Identification of Species and Subspecies of Lactic Acid Bacteria Present in Spanish Cheeses Type "Torta" by MALDI-TOF MS and pheS gene Analyses.

Several artisanal cheeses are elaborated in European countries, being commonly curdled with rennets of animal origin. However, in some Spanish regions some cheeses of type "Torta" are elaborated using Cynara cardunculus L. rennets. Two of these cheeses, "Torta del Casar" and "Torta de Trujillo", are elaborated in Cáceres province with ewe's raw milk and matured over at least 60 days without starters. In this work, we identified the lactic acid bacteria present in these cheeses using MALDI-TOF MS and pheS gene analyses, which showed they belong to the species Lactobacillus curvatus, Lactobacillus diolivorans, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactococcus lactis and Leuconostoc mesenteroides. The pheS gene analysis also allowed the identification of the subspecies La. plantarum subsp. plantarum, La. paracasei subsp. paracasei and Le. mesenteroides subsp. jonggajibkimchii. Low similarity values were found in this gene for some currently accepted subspecies of Lc. lactis and for the two subspecies of La. plantarum, and values near to 100% for the subspecies of Le. mesenteroides and La. paracasei. These results, which were confirmed by the calculated ANIb and dDDH values of their whole genomes, showed the need to revise the taxonomic status of these species and their subspecies.

1. Introduction

Lactic acid bacteria (LAB) encompass Gram positive cocci and rods distributed in different genera, species and subspecies belonging to different families from the order Lactobacillales [1]. Many of these bacteria are considered probiotics due to their beneficial effects for human health [2] and they are present in fermented foods [3].

Cheeses, including artisanal ones, are commonly curdled with rennet of animal origin, however, the Spanish agronomic writer Columela (4–70 AD) mentioned in his book entitled De Re Rustica that cheese can be curdled with the thistle flowers. This practice is currently maintained in some Spanish regions, where the cheeses of type “Torta” are elaborated using Cynara cardunculus L. rennets. The best known of these cheeses is the “Torta del Casar” elaborated in Cáceres province with ewe’s raw milk and matured over at least 60 days without starters.

The LAB present in “Torta del Casar” cheese were initially identified using phenotypic traits [4], and more recently through the analysis of the 16S rRNA gene sequences [5], which was the methodology also used for the identification of these bacteria in other European artisanal cheeses [6,7,8,9,10,11].

However, the 16S rRNA gene has limitations in differentiating among closely related species and subspecies of LAB needing additional techniques, such as the sequencing of protein-coding genes or MALDI-TOF MS [12]. The latter technique has been used to identify the LAB from a French artisanal cheese, showing the presence of species such as La. plantarum and La. paracasei, which encompass several subspecies [13].

The usefulness of MALDI-TOF MS to differentiate some subspecies of La. paracasei, La. plantarum and Lc. lactis has been shown in some works [14,15,16], but the identification at subspecies level should be assessed by the sequencing of protein-coding genes, which have a higher discriminating power than the 16S rRNA gene among closely related taxa. In the case of LAB, the pheS gene has been used, combined with MALDI-TOF MS, for their identification in some fermented foods [17,18], but, to date, these two techniques have not been used together to identify LAB in cheese samples.

Therefore, the first aim of this work was to identify the LAB isolated from two cheeses of type “Torta” elaborated in two different sites (Casar and Trujillo) in Cáceres province in Spain through MALDI-TOF MS and pheS gene analyses. The second aim was to analyse the results obtained with these two techniques compared to those of whole-genome analysis for the differentiation of the subspecies currently accepted within several species of LAB.

2. Materials and Methods

2.1. Strains Isolation

The strains were isolated from ripened cheeses type “Torta” named “Torta del Casar” (Doña Engracia Torta del Casar, Casar de Cáceres, Spain) and “Torta de Trujillo” (or “Retorta de Trujillo”) (Quesería Finca Pascualete, Trujillo, Spain), both elaborated in Cáceres province. For strains’ isolation, we followed the methodology described by Ordiales et al. [5] using MRS agar (Sigma Co., St. Louis, MO., USA) for strain isolation. The inoculated plates were incubated at 20 °C for 48h.

2.2. MALDI-TOF MS Performing and Data Analysis

The sample preparation and the MALDI-TOF MS analysis were carried out as was previously published [19] using a matrix of saturated solution of α-HCCA (Bruker Daltonics, Bremen, Germany) in 50% acetonitrile and 2.5% trifluoracetic acid. We used amounts of biomass between 5 and 100 mg to obtain the spectra as indicated by the manufacturer. The calibration masses were the Bruker Bacterial Test Standards (BTS), which were as follows (masses as averages): RL36, 4365.3 Da; RS22, 5096.8 Da; RL34, 5381.4 Da; RL33meth, 6255.4 Da; RL29, 7274.5 Da; RS19, 10,300.1 Da; RNase A, 13,683.2 Da and myoglobin, 16,952.3 Da.

The score values proposed by the manufacturer are the following: a score value between 2.3 and 3.00 indicates highly probable species identification; a score value between 2.0 and 2.299 indicates secure genus identification and probable species identification, a score value between 1.7 and 1.999 indicates probable genus identification, and a score value <1.7 indicates no reliable identification.

Cluster analysis was performed based on a comparison of strain-specific main spectra, created as described above. The dendrogram was constructed by the statistical toolbox of Matlab 7.1 (MathWorks Inc., Natick, MA, USA) integrated in the MALDI Biotyper 3.0 software. The parameter settings were: ‘Distance Measure=Euclidean’ and ‘Linkage=Complete’. The linkage function is normalized according to the distance between 0 (perfect match) and 1000 (no match).

