Novel sequence types of Lactococcus lactis subsp. lactis obtained from Brazilian dairy production environments

Journal Pre-proof Novel sequence types of Lactococcus lactis subsp. lactis obtained from Brazilian dairy production environments Mayra Carla de Freitas Martins, Andressa Fusieger, Rosângela de Freitas, Florence Valence, Luís Augusto Nero, Antônio Fernandes de Carvalho PII:

S0023-6438(20)30134-1

DOI:

https://doi.org/10.1016/j.lwt.2020.109146

Reference:

YFSTL 109146

To appear in:

LWT - Food Science and Technology

Received Date: 23 December 2019 Revised Date:

5 February 2020

Accepted Date: 10 February 2020

Please cite this article as: Carla de Freitas Martins, M., Fusieger, A., de Freitas, Rosâ., Valence, F., Nero, Luí.Augusto., Fernandes de Carvalho, Antô., Novel sequence types of Lactococcus lactis subsp. lactis obtained from Brazilian dairy production environments, LWT - Food Science and Technology (2020), doi: https://doi.org/10.1016/j.lwt.2020.109146. 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. © 2020 Published by Elsevier Ltd.

Author contributions AFC, FV and LAN were responsible for the conceptualization and design of the study, AFC was responsible for funding acquisition and supervision, ACFM, AF and RF conducted the laboratory analysis and validated the results, MACFM, AF, RF, FV, LAN and AFC were responsible for the data analysis, AF, MACFM and RF wrote the original draft of the manuscript, AF, FV, LAN and AFC reviewed and edited the manuscript.

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Novel Sequence Types of Lactococcus lactis subsp. lactis obtained from Brazilian dairy

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production environments

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Mayra Carla de Freitas Martinsa,#, Andressa Fusiegera,#, Rosângela de Freitasa, Florence Valenceb,

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Luís Augusto Neroc,*, Antônio Fernandes de Carvalhoa,*

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a

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Universitário, s/n, CEP 36570-900 Viçosa, MG, Brazil

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b

Departamento de Tecnologia de Alimentos, Universidade Federal de Viçosa, Campus

UMR1253 Science et Technologie du Lait et de l’Œuf, INRA, Agrocampus Ouest, 65 rue de Saint

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Brieuc, 35000 Rennes, France

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c

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36570-900 Viçosa, MG, Brazil

Departamento de Veterinária, Universidade Federal de Viçosa, Campus Universitário, s/n, CEP

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#

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* Corresponding author at: Campus Universitário, s/n, Centro – Departamento de Tecnologia de

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Alimentos, Universidade Federal de Viçosa – Viçosa, Viçosa, MG 36570-900, Brazil.

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E-mais address: [email protected] (M.C.F. Martins), [email protected] (A.

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Fusieger), [email protected] (R. de Freitas), [email protected] (F. Valence),

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[email protected] (L.A. Nero), [email protected] (A.F. Carvalho).

Contributed equally

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Abstract

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Lactococcus lactis subsp. lactis are widely used by the dairy industry in fermentation processes.

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This study aimed to characterize the genetic diversity of L. lactis subsp. lactis isolated from

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Brazilian dairy environments. A collection of 23 isolates of L. lactis subsp. lactis was subjected to

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rep-PCR (GTG5) and PFGE (SmaI) to determine their genetic profiles. rep-PCR allowed a

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maximum similarity of 97.2% among strains, while PFGE grouped isolates in four clusters, and it

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was possible to identify isolates with 100% of similarity. The selected strains were also subjected to

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MLST (pepXP, pgk, glyA, recN, bcaT and pdp), resulting in the characterization of 11 STs; nine of

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these STs were firstly described in the present study. ST grouping allowed for the characterization

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of 2 CC: CC1 with 3 isolates and CC2 with 2 isolates. The remaining STs were distributed as

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singletons. These results show that L. lactis subsp. lactis obtained from Brazilian dairy

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environments present a high genetic diversity, highlighting the relevance of further studies to

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characterize their beneficial potential to be exploited by the food industry.

