has been shown to be directly correlated with the presence of a single gene, tet(W) (Gueimonde et al., 2010). The tet(W) gene is widely distributed in B. animalis subsp. lactis. Studies by Gueimonde et al. (Gueimonde et al., 2010),

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Masco et al. (Masco, Van Hoorde, De Brandt, Swings, & Huys, 2006), and Aires et al. (Aires, Doucet-Populaire, & Butel, 2007) consistently found tet(W) in all strains they tested. Gueimonde et al. (Gueimonde et al., 2016) also determined that "tet(W) is necessary and sufficient for the tetracycline resistance seen in B. animalis subsp. lactis." Noting the presence of the transposase gene, the authors, nevertheless, concluded that there is no evidence that tet(W) in B. animalis subsp. lactis is transmissible. The tet(W) is chromosomally located, and it is not associated with the conjugative transposon TnB1230, found in some other tet(W)-positive bacteria (Kastner et al., 2006; Masco, Van Hoorde, De Brandt, Swings, & Huys, 2006; Mättö et al., 2007). Aires et al. (Aires, Doucet-Populaire, & Butel, 2007) reported that attempted parallel conjugation of tet(W) among Bifidobacterium isolates failed to produce any transconjugants. It is noteworthy that B. lactis AD011 has no plasmid capable of transmitting antibiotic resistance genes. EFSA cutoffs are not available for the following 13 antibiotics: penicillin, carbenicillin, methicillin, dicloxacillin, kanamycin, neomycin, cephalothin, polymyxin B, metronidazole, rifampicin, phosphomycin, mupirocin, and trimethoprim-sulfamethoxazol (EFSA, 2012). The MICs of B. lactis AD011 for penicillin, carbenicillin, methicillin, dicloxacillin, kanamycin, neomycin, cephalothin, polymyxin B, metronidazole, rifampicin, phosphomycin, and trimethoprim-sulfamethoxazol were 0.25, 2, 2, 8, 1,024, 512, 32, 256, 256, 2, 64, and <0.5, respectively; these values were comparable to or lower than the MICs for other GRAS strains (B. breve M-16V and B. lactis BB-12) (Kim et al., 2018). The MIC values of B. lactis AD011 for mupirocin was significantly lower than other GRAS strains (32 vs. >128) and that for metronidazole was significantly higher than those of other GRAS strains (256 vs. 4–8). The MIC value of B. breve M-16V for polymyxin B was significantly higher than B. lactis AD011 and BB12 strains (1,024 vs. 256). Ampicillin, vancomycin, gentamicin, and erythromycin are frequently

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used antibiotics in pediatric patients. For B. longum BORI, none of these pediatric antibiotics had MIC values exceeding EFSA breakpoints (EFSA, 2012). Based on EFSA cut-off values, B. lactis AD011 MIC values were comparable to other GRAS strains (B. lactis BB-12, HN019, Bl-04, B420, Bi-07, and Bf-6 strains, and B. breve M-16V) which have received FDA no question letters (FDA, 2019b–f). MIC values for these and other bacterial strains are presented in Table 4. The available information on the antibiotic resistance pattern of B. lactis AD011 indicates that overall antibiotic susceptibilities of the strain are similar to patterns of other GRAS strains of bifidobacterial species, and the strain is not likely to have transmissible antibiotic resistance genes. In addition, B. lactis AD011 does not contain plasmid capable of transmitting antibiotic resistance genes. These findings indicate that the use of B. lactis AD011 in foods does not present antibiotic resistance concerns. Because the antibiotic susceptibility tests showed AD011 has resistance to tetracycline (MIC of 16 μg/mL; Table 5), a tetracycline resistance transferability assay was conducted for further safety analysis of AD011. AGBG1 was utilized as the recipient probiotics cell strain due to its significant antibiotics sensitivity and susceptibility to tetracycline. As shown in Table 5, AD011 did not transfer antibiotic resistance to its recipient (AGBG1). AGBG1 cells, which are significantly susceptible to tetracycline, grew well in conventional MRS medium (>9 log CFU/mL); however, AGBG1 did not grow in the MRS medium containing tetracycline or the broth that was co-cultivated with AD011. In contrast, AD011 showed resistance to 16 μg/mL tetracycline in this study. These data indicate that the tetracycline resistance of AD011 was not transferred to the tetracycline sensitive recipient strains under the test conditions.