2.3. Phylogenetic Analysis of pheS Gene

The amplification and sequencing of pheS gene was carried out as indicated by Doan et al. [17] using the primers pheS-21-F (5’-CAYCCNGCHCGYGAYATGC-3’) and pheS-23-R (5’-GGRTGRACCATVCCNGCHCC-3’). The sequences obtained were compared with those from the GenBank using the BLASTN program [20]. The obtained sequences and those of related bacteria retrieved from GenBank were aligned using the Clustal W program [21]. The phylogenetic distances were calculated according to Kimura´s two-parameter model [22]. The phylogenetic trees were inferred using the neighbour joining model [23] and MEGA 7.09 [24] was used for all the phylogenetic analyses.

2.4. Genome Analysis of the Subspecies from the Species Identified in this Study

The Average nucleotide identity blast (ANIb) and Digital DNA–DNA hybridization (dDDH) was calculated using the JSpecies service [25] (http://imedea.uib-csic.es/jspecies/) and dDDH values were calculated using the genome-to-genome distance calculator website service from DSMZ (GGDC 2.1) [26] (http://ggdc.dsmz.de/ggdc.php/). These values were calculated using the formula two at the GGDC website because it is the only function appropriate to analyse draft genomes [27].

3. Results

3.1. MALDI-TOF MS Analysis

The results of this analysis showed that the isolated strains belong to different genera and species of LAB, namely La. curvatus, La. diolivorans, La. paracasei, La. plantarum, La. rhamnosus, Le. mesenteroides and Lc. lactis. All our strains matched with score values near or higher than 2.0 with strains of these species available in the Biotyper 3.0 database (Table 1). Nevertheless, in most cases, the first matching strain is not the strain type of the identified species, and therefore the identification must be confirmed by gene analysis. In order to select representative strains for this analysis, we grouped the isolated strains through mathematical analysis of their, and the resulting dendrogram is shown in Figure 1.

Table 1

Results obtained using MALDI-TOF MS analysis.