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Keywords: genetic profiles; Lactococcus; MLST; rep-PCR; PFGE

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1. Introduction

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Lactococcus lactis is lactic acid bacteria (LAB) used worldwide as starter cultures in the dairy

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industry, particularly to produce hard and semi-hard cheeses. Among the currently four L. lactis

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described subspecies, L. lactis subsp. lactis and L. lactis subsp. cremoris are of particular interest as

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starter cultures due to their technological potential, like acidifying ability, maltose fermentation and

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grow at different NaCl concentrations (Rademaker et al., 2007; Bachmann et al., 2009; Pérez et al.,

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2011) . In addition, the bio-variety diacetylactis deserves special attention due to its production of

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aromatic compounds (diacetyl and acetoin), which are highly desirable in specific fermented dairy

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products (Kempler & McKay, 1981).

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L. lactis is found in a wide variety of plant and animal environments. With their ability to colonize

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distinct biotypes and to adapt to various niches in the dairy production, L. lactis strains are present

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in the raw milk of different animal species, including cow, sheep and goat (Nomura et al., 2006;

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Perin & Nero, 2014). L. lactis can also be found in plant niches, such as grasses and silages (Yang

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et al., 2010; Khota et al., 2016). The natural presence of L. lactis in these environments leads to its

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consequently presence in the microbiota of raw milk and its products, like cheeses (Dal Bello et al.,

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2010; Pangallo et al., 2014; Martins et al., 2018).

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Molecular methods have increasingly been adopted for proper characterization of bacterial isolates.

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PCR-based, enzymatic restriction, and sequencing methods have been used to identify L. lactis

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subspecies (Pu et al., 2002; Yu et al., 2015). DNA fingerprinting analysis, including rep-PCR and

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pulsed-field gel electrophoresis (PFGE), are considered to be methods of reference when considered

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to characterize intraspecies diversity, because they are widely used for population genetic studies

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(Rademaker et al., 2007; Passerini et al., 2010). The diversity of L. lactis communities obtained

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from different dairy production environments has expanded with the use of molecular analyses,

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such as multi-locus sequence typing (MLST). MLST makes it possible to describe population

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structure and phylogeny with a limited number of genes sequenced; it is a tool used to assess the

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ecology within the microbial populations and their genetic evolution (Passerini et al., 2010).

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Because of the relevance of L. lactis to the dairy industry and the constant search for novel strains

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with technological features, in this study we aimed to characterize the genetic diversity of L. lactis

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subsp. lactis isolated from dairy production environment in Brazil.

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2. Material and Methods

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2.1. Isolates and further subspecies identification

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From the bacterial culture collection of InovaLeite (Laboratory of Milk and Dairy Products,

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Universidade Federal de Viçosa), identified based on partial sequencing of 16S rRNA (Felske et al.,

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1997), Lactococcus spp. isolates (n = 23) were selected an subjected to DNA extraction using the

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Genomic Wizard DNA Purification Kit (Promega Inc., Madison, WI, USA). Table 1 shows the

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origin of the isolates, as well as their identities based on 16S rRNA sequencing.

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The selected isolates were subjected to a PCR assay for identification of L. lactis, using the primers

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1RL (5’-TTT GAG AGT TTG ATC CTG G-3’) and LacreR (5’-GGG ATC ATC TTT GAG TGA

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T-3’) (Pu et al., 2002). Reactions were composed of 12.5 µL of Go Taq Green Master Mix 2x

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(Promega), 60 pMol of each pair of primers, 2 µL of DNA (80 ng/µL) and ultra-pure PCR water

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(Promega) to reach a final volume of 25 µL. PCR conditions were: (1) 95 °C for 5 min, (2) 35

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cycles at 95 °C for 30 s, 45 °C for 30 s and 72 °C for 30 s, and (3) a final extension step at 72 °C for

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10 min. PCR products were electrophoresed on 1% agarose gels (w/v), stained using GelRed

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(Biotium Inc., Hayward, CA, USA) and visualized using a transilluminator LPIX (Loccus

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Biotecnologia, São Paulo, SP, Brazil). A band of 238 bp was the indicative of L. lactis. For

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subspecies identification, 10 µL of the PCR products obtained for species identification were

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digested with 1U of MboII (Invitrogen Thermo Fisher Scientific, Massachusetts, USA) at 37 °C for

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2 h (Pu et al., 2002), and subjected to electrophoresis on 1.5% agarose gels (w/v). PCR products 4

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with 238 bp were indicative of L. lactis subsp. lactis, and PCR products with 134 bp were indicative

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of L. lactis subsp. cremoris. L. lactis subsp. lactis ATCC 13675 and L. lactis subsp. cremoris ATCC

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19257 were used as controls in all PCR assays.