4. Conclusions

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To evaluate the safety of AD011, the following tests were conducted: (i) Ammonia and (ii) biogenic amine production, (iii) hemolytic and (iv) mucin degradation activity, (v) virulence gene searching, (vi) antibiotic susceptibility (vii) antibiotic resistance transferability, (viii) whole genomic sequence to compare genomic sequences of AD011 and other commercially available B. lactis strains, and (iix) virulence analysis. AD011 was not observed to produce clinically significant levels of biogenic amines or ammonia. Hemolytic and mucin degradation activities were not detected, and functional assays indicated that B. lactis AD011 exhibits antibiotic susceptibility to 20 antibiotics. Tetracycline resistance was demonstrated and the MIC value for tetracycline was higher than that established by EFSA, but comparable to those of other GRAS strains which have received US FDA ‘no question’ letters. AD011 was not observed to contain plasmid capable of transmitting antibiotic resistance genes. The genome of B. lactis AD011 does not contain regions with significant homology to known toxigenic or pathogenic genes. It was shown that B. lactis AD011, BB-12, and Bl-04 are very similar with genome sequence homology of 99.85% and 99.93% by ANI value and 99.99% by TNA value. The data in this work indicate that B. lactis AD011 is safe and is suitable for human use as a probiotic ingredient applicable to infant formulas, conventional foods, and/or dietary supplements.

Acknowledgments: This work was supported by the Bio &Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2017M3A9F3041747 & 2017R1A2B2012390) and by the High Valueadded Food Technology Development Program funded by the Ministry of Agriculture, Food and Rural Affairs(MAFRA)(317043-3), Korea.

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Conflicts of Interest Seockmo Ku, Deokyeong Choe and Tony V. Johnston declare no conflicts of interest. SuyoungYang, Hyun Ha Lee and Myeong Soo Park are directly employed by BIFIDO Co., Ltd. as a researchers and CTO, respectively. Geun Eog Ji and Myeong Soo Park hold BIFIDO Co., Ltd. stocks.

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Figure captions

Fig. 1. Anaerobic growth of L. ivanovii ATCC 19119 (beta hemolytic) and B. lactis AD011 on blood agar. Complete and no hemolysis phenomena were observed around the colonies of ATCC 19119 and AD011, respectively.

Fig. 2. The effect of different concentrations (0, 0.5 and 1% [w/v]) of mucin and dextrose on B. lactis AD011 in modified MRS, evaluated as optical density (550 nm) and recorded after 12 to 48 h.

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Fig. 1.

Fig.2.

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Table 1 Comparison of ammonia production by batch fermentation using four kinds of microbial cells in BHI medium for 5 days. Data are expressed as the mean ± SD (n = 3).

aND

Bacterial strains

Ammonia (μg/mL)

Bifidobacterium animalis subsp. lactis AD011

NDa

Bifidobacterium bifidum BGN4

ND

Bifidobacterium longum BORI

ND

Enterococcus faecium KCTC 13225

109.3 ± 7

= not detected.

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Table 2 Cultivation of B. lactis AD011in whole milk and MRS in a batch culture and enumeration of four kinds of biogenic amines in media (n = 3). Biogenic amines (µg/mL)

aND

Bifidobacterium animalis subsp. lactis AD011 Milk medium

MRS medium

Cadaverine

NDa

ND

Histamine

ND

ND

Putrescine

ND

ND

Tyramine

ND

ND

= not detected.

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Table 3 Genetics of AD011 in Comparison with Other B. lactis Strains Designated as GRAS by the FDA. Original/User's Label

B. lactis AD011 (current notice)

B. lactis BB-12 (GRN 49)

B. lactis Bl-04 (GRN 445)

Project accession

GCA_000021425.1 GCA_000025245.1 GCA_000022705.1

Status

COMPLETE

COMPLETE

COMPLETE

No. of contigs

1

1

1

Plasmids

0

0

0

Genome size (bp)

1,933,695

1,942,198

1,938,709

DNA G+Cb content (%)

60.49

60.48

60.48

No. of CDSsc

1,577

1,567

1,561

No. of rRNA genes

7

12

12

No. of tRNA genes

52

52

52

Mean of CDS lengths (bpa)

1,067.5

1074.5

1076.8

Median of CDS lengths (bp)

936

948

951

Mean of intergenic lengths (bp)

159.9

159

159.1

Median of intergenic lengths (bp) 113

111

111

Homology with AD011 by OrthoANId

NAe

99.85%

99.93%

Homology with AD011 by TNA

NA

99.99%

99.99%

abp = base pair; bG+C = guanine + cytosine; cCDSs = coding sequences; dANI = average nucleotide

identity; eNA = not applicable.