Torta del Casar
Strains	Closest Taxa	Score Values	Groups
CCDET 01	La. paracasei subsp. paracasei DSM 20006	2.502	VIIB
	La. paracasei subsp. paracasei DSM 5622T	2.194
	La. paracasei subsp. tolerans DSM 20258T	1.960
CCDET 04	La. diolivorans DSM 14421T	2.228	IIB
CCDET 05	La. paracasei subsp. paracasei DSM 20006	2.504	VIIB
	La. paracasei subsp. paracasei DSM 5622T	2.193
	La. paracasei subsp. tolerans DSM 20258T	2.174
CCDET 07	La. plantarum DSM 2601	2.478	IV
	La. plantarum subsp. argentoratensis DSM 16365T	2.322
	La. plantarum subsp. plantarum DSM 20174T	2.037
CCDET 09	La. paracasei subsp. paracasei DSM 20006	2.511	VIIB
	La. paracasei subsp. paracasei DSM 5622T	2.128
	La. paracasei subsp. tolerans DSM 20258T	1.476
CCDET 10	La. paracasei subsp. paracasei DSM 20006	2.483	VIIB
	La. paracasei subsp. paracasei DSM 5622T	2.097
	La. paracasei subsp. tolerans DSM 20258T	2.051
CCDET 11	La. paracasei subsp. paracasei DSM 20244	2.517	VIIB
	La. paracasei subsp. paracasei DSM 5622T	2.063
	La. paracasei subsp. tolerans DSM 20258T	1.911
CCDET 12	La. paracasei subsp. paracasei DSM 20006	2.433	VIIB
	La. paracasei subsp. tolerans DSM 20258T	2.113
	La. paracasei subsp. paracasei DSM 5622T	2.018
CCDET 13	La. diolivorans DSM 14421T	2.218	IIB
CCDET 14	La. paracasei subsp. paracasei DSM 2649	2.43	VIIB
	La. paracasei subsp. paracasei DSM 5622T	2.047
	La. paracasei subsp. tolerans DSM 20258T	1.773
CCDET 15	La. paracasei subsp. paracasei DSM 20006	2.531	VIIB
	La. paracasei subsp. paracasei DSM 5622T	2.053
	La. paracasei subsp. tolerans DSM 20258T	1.542
CCDET 16	La. paracasei subsp. paracasei DSM 20006	2.545	VIIB
	La. paracasei subsp. tolerans DSM 20258T	2.147
	La. paracasei subsp. paracasei DSM 5622T	2.112
CCDET 18	La. paracasei subsp. paracasei DSM 20006	2.463	VIIB
	La. paracasei subsp. paracasei DSM 5622T	2.107
	La. paracasei subsp. tolerans DSM 20258T	1.911
CCDET19	La. paracasei subsp. paracasei DSM 20244	2.500	VIIA
	La. paracasei subsp. tolerans DSM 20258T	2.309
	La. paracasei subsp. paracasei DSM 5622T	2.103
CCDET20	Le. mesenteroides subsp. dextranicum DSM 20187	2.062	IA
	Le. mesenteroides subsp. mesenteroides DSM 20343T	1.683
	Le. mesenteroides subsp. cremoris DSM 20346T	1.648
CCDET21	La. paracasei subsp. paracasei DSM 20006	2.337	VIIB
	La. paracasei subsp. tolerans DSM 20258T	2.174
	La. paracasei subsp. paracasei DSM 5622T	1.952
CCDET 22	La. paracasei subsp. paracasei DSM 20006	2.380	VIIB
	La. paracasei subsp. paracasei DSM 5622TLa. paracasei subsp. tolerans DSM 20258T	2.0401.752
CCDET 23	La. paracasei subsp. paracasei DSM 20244	2.397	VIIB
	La. paracasei subsp. paracasei DSM 5622T	1.938
	La. paracasei subsp. tolerans DSM 20258T	1.800
CCDET 24	La. paracasei subsp. paracasei DSM 20312	2.355	VIIB
	La. paracasei subsp. paracasei DSM 5622T	2.038
	La. paracasei subsp. tolerans DSM 20258T	1.897
CCDET 25	La. paracasei subsp. paracasei DSM 20006	2.544	VIIB
	La. paracasei subsp. paracasei DSM 5622T	2.115
	La. paracasei subsp. tolerans DSM 20258T	2.092
CCDET 26	La. paracasei subsp. paracasei DSM 20006	2.476	VIIB
	La. paracasei subsp. paracasei DSM 5622T	2.165
	La. paracasei subsp. tolerans DSM 20258T	2.033
CCDET 27	La. plantarum subsp. plantarum DSM 12028	2.177	IV
	La. plantarum subsp. argentoratensis DSM 16365T	2.131
	La. plantarum subsp. plantarum DSM 20174T	1.963
CCDET 28	La. paracasei subsp. paracasei DSM 20244	2.386	VIIB
	La. paracasei subsp. tolerans DSM 20258T	2.097
	La. paracasei subsp. paracasei DSM 5622T	2.097
CCDET 29	La. paracasei subsp. paracasei DSM 46331	2.432	VIIA
	La. paracasei subsp. tolerans DSM 20258T	2.224
	La. paracasei subsp. paracasei DSM 5622T	2.072
CCDET 30	La. paracasei subsp. paracasei DSM 20006	2.492	VIIB
	La. paracasei subsp. paracasei DSM 5622T	2.157
	La. paracasei subsp. tolerans DSM 20258T	2.003
CCDET 32	La. paracasei subsp. paracasei DSM 20006	2.513	VIIB
	La. paracasei subsp. paracasei DSM 5622T	2.113
	La. paracasei subsp. tolerans DSM 20258T	1.540
CCDET 34	La. paracasei subsp. paracasei DSM 20244	2.444	VIIB
	La. paracasei subsp. tolerans DSM 20258T	2.160
	La. paracasei subsp. paracasei DSM 5622T	2.120
CCDET 35	La. paracasei subsp. paracasei DSM 20006	2.475	VIIB