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2.3. Genetic profiles of Lactococcus lactis

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2.3.1. rep-PCR

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Rep-PCR was performed according to Dal Bello et al. (2010). PCR reactions contained 12.5 µL of

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Go Taq Green Master Mix 2x (Promega), 50 pMol of the single primer (GTG)5, 2 µL of DNA (50

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ng/µL) and ultra-pure PCR water (Promega) to obtain a final volume of 25 µL. PCR conditions

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were: (1) 5 min at 95 °C, (2) 30 cycles for 30 s at 95 °C; 30 s at 40 °C and 8 min at 65 °C, and (3) a

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final extension for 16 min at 65 °C. The PCR products were analyzed in 2% (w/v) agarose gels for

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6 h at a constant voltage of 75 V, in 0.5 × Tris/Borate/EDTA buffer (TBE). The gels were stained

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using GelRed (Biotium) and recorded using a transilluminator LPIX (Loccus). The genetic

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fingerprints were analyzed using BioNumerics 6.6.11 (Applied Maths, Kortrijk, Belgium). The

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similarities among profiles were calculated using the Pearson correlation, and a dendrogram was

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constructed using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA).

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2.3.2. PFGE

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Bacterial cultures and agarose plugs were prepared, as described previously by Lortal et al. (1997).

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The plugs were equilibrated for 1 h at 4 °C in a restriction buffer (Cut SmartTM buffer, New

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England Biolabs, Evry Cedex, France). Restriction enzyme digestion with 15 units of enzyme SmaI

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(Promega) was performed at 25 °C for 4 h. Electrophoresis was carried out in 0.5 × TBE buffer (45

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mM L−1, 45 mM boric acid, 1 mM EDTA, pH 8, Sigma) in a 1% (w/v) agarose gel (PFGE certified

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agarose, Bio-Rad) with a pulse time of 2 to 20 s, voltage of 6 V/cm, for 21 h at 14 °C, using a

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CHEF-DR III apparatus (Bio-Rad) (Luiz et al., 2016). The gel was stained with GelRed (3 × in 0.1

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M NaCl solution) (Biotium) and visualized under UV light. Band profiles were analyzed using 5

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BioNumerics, version 6.6.11 (Applied Maths). Comparisons among the normalized band profiles

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were made using the Pearson coefficient and the UPGMA clustering algorithm.

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2.3.3. MLST

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Based on the genetic profiles obtained by rep-PCR and PFGE, L. lactis subsp. lactis isolates were

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selected and subjected to MLST. DNA extraction was carried out as described above and the DNA

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sequences were analyzed according to the intragenic regions of the genome encoding the X-prolyl-

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dipeptidyl aminopeptidase (pepXP), phospho-glycerate kinase (pgk), serine hydroxymethyl-

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transferase (glyA), ATPase involved in DNA repair (recN), branched-chain-amino-acid (bcaT), and

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pyrimidine-nucleoside phosphorylase (pdp) were performed, as described by Passerini et al. (2010).

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PCR conditions were: (1) 3 min at 94 °C, (2) 30 cycles at 94 °C for 45 s, 55 °C for 1 min, and (3)

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72 °C for 1 min. The PCR products were sequenced by Macrogen Inc and all sequences were

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analyzed using CLC Sequence Viewer 6.0 (Qiagen, Aarhus, Denmark). For each locus, the

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sequences obtained for all isolates were compared to those in the L. lactis subsp. lactis MLST

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database (http://www-mlst.biotoul.fr/Lactococcuslactissubsplactis/), and allele numbers were

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assigned to each unique sequence. Each isolate was defined by an allele profile or sequence type

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(ST) derived from the combination of numbers corresponding to the alleles at the analyzed loci

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(Table 2). The same ST was used for several strains that shared the same allelic profiles. The allelic

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profiles identified for the strains were clustered using e-BURST software (http://eburst.mlst.net/).