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1

Table 4 Antimicrobial susceptibility of B. lactis AD011 and Other Bifidobacterium spp. (MIC values, µg/mL).

No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

2

Antibiotics Ampicillin sodium salt Gentamicin sulfate Streptomycin sulfate salt Tetracycline Erythromycin Vancomycin hydrochloride Chloramphenicol Clindamycin hydrochloride Penicillin G Carbenicllin disodium salt Methicillin Dicloxacillin sodium salt hydrate Kanamycin sulfate Neomycin sulfate Cephalothin sodium salt Polymyxin B sulfate salt Metronidazole Rifampicin Phosphomycin disodium salt Mupirocin TrimethoprimSulfamethoxazole

3

a

4

NA = not applicable.

EFSAa

B. lactis strains GRN 445b AD011 HN019 Bl-04 Bi-07 B420

2 64 128 8 1 2 4 1 NR NR NR

0.5 256 128 16 0.063 <0.25 2 <0.032 0.25 2 2

NR

8

NR NR NR NR NR NR NR NR

1,024 512 32 256 256 2 64 32

NR

<0.5

Cut-off of Bifidobacterium

0.12 64 64 32 0.06 0.5 2

256

0.5 64 8 16 0.05 1 2 <0.03

512

0.5 256 8 0.12 <0.03 0.25 2 2

64

0.25 64 64 16 0.05 0.5 2 0.05

256

GRN 377c Bf-6 0.25 64 32–64 4–16 0.032–0.5 0.5–1 1–2 <0.03–0.06 0.5

256

2

Kim et al. (2018)d

GRN 453, 454, and 455e

B. lactis BB-12 B. breve M-16V B. breve M-16V 0.125 128 128 16 0.125 0.5 2 <0.032 0.125 2 2

0.25 128 256 16 0.125 0.5 2 0.063 0.25 4 8

0.125–0.25 32–128 14–128 0.5–2.0 0.016–0.25 0.25–0.5 1–2 0.032–0.125 <1.52 NA NA

4

8

NA

1,024 512 8 256 4 2 64 >128

1,024 1,024 16 1,024 8 1 32 >128

1

2

>256 NA 15.6–125 15.6–31.3 NA NA 32–128

Data from EFSA (2012); b Data from FDA (2019b); c Data from FDA (2019c); d Data from Kim et al. (2018); e Data from FDA (2019d–f); NR = not required;

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5

Table 5 Transferability of tetracycline resistance from donor (B. lactis AD011) to recipient (L.

6

fermentum AGBG1) (log CFU/mL).

Antibiotics Nonea T12b

L. fermentum AGBG1 (Aerobic)

B. lactis AD011 (Anaerobic)

9.6 NDc

9.4±0.1 9.4

L. fermentum AGBG1 + B. lactis AD011 Aerobic

Anaerobic

9.6 ND

9.5 8.8±0.1

7 None = No antibiotics were included in the counting agar medium; bT12 = Tetracycline (12

8

a

9

μg/mL) was included in the counting agar medium. cND = Not detected

10

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Conflict of Interest and Authorship Conformation Form Please check the following as appropriate: o

All authors have participated in (a) conception and design, or analysis and interpretation of the data; (b) drafting the article or revising it critically for important intellectual content; and (c) approval of the final version.

o

This manuscript has not been submitted to, nor is under review at, another journal or other publishing venue.

o

The authors have no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript

o

The following authors have affiliations with organizations with direct or indirect financial interest in the subject matter discussed in the manuscript:

Author’s name Myeong Soo Park Suyoung Yang Hyun Ha Lee Seockmo Ku Deokyeong Choe Tony V. Johnston Geun Eog Ji

Affiliation BIFIDO Co., Ltd, South Korea BIFIDO Co., Ltd, South Korea BIFIDO Co., Ltd, South Korea Middle Tennessee State University Middle Tennessee State University Middle Tennessee State University Seoul National University, South Korea