	La. paracasei subsp. paracasei DSM 5622T	2.080
	La. paracasei subsp. tolerans DSM 20258T	1.962
CCDET 38	La. paracasei subsp. paracasei DSM 20006	2.452	VIIB
	La. paracasei subsp. paracasei DSM 5622T	2.112
	La. paracasei subsp. tolerans DSM 20258T	2.083
CCDET 39	La. paracasei subsp. paracasei DSM 20006	2.494	VIIB
	La. paracasei subsp. paracasei DSM 5622T	2.059
	La. paracasei subsp. tolerans DSM 20258T	1.847
CCDET 42	La. paracasei subsp. paracasei DSM 20006	2.223	VIIB
	La. paracasei subsp. paracasei DSM 5622T	1.871
	La. paracasei subsp. tolerans DSM 20258T	1.854
CCDET 43	La. paracasei subsp. paracasei DSM 20244	2.348	VIIB
	La. paracasei subsp. paracasei DSM 5622T	2.035
	La. paracasei subsp. tolerans DSM 20258T	1.990
CCDET 44	La. paracasei subsp. paracasei DSM 20244	2.339	VIIB
	La. paracasei subsp. tolerans DSM 20258T	2.106
	La. paracasei subsp. paracasei DSM 5622T	1.998
CCDET 45	La. paracasei subsp. paracasei DSM 20244	2.353	VIIB
	La. paracasei subsp. paracasei DSM 5622T	2.061
	La. paracasei subsp. tolerans DSM 20258T	2.053
CCDET 46	La. diolivorans DSM 14421T	2.235	IIB
CCDET51	La. paracasei subsp. paracasei DSM 20244	2.437	VIIA
	La. paracasei subsp. tolerans DSM 20258T	2.054
	La. paracasei subsp. paracasei DSM 5622T	2.054
CCDET52	Le. mesenteroides subsp. dextranicum DSM 20187	2.000	IA
	Le. mesenteroides subsp. cremoris DSM 20346T	1.690
	Le. mesenteroides subsp. mesenteroides DSM 20343T	1.374
CCDET53	Le. mesenteroides subsp. mesenteroides DSM 20241	2.120	IA
	Le. mesenteroides subsp. mesenteroides DSM 20343T	2.106
	Le. mesenteroides subsp. cremoris DSM 20346T	1.961
CCDET 54	La. diolivorans DSM 14421T	2.003	IIB
CCDET 55	La. diolivorans DSM 14421T	1.911	IIB
CCDET 56	La. diolivorans DSM 14421T	2.093	IIB
CCDET 57	La. diolivorans DSM 14421T	2.100	IIB
CCDET 58	La. diolivorans DSM 14421T	2.149	IIB
CCDET 59	La. diolivorans DSM 14421T	2.106	IIB
CCDET 61	La. paracasei subsp. paracasei DSM 20006	2.353	VIIA
	La. paracasei subsp. paracasei DSM 5622T	2.170
	La. paracasei subsp. tolerans DSM 20258T	2.101
CCDET62	La. paracasei subsp. paracasei DSM 20244	2.536	VIIA
	La. paracasei subsp. tolerans DSM 20258T	2.157
	La. paracasei subsp. paracasei DSM 5622T	2.148
CCDET 63	La. paracasei subsp. paracasei DSM 8741	2.383	VIIA
	La. paracasei subsp. paracasei DSM 5622T	2.100
	La. paracasei subsp. tolerans DSM 20258T	2.082
CCDET64	La. paracasei subsp. paracasei DSM 20244	2.493	VIIA
	La. paracasei subsp. tolerans DSM 20258T	2.149
	La. paracasei subsp. paracasei DSM 5622T	2.092
CCDET65	Le. mesenteroides subsp. dextranicum DSM 20187	2.072	IA
	Le. mesenteroides subsp. cremoris DSM 20346T	1.692
	Le. mesenteroides subsp. mesenteroides DSM 20343T	1.454
CCDET66	Le. mesenteroides subsp. dextranicum DSM 20187	2.071	IA
	Le. mesenteroides subsp. cremoris DSM 20346T	1.633
	Le. mesenteroides subsp. mesenteroides DSM 20343T	1.355
CCDET67	La. paracasei subsp. paracasei DSM 20244	2.468	VIIA
	La. paracasei subsp. tolerans DSM 20258T	2.233
	La. paracasei subsp. paracasei DSM 5622T	2.047
CCDET68	Le. mesenteroides subsp. mesenteroides DSM 20343T	2.204	IA
	Le. mesenteroides subsp. dextranicum DSM 20187	2.089
	Le. mesenteroides subsp. cremoris DSM 20346T	1.953
Torta de Trujillo
Strains	Closest taxa	Score values	Groups
TRRT01	La. rhamnosus CIP A157T	2.362	VI
TRRT02	Lc. lactis subsp. lactis DSM 20661	2.433	III
	Lc. lactis subsp. lactis DSM 20481T	2.149
	Lc. lactis subsp. cremoris DSM 20069T	1.913
TRRT03	La. rhamnosus CIP A157T	2.389	VI
TRRT04	La. curvatus DSM 20499	2.430	V
	La. curvatus DSM 20019T	2.007
TRRT05	La. paracasei subsp. paracasei DSM 20006	2.393	VIIB
	La. paracasei subsp. tolerans DSM 20258T	2.193
	La. paracasei subsp. paracasei DSM 5622T	2.109
TRRT06	Le. mesenteroides subsp. mesenteroides DSM 20343T	2.368	IB
	Le. mesenteroides subsp. dextranicum DSM 20187	2.097
	Le. mesenteroides subsp. cremoris DSM 20346T	1.963
TRRT07	Le. mesenteroides subsp. mesenteroides DSM 20343T	2.380	IB
	Le. mesenteroides subsp. cremoris DSM 20346T	2.221
	Le. mesenteroides subsp. dextranicum DSM 20187	2.026
TRRT08	Lc. lactis subsp. lactis DSM 20661	2.373	III
	Lc. lactis subsp. lactis DSM 20481T	2.209
	Lc. lactis subsp. cremoris DSM 20069T	1.848