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3. Results and Discussion

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The protocol proposed by Pu et al. (2002) was adopted as 16s rRNA sequencing of the original

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culture collection did not yield results with enough quality to allow the identification at species and

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subspecies levels, and all selected isolates were identified as L. lactis subsp. lactis. The presence of

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L. lactis in different sources within dairy production environments has already been demonstrated in 6

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similar studies. Perin & Nero (2014) identified L. lactis strains in raw goat milk from Minas Gerais

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state, Brazil. Luiz et al. (2016) reported their presence in raw cow milk and grazing soil from the

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rural areas Campo das Vertentes region, in the same Brazilian state. Additional studies have

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recorded L. lactis strains isolated from artisanal cheeses, raw milk, grass and silage (Nomura et al.,

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2006; Dal Bello et al., 2010; Khota et al., 2016; Martins et al., 2018). Scientific data indicate that

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pasture, grass, and plants commonly found in dairy production environments are common habitats

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of L. lactis, indicating the adaptability of the genus to these various conditions (Kelly, Ward, &

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Leahy, 2010; Cavanagh et al., 2015). According to Guchte et al. (2002), L. lactis shows an excellent

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ability to adapt to different environments and can often survive extreme pH conditions, different

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nutrient availabilities and competition with other microorganisms, despite being characterized as

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fastidious in some specific culture conditions.

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Based on rep-PCR analysis, isolates did not share any identical genetic profile: the similarity varied

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from 15.9 to 97.2%. Genetic profiles obtained by rep-PCR allowed to group the isolates in two

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major clusters: I, with 6 isolates and similarity of 36.5%, and II, with 17 isolates and similarity of

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41.9% (Figure 1). The formed clusters presented low homology to each other (15.9%), indicating a

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high level of diversity among L. lactis subsp. lactis strains. The highest homology identified was

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between isolates 55 and 56 (97.2%), both from artisanal cheeses produced in the Amazonian region.

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Isolates from different sample origin and Brazilian regions also presented homology higher than

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90%, indicating potential common origins (58 and 61, 91.9%; 2 and 50, 92.9%; Figure 1), but other

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isolates with also high similarity (higher than 90%) were obtained from a same sample origin,

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characterizing them as potentially clones (Figure 1). Rep-PCR using (GTG)5 primer was selected

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because it has been shown to be suitable for characterizing lactococci (Dal Bello et al., 2010; Perin

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& Nero, 2014). However, isolates from milk and cheese were expected to demonstrate greater

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similarity than those from silage due to potential losses, mutations and gene acquisitions that occur

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to allow isolates to adapt to new habitats (Siezen et al., 2011; Laroute et al., 2017).

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Based on PFGE, 19 pulsotypes were characterized, being grouped in four major clusters (homology

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from 36.9 to 59.2%, Figure 2). Just one isolate, 27 (goat milk), did not cluster to any group and

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presented low homology with all isolates (15.5%) (Figure 2); interestingly, isolate 27 presented

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high similarity with isolate 35 by rep-PCR (Figure 1). Also, isolates that presented identical

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pulsotypes by PFGE did not share high homology by rep-PCR (isolates 22 and 56, 9 and 24, 17 and

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23, 38 and 61). As PFGE and rep-PCR have different approaches (PFGE is based on digestion of

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whole DNA, and rep-PCR is based on the amplification of specific DNA regions), the recorded

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profiles should be different. Random genetic events, including point mutations and DNA insertions

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and deletions can alter PFGE patterns, while rep-PCR profiles would not be necessarily affected

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(Tenover et al., 1995). PFGE analysis’ discriminatory capacity is directly linked to restriction

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enzyme choice, as SmaI in this and other studies (Psoni et al., 2007; Pillidge et al., 2009; Terzić-

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Vidojević et al., 2015; Bozoudi et al., 2016; Domingos-Lopes et al., 2017). The discriminatory

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power of PFGE can be enhanced by using additional restriction enzymes (Fernández et al., 2011).

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When the genetic profiles obtained by the PFGE and rep-PCR were both taken into account, it was

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determined that the 23 L. lactis subsp. lactis isolates could be identified as single strains, and

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therefore they were subjected to MLST analysis.