TRRT09	Lc. lactis subsp. lactis DSM 20661	2.283	III
	Lc. lactis subsp. lactis DSM 20481T	2.214
	Lc. lactis subsp. cremoris DSM 20069T	1.896
TRRT10	Lc. lactis subsp. lactis DSM 20661	2.236	III
	Lc. lactis subsp. lactis DSM 20481T	2.198
	Lc. lactis subsp. cremoris DSM 20069T	1.771
TRRT11	La. rhamnosus CIP A157T	2.366	VI
TRRT12	Lc. lactis subsp. lactis DSM 20661	2.310	III
	Lc. lactis subsp. lactis DSM 20481T	2.150
	Lc. lactis subsp. cremoris DSM 20069T	1.983
TRRT13	Lc. lactis subsp. lactis DSM 20661	2.392	III
	Lc. lactis subsp. lactis DSM 20481T	2.255
	Lc. lactis subsp. cremoris DSM 20069T	1.988
TRRT14	Lc. lactis subsp. lactis DSM 20661	2.371	III
	Lc. lactis subsp. lactis DSM 20481T	2.223
	Lc. lactis subsp. cremoris DSM 20069T	1.901
TRRT15	La. rhamnosus CIP A157T	2.324	VI
TRRT16	Lc. lactis subsp. lactis DSM 20661	2.456	III
	Lc. lactis subsp. lactis DSM 20481T	2.228
	Lc. lactis subsp. cremoris DSM 20069T	1.868
TRRT17	La. rhamnosus CIP A157T	2.360	VI
TRRT18	Lc. lactis subsp. lactis DSM 20661	2.521	III
	Lc. lactis subsp. lactis DSM 20481T	2.215
	Lc. lactis subsp. cremoris DSM 20069T	1.998
TRRT19	Lc. lactis subsp. lactis DSM 20661	2.514	III
	Lc. lactis subsp. lactis DSM 20481T	2.196
	Lc. lactis subsp. cremoris DSM 20069T	1.927
TRRT20	Lc. lactis subsp. lactis DSM 20661	2.461	III
	Lc. lactis subsp. lactis DSM 20481T	2.157
	Lc. lactis subsp. cremoris DSM 20069T	1.992
TRRT21	Lc. lactis subsp. lactis DSM 20661	2.538	III
	Lc. lactis subsp. lactis DSM 20481T	2.226
	Lc. lactis subsp. cremoris DSM 20069T	2.045
TRRT22	Lc. lactis subsp. lactis DSM 20661	2.345	III
	Lc. lactis subsp. lactis DSM 20481T	2.286
	Lc. lactis subsp. cremoris DSM 20069T	1.955
TRRT23	La. rhamnosus CIP A157T	2.426	VI
TRRT24	Lc. lactis subsp. lactis DSM 20661	2.468	III
	Lc. lactis subsp. lactis DSM 20481T	2.243
	Lc. lactis subsp. cremoris DSM 20069T	2.036
TRRT25	La. paracasei subsp. paracasei DSM 20006	2.425	VIIB
	La. paracasei subsp. tolerans DSM 20258T	2.181
	La. paracasei subsp. paracasei DSM 5622T	2.144
TRRT26	La. rhamnosus CIP A157T	2.357	VI
TRRT28	Le. mesenteroides subsp. mesenteroides DSM 20343T	2.358	IB
	Le. mesenteroides subsp. dextranicum DSM 20187	2.090
	Le. mesenteroides subsp. cremoris DSM 20346T	2.044
TRRT30	La. curvatus DSM 20499	2.340	V
	La. curvatus DSM 20019T	2.116
TRRT31	La. rhamnosus CIP A157T	2.433	VI
TRRT32	La. rhamnosus CIP A157T	2.350	VI
TRRT33	La. paracasei subsp. paracasei DSM 20006	2.435	VIIB
	La. paracasei subsp. paracasei DSM 5622T	2.136
	La. paracasei subsp. tolerans DSM 20258T	2.103
TRRT34	La. curvatus DSM 20499	2.405	V
	La. curvatus DSM 20019T	2.166
TRRT35	La. rhamnosus CIP A157T	2.367	VI
TRRT36	Le. mesenteroides subsp. mesenteroides DSM 20343T	2.389	IB
	Le. mesenteroides subsp. dextranicum DSM 20187	2.035
	Le. mesenteroides subsp. cremoris DSM 20346T	1.985
TRRT37	La. paracasei subsp. paracasei DSM 20006	2.448	VIIB
	La. paracasei subsp. tolerans DSM 20258T	2.114
	La. paracasei subsp. paracasei DSM 5622T	2.000
TRRT38	Le. mesenteroides subsp. mesenteroides DSM 20343T	2.359	IB
	Le. mesenteroides subsp. dextranicum DSM 20187	2.131
	Le. mesenteroides subsp. cremoris DSM 20346T	1.908
TRRT39	La. curvatus DSM 20499	2.234	V
	La. curvatus DSM 20019T	2.118
TRRT40	La. rhamnosus CIP A157T	2.361	VI
TRRT41	La. rhamnosus CIP A157T	2.354	VI
TRRT42	Le. mesenteroides subsp. mesenteroides DSM 20343T	2.308	IB
	Le. mesenteroides subsp. dextranicum DSM 20187	2.004
	Le. mesenteroides subsp. cremoris DSM 20346T	1.982
TRRT43	La. paracasei subsp. paracasei DSM 20006	2.362	VIIB
	La. paracasei subsp. paracasei DSM 5622T	2.234
	La. paracasei subsp. tolerans DSM 20258T	2.182
TRRT44	La. rhamnosus CIP A157T	2.405	VI
TRRT46	La. paracasei subsp. paracasei DSM 20006	2.459	VIIB
	La. paracasei subsp. tolerans DSM 20258T	2.221
	La. paracasei subsp. paracasei DSM 5622T	2.000
TRRT47	Le. mesenteroides subsp. mesenteroides DSM 20343T	2.322	IB
	Le. mesenteroides subsp. dextranicum DSM 20187	2.041
	Le. mesenteroides subsp. cremoris DSM 20346T	1.864
TRRT48	La. paracasei subsp. paracasei DSM 20006	2.242	VIIB
	La. paracasei subsp. tolerans DSM 20258T	1.983
	La. paracasei subsp. paracasei DSM 5622T	1.658

Figure 1

Cluster analysis of MALDI-TOF MS spectra of strains isolated in this study. Distance is displayed in relative units. Representative strains of each group selected for pheS gene analysis are marked in bold.

The strains were distributed into seven groups with similarity values lower than 2, which correspond to the different species identified in this study (Figure 1). Group I encompasses strains that matched with score values higher than 2.0 with Le. mesenteroides strains and was divided into two subgroups. The strains from the subgroup IA matched with the type strains of Le. mesenteroides subsp. mesenteroides DSM 20343T and Le. mesenteroides subsp. cremoris DSM 20346T and with the non-type strain of Le. mesenteroides subsp. dextranicum DSM 20187 with score values lower than 2.3, whereas those from the subgroup IB matched with the type strain of Le. mesenteroides subsp. mesenteroides DSM 20343T with score values higher than 2.3 (Table 1).

Group II encompasses strains that matched with the type strain of La. diolivorans DSM 14421T and comprised the independent branch IIA and the subgroup IIB (Figure 1). The strain CCDET 55 formed an independent branch and matched with the type strain of La. diolivorans DSM 14421T with a score value lower than 2.0, whereas the strains from subgroup IIB matched with score values higher than 2.0 and lower than 2.3 with the same type strain (Table 1).

Group III encompasses strains that matched with score values higher than 2.0 with Lc. lactis strains (Figure 1). All strains isolated in this study matched with the non-type strain Lc. lactis subsp. lactis DSM 20661, with score values near to or higher than 2.3 with Lc. lactis subsp. lactis DSM 20481T with score values lower than 2.3 and with Lc. lactis subsp. cremoris DSM 20069T with score values lower than 2.0 in most of cases (Table 1).

Group IV encompasses two strains that matched with score values higher than 2.0 with La. plantarum strains (Figure 1). The strain CCDET07 matched with score values higher than 2.3 with the non type strain La. plantarum DSM 2601 and with the type strain of La. plantarum subsp. argentoratensis DSM 16365T, whereas these values were lower than 2.3 with respect to the type strain of La. plantarum subsp. plantarum DSM 20174T. The strain CCDET27 matched with score values higher than 2.0 with respect to the non-type strain La. plantarum DSM 12028 and with the type strain of La. plantarum subsp. argentoratensis DSM 16365T, whereas these values were lower than 2.0 with respect to the type strain of La. plantarum subsp. plantarum DSM 20174T (Table 1).

Group V encompasses strains matching with score values higher than 2.0 with La. curvatus strains (Figure 1). The higher score values, near or higher than 2.3, were found with respect to the non-type strain DSM 20499, whereas these values were lower than 2.3 with respect to the type strain of La. curvatus DSM 20499T (Table 1).

Group VI encompasses strains that matched with the type strain of La. rhamnosus CIP A157T with score values higher than 2.3 in all cases (Figure 1, Table 1).

Finally, group VII encompasses strains that matched with score values higher than 2.0 with La. paracasei strains (Figure 1). This group was divided into two subgroups whose strains mostly matched with score values higher than 2.3 with different non-type strains of La. paracasei subsp. paracasei (DSM 20006, DSM 20244, DSM 2649, DSM 20312 or DSM 8741). Only the strain CCDET19 matched with values higher than 2.3 with respect to the type strain of La. paracasei subsp. tolerans DSM 20258T and the remaining strains matched with La. paracasei subsp. paracasei DSM 5622T and/or La. paracasei subsp. tolerans DSM 20258T with score values lower, near or higher than 2.0, but in all cases lower than 2.3 (Table 1).

Since the type strains of several subspecies identified in this study are included in the Biotyper 3.0 database, we calculated the score values between the subspecies from the same species (Table 2). Score values higher than 2.3, typically found in strains from the same species, were presented by the type strains of the subspecies plantarum and argentoratensis of La. plantarum (2.424) and by those of the subspecies mesenteroides and cremoris of Le. mesenteroides (2.456). However, score values lower than 2.3, which can be found in strains of different species, were found between by the type strains of the subspecies lactis and cremoris of Lc. lactis (2.174) and by those of the subspecies paracasei and tolerans of La. paracasei (1.846). These results show the need to carry out genetic analyses to verify the taxonomic status of these subspecies.

Table 2

Results of the comparison of the type strains of subspecies from different species of LAB identified in this study obtained with different methodologies.

Strains	Closest Species	Score Values MALDI-TOF	pheS Gene Similarity (%)	ANIb (%)	dDDH (%)
La. plantarum subsp plantarum ATCC 14917T (DSM 20174T)	La. plantarum subsp. argentoratensis DSM 16365T	2.424	90.5%	94.9	62.9
La. paracasei subsp paracasei DSM 5622T	La. paracasei subsp. tolerans DSM 20258T	1.846	99.5	97.9	84.9
Le. mesenteroides subsp mesenteroides ATCC 8293T (DSM 20343T)	Le. mesenteroides subsp. cremoris ATCC 19254T (DSM 20346T)	2.456	99.5	98.1	90.9
Le. mesenteroides subsp mesenteroides ATCC 8293T (DSM 20343T)	Le. mesenteroides subsp. dextranicum DSM 20484T	nd	99.2	98.2	91.9
Le. mesenteroides subsp mesenteroides ATCC 8293T (DSM 20343T)	Le. mesenteroides subsp. jonggajibkimchii DRC1506T	nd	99.7	98.4	90.1
Le. mesenteroides subsp. cremoris ATCC 19254T (DSM 20346T)	Le. mesenteroides subsp. dextranicum DSM 20484T	nd	99.7	98.5	91.5
Le. mesenteroides subsp. cremoris ATCC 19254T (DSM 20346T)	Le. mesenteroides subsp. jonggajibkimchii DRC1506T	nd	99.7	98.1	88.5
Le. mesenteroides subsp. dextranicum DSM 20484T	Le. mesenteroides subsp. jonggajibkimchii DRC1506T	nd	99.5	98.4	90.1
Lc.lactis subsp lactis ATCC 19435T (DSM 20481T)	Lc. lactis subsp. cremoris NBRC 100676T (DSM 20069T)	2.174	92.2	86.7	32.7
Lc.lactis subsp lactis ATCC 19435T (DSM 20481T)	Lc. lactis subsp. hordniae CCUG 32210T	nd	99.2	96.7	79.9
Lc.lactis subsp lactis ATCC 19435T (DSM 20481T)	Lc. lactis subsp. tructae DSM 21502T	nd	92.5	86.1	31.7
Lc. lactis subsp. cremoris NBRC 100676T (DSM 20069T)	Lc. lactis subsp. hordniae CCUG 32210T	nd	91.5	86.0	31.4
Lc. lactis subsp. cremoris NBRC 100676T (DSM 20069T)	Lc. lactis subsp. tructae DSM 21502T	nd	98.5	97.5	83.6
Lc. lactis subsp. hordniae CCUG 32210T	Lc. lactis subsp. tructae DSM 21502T	nd	91.8	85.9	31.6

nd: no data because the type strains of some subspecies are not included in the Biotyper 3.0 database.