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MLST typing of the L. lactis subsp. lactis strains revealed the existence of 11 STs, thus indicating a

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high level of heterogeneity among them (Table 2), as indicated by rep-PCR and PFGE (Figures 1

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and 2). New alleles were obtained for the loci studied, excluding Bcat, that did not present any new

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locus. For the obtained 11 STs, only two (14 and 65) had been previously described (Passerini et al.,

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2010; Luiz et al., 2016). Examination of the strains’ ST distribution using e-BURST software did

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not indicate a common ancestor among the organisms in the collection (Figure 3). Most STs

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presented a distribution type called singletons, which characterize STs that were isolated in the

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diagram because they differed in more than 2 loci of the 6 studied. These were therefore considered

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to be distant profiles of the other strains analyzed. Two clonal complexes were formed, CC1

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composed of 3 strains of STs 109, 111, 108 and CC2 composed of 2 strains with STs 110 and 107 8

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(Fig. 3). The isolates that make up CC1 include two strains from artisanal cheese (Amazonian

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region) and one strain from grass silage. CC2 included 2 strains from buffalo milk and peanut

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silage. The relationships observed in these two complexes indicate the genetic proximity that can

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occur between isolates taken from milk and silage. The lack of correlation between strains with low

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degrees of PFGE similarity that demonstrated MLST genetic relations has been noted by other

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authors (Picozzi et al., 2010; Freitas et al., 2015).

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Some isolates presented 100% similarity by PFGE analysis with different STs, and a distant

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relationship according to ST analysis. These results can be explained simply by the different

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approaches from these methods: MLST can identify small mutations in constitutive genes, while

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PFGE identifies significant rearrangements in the whole genome. The speed with which these

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genetic modifications are evaluated by both methods are different and therefore difficult to

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compare. Nevertheless, in some cases, there is a direct correlation between the isolates found to

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have high similarity by PFGE and isolates with the same ST as reported by Luiz et al. (2016).

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In addition to the isolates grouped as CC, STs were distributed in the form of singletons because

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they had a more distant relationship from the other isolates. STs 103 and 105 were distributed as

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singletons and were composed of only 1 isolate each, both from grass silage (Table 2, Fig. 3). STs

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104 and 106 were formed by 7 and 5 isolates, respectively (Table 2, Fig. 3). Only cow and goat

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milk isolates were characterized as ST 104. All isolates characterized as ST 106 come from

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artisanal cheeses (Amazonian region) (Table 2, Fig. 3). In this case, the grouping may be directly

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related to the region in which the cheeses were obtained. It is believed that despite a variability in

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isolates from the same region, the variability is lower when compared to isolates from other regions

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and/or habitats. Despite the differences between the MLST and PFGE, both methods are

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complementary and can be used together for L. lactis with high discriminatory results (González-

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Arenzana et al., 2014).

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4. Conclusion 9

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After studying L. lactis subsp. lactis strains obtained from Brazilian dairy production environments,

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we characterized their molecular diversity as a first step in the selection of novel starter cultures.

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Novel sequence types were described based on MLST analysis, indicating that this approach can be

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a useful tool to characterize isolates with potential use as starter cultures.

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Declaration of Competing Interest

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The authors declared no conflict of interest.

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Acknowledgements

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We are thankful for the financial support provided by the Brazilian agencies: Conselho Nacional de

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Desenvolvimento Científico e Tecnológico (CNPq, Brasília, DF, Brazil), Fundação de Amparo à

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Pesquisa do Estado de Minas Gerais (FAPEMIG, Belo Horizonte, MG, Brazil), and Coordenação

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de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brasília, DF, Brazil, Financial code

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001).

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17

Table 1. Identification of Lactococcus spp. isolates obtained in Brazilian dairy environment.

a

id

geographical region

original sample

identificationa

identity (%)

acession no.b

INOVALEITE id

2

Southeastern Pará

artisanal cheese (Amazon)

Lactococcus spp.

99.71

CP033606.1

Q4C8

8

Northern Pará

cream milk (cow)

Lactococcus spp.

98.36

KT261220.1

LVTCC8MRS

9

Northern Pará

artisanal cheese (Marajó)

Lactococcus spp.

99.57

CP033606.1

Q13C4

17

Southeastern Minas Gerais

grass silage (dairy farm)

Lactococcus spp.

99.12

CP042408.1

SBR4

18

Southeastern Minas Gerais

cow milk

Lactococcus spp.

99.90

CP033607.1

LVA2VACA

19

Southeastern Minas Gerais

grass silage (dairy farm)

Lactococcus spp.

99.03

MK611133.1

SBR1

20

Southeastern Pará

artisanal cheese (Amazon)

Lactococcus spp.