3.2. pheS Gene Analysis

The analysis of partial sequences of pheS gene of representative strains of different MALDI-TOF MS groups are shown in Figure 2 and Table 2 and Table 3. The results of this analysis confirmed the identification obtained after MALDI-TOF MS analysis at genus and species levels for all strains isolated in this study.

Figure 2

(A) Neighbour-joining phylogenetic unrooted tree based on pheS gene partial sequences (400 nt) showing the taxonomic location of representative strains from different groups of MALDI-TOF MS within the genus Lactobacillus. (B) Neighbour-joining phylogenetic unrooted tree based on pheS gene partial sequences (400 nt) showing the taxonomic location of representative strains from different groups of MALDI-TOF MS within the genera Lactobacillus and Leuconostoc. Bootstrap values calculated for 1000 replications are indicated. Bar, 5 nt substitution per 1000 nt. Accession numbers from Genbank are given in brackets.

Table 3

Results obtained using MALDI-TOF MS and pheS gene analyses.

MALDI-TOF MS Group	Number of Strains	Selected Strains	Closest Taxa	Score Values	pheS Gene Similarity (%)
Group IA	6 from “Torta del Casar”	CCDET66,	Le. mesenteroides subsp. mesenteroides DSM 20343T	1.3–2.2	99.2
	1 from “Torta de Trujillo”	CCDET68	Le. mesenteroides subsp. cremoris DSM 20346T	1.6–2.0	99.7
Group IB *	6 from “Torta de Trujillo”	TRRT07,	Le. mesenteroides subsp. mesenteroides DSM 20343T	2.3–2.4	99.7
		TRRT36	Le. mesenteroides subsp. cremoris DSM 20346T	1.8–2.2	99.5
Branch IIA	1 from “Torta del Casar”	CCDET55	La. diolivorans DSM 14421T	1.9	99.3
Group IIB	9 from “Torta del Casar”	CCDET04, CCDET57	La. diolivorans DSM 14421T	1.9–2.2	99.3
Group III	13 from “Torta de Trujillo”	TRRT10,	Lc. lactis subsp. lactis DSM 20481T	1.9–2.3	99.5
		TRRT20	Lc. lactis subsp. cremoris DSM 20069T	1.7–2.1	99.0
Group IV	2 from “Torta del Casar”	CCDET07	La. plantarum subsp. plantarum DSM 20174T	2.1–2.3	100
			La. plantarum subsp. argentoratensis DSM 16365T	1.9–2.0	90.8
Group V	4 from “Torta de Trujillo”	TRRT34	La. curvatus DSM 20019T	2.0–2.2	99.2
Group VI	13 from “Torta de Trujillo”	TRRT03, TRRT32	La. rhamnosus CIP A157T	2.3–2.4	100
VIIA	8 from “Torta del Casar”	CCDET29	La. paracasei subsp. paracasei DSM 5622T	2.0–2.1	99.5
			La. paracasei subsp. tolerans DSM 20258T	2.0–2.3	99.5
VIIA	8 from “Torta del Casar”	CCDET51	La. paracasei subsp. paracasei DSM 5622T	2.0–2.1	100
			La. paracasei subsp. tolerans DSM 20258T	2.0–2.3	99.7
VIIB	22 from “Torta del Casar”	CCDET16	La. paracasei subsp. paracasei DSM 5622T	1.8–2.3	100
	7 from “Torta de Trujillo”
			La. paracasei subsp. tolerans DSM 20258T	1.9–2.2	99.7
VIIB	22 from “Torta del Casar”	TRRT46	La. paracasei subsp. paracasei DSM 5622T	1.8-2.3	99.5
	7 from “Torta de Trujillo”		La. paracasei subsp. tolerans DSM 20258T	1.9-2.2	99.5

* These strains presented 100% similarity with respect to L. mesenteroides subsp. jonggajibkimchii which is not included in Biotyper 3.0.

According to the results of the pheS gene analysis, several strains were identified with high similarity values as Lactobacillus species that, to date, do not encompasses subspecies (Figure 2A, Table 3). The strains TRRT03, TRRT32 representative of group VI, were identified as La. rhamnosus with 100% similarity. The strain CCDET55, representative of subgroup IIA, and the strains CCDET04, CCDET57, representative of subgroup IIB, were identified as La. diolivorans with 99.3% similarity. The strain TRRT34, representative of group V, was identified as La. curvatus with 99.2% similarity.

After the pheS gene analysis, the remaining strains were identified with high similarity values with LABs of species that contain two or more subspecies (Figure 2A, Table 3). This happened in the case of the strain CCDET07, representative of group IV, which was identified as La. plantarum which currently encompasses two subspecies, L. plantarum subsp. plantarum and L. plantarum subsp. argentoratensis, whose type strains showed 90.5% similarity in their pheS gene sequences (Table 2). The strain CCDET07, representative of group II, can be assigned to the subspecies plantarum, since it presented 100% similarity with respect to the type strain of this subspecies and 90.5% similarity with respect to the type strain of the subspecies argentoratensis (Figure 2B, Table 3).