97.96

KX880977.1

Q1C5

22

Southeastern Pará

artisanal cheese (Amazon)

Lactococcus spp.

98.60

KX880975.1

Q1C10

23

Southeastern Minas Gerais

peanuts silage (dairy farm)

Lactococcus spp.

99.73

CP033606.1

SAM12

24

Southeastern Pará

artisanal cheese (Amazon)

Lactococcus spp.

99.73

CP033606.1

Q5C6

25

Southeastern Pará

artisanal cheese (Amazon)

Lactococcus spp.

99.15

CP042408.1

Q6C2

27

Southeastern Minas Gerais

goat milk

Lactococcus spp.

99.46

CP042408.1

LCA2

35

Southeastern Minas Gerais

goat milk

Lactococcus spp.

99.81

CP042408.1

LCA4

37

Northern Pará

artisanal cheese (Marajó)

Lactococcus spp.

99.47

KX880977.1

Q15C3

38

Southeastern Minas Gerais

cow milk

Lactococcus spp.

99.91

CP033606.1

LVA2.2

39

Southeastern Pará

artisanal cheese (Amazon)

Lactococcus spp.

99.73

CP042408.1

Q1C7

43

Central Minas Gerais

buffalo milk

Lactococcus spp.

99.67

CP033606.1

BUF1

50

Southeastern Minas Gerais

grass silage (dairy farm)

Lactococcus spp.

99.91

CP042408.1

SBR3

55

Southeastern Pará

artisanal cheese (Amazon)

Lactococcus spp.

99.73

CP033606.1

Q1C2

56

Southeastern Pará

artisanal cheese (Amazon)

Lactococcus spp.

99.47

MK889240.1

Q1C4

58

Southeastern Minas Gerais

goat milk

Lactococcus spp.

99.71

CP042408.1

LCA5

61

Southeastern Minas Gerais

cow milk

Lactococcus spp.

99.46

CP033606.1

LVA2.1

65 Southeastern Minas Gerais goat milk identification by 16S rRNA sequencing;

b

accession

Lactococcus spp. number of sequence

of

99.72 closest

relative

CP033606.1 LCA1 found with Blast search.

Table 2. Alleles and STs obtained in MLST analysis of L. lactis subsp. lactis strains. codes

alleles ST

Bcat

Glya

Pdp Pepxp

Pgk Recn

2

65

8

12

9

22

22

14

8

65

8

12

9

22

22

14

9

14

4

4

3

4

6

6

17

103*

11

17

32

6

10

33

18

104*

8

12

14

1

14

14

19

105*

11

17

3

32

33

33

20

106*

8

12

9

11

33

34

22

106*

8

12

9

11

33

34

23

107*

11

17

33

6

3

35

24

108*

2

33

34

6

34

36

25

109*

2

33

34

6

35

36

27

104*

8

12

14

1

14

14

35

104*

8

12

14

1

14

14

37

65

8

12

9

22

22

14

38

104*

8

12

14

1

14

14

39

106*

8

12

9

11

33

34

43

110*

11

17

33

6

3

33

50

111*

11

17

6

6

36

33

55

106*

8

12

9

11

33

34

56

106*

8

12

9

11

33

34

58

104*

8

12

14

1

14

14

61

104*

8

12

14

1

14

14

65 104* * novel STs

8

12

14

1

14

14

Figure captions

Fig. 1. Dendrogram generated after cluster analysis of rep-PCR fingerprints of L. lactis subsp. lactis strains obtained in Brazilian dairy environment. Fig. 2. Dendrogram based on UPGMA clustering of SmaI PFGE profile of L. lactis subsp. lactis strains obtained in Brazilian dairy environment. Fig. 3. e-BURST diagram "population snapshot" of 23 L. lactis subsp. lactis strains obtained in Brazilian dairy environment. Two different clonal complexes (CC1-CC2) were formed. The

size

of

the

points

is

proportional

to

the

number

of

strains.

Fig. 1.

Fig. 2.

Fig. 3.

Highlights

Molecular identification of L. lactis subsp. lactis isolated from dairy environment Presence and genetic diversity of L. lactis subsp. lactis in different sources MLST typing of the strains revealed the existence of 11 STs New alleles were obtained for the loci studied First step in the selection of new starter cultures for use in the dairy industry