The representative strains from group I were identified with pheS gene similarity values higher than 99.2% with the species Le. mesenteroides, whose subspecies mesenteroides, cremoris, dextranicum and jonggajibkimchii showed values ranging from 99.2% to 99.7% (Figure 2B, Table 2). The strains CCDET66, CCDET68 representative of subgroup IA were slightly more closesly related to the type strain of Le. mesenteroides subsp. cremoris, with 99.7% similarity, than to the type strains of the remaining subspecies, with similarity values ranging from 99.2% to 99.5%. The strains TRRT07, TRRT36, representative of subgroup IB, presented 100% similarity with respect to the type strain of Le. mesenteroides subsp. jonggajibkimchii, and values ranging from 99.5% to 99.7% with respect to the type strains of the other three subspecies. Therefore, the strains from the group IB can be assigned to the subspecies Le. mesenteroides subsp. jonggajibkimchii, whereas it is difficult to assign those of group IA to any of the subspecies from Le. mesenteroides (Figure 2B, Table 3).

The representative strains from group III were identified with pheS gene similarity values higher than 99.0% as Lc. lactis, whose subspecies formed two clearly separated clusters with less than 93% similarity (Figure 2B, Table 2). Cluster I contains the subspecies lactis and hordniae showing 99.2% similarity and cluster II the subspecies cremoris and tructae, showing 98.5% similarity (Table 2). The strains TRRT10, TRRT20, representative of group III, belong to cluster II and, since they presented 99.0% and 99.5% similarity, respectively, to the subspecies lactis and hordniae, it is difficult to assign the strains of group III to any of these two subspecies (Figure 2B, Table 3).

The representative strains from group VII with pheS gene similarity values higher than 99.5% were identified as La. paracasei, which contains two subspecies, paracasei and tolerans, showing 99.5% similarity between their type strains (Figure 2A, Table 2). The representative strains for both subgroups VIIA and VIIB were divided into two subclusters with 99.5% similarity, each one containing strains of these both subgroups (Figure 2A). The strains CCDET51 and CCDET16 can be assigned to the subspecies paracasei since they showed 100% similarity with the type strain of this subspecies, however, the strains CCDET29 and CCDET46 cannot be assigned to these subspecies because they showed 99.5% similarity with respect to their type strains (Figure 2A, Table 3).

Therefore, the identification at species level obtained by MALDI-TOF MS was confirmed by pheS gene sequencing. Moreover, the pheS gene analysis supports the identification at subspecies level for some strains isolated in this work, but it is remarkable that several others cannot be assigned to any subspecies because they formed subclusters whose similarity values are similar to those found among the currently accepted subspecies of LAB identified in this study.

Collectively, the data from MALDI-TOF MS and pheS gene analyses showed that most of the strains isolated from “Torta del Casar” belong to the species La. paracasei, which was also present in “Torta de Trujillo” and that the species Le. mesenteroides was present in both cheeses in similar proportions. However, other species only were found in one of the two cheeses, La. diolivorans and La. plantarum in “Torta del Casar”, and La. curvatus, La. rhamnosus and Lc. lactis in “Torta de Trujillo” (Figure 3, Table 2).

Figure 3

Pie charts showing the distribution of the different species of LAB in the two cheeses type “Torta” analysed in this study.

3.3. Taxonomic Status of the Subspecies from the Species Identified in this Study

The pheS gene analysis showed that similarity values ranging from 98.5% to 99.7% are presented by the type strains of the subspecies of La. paracasei and Le. mesenteroides, whereas values lower than 93% were found between the type strains of the subspecies of La. plantarum and those of some subspecies of Lc. lactis (Table 2). These results should be compared with those obtained after whole genome analysis, taking into account the threshold values of ANIb and dDDH for bacterial species differentiation (95%~96% and 70%, respectively) [28] and the dDDH cut-off values for bacterial subspecies differentiation (79%~80%) [29].

The whole genomes of the type strains of all subspecies found in this study are available in Genbank and we calculated the ANIb and dDDH values for all of them, whether or not they are present in the Biotyper 3.0 database (Table 2). In agreement with the results of both pheS gene and MALDI-TOF MS analyses, the type strains of the subspecies mesenteroides and cremoris of Le. mesenteroides showed ANIb and dDDH values typical of the same species, 98.1% and 90.9%, respectively (Table 2). Concerning the subspecies dextranicum and jonggajibkimchii, whose type strains are not in Biotyper 3.0 database, in agreement with the results of pheS gene analysis, their ANIb and dDDH values, between them and with respect to the remaining two subspecies, were higher than those proposed for bacterial species differentiation (Table 2).

In agreement with the results of both pheS gene and MALDI-TOF MS analyses, the type strains of the subspecies lactis and cremoris of Lc. lactis showed ANIb and dDDH values typical of different species, 86.7% and 33.1 %, respectively (Table 2). These results confirmed that the type strains of the subspecies cremoris and lactis belong to different species, making it necessary to reclassify the subspecies cremoris into a different, novel species. Nevertheless, it is also necessary to analyse the two subspecies of Lc. lactis that are not present in the Biotyper 3.0 database, as the pheS gene analysis showed that they belong to two divergent clusters, one of them containing the type strains of the subspecies lactis and hordniae and the other containing the type strains of the subspecies cremoris and tructae. Taking into account the ANIb and dDDH values found among the type strains of these subspecies, the subspecies hordniae should be maintained within the species Lc. lactis, and the subspecies tructae, together with the subspecies cremoris, should be transferred to a novel species (Table 2).

In agreement with the results of the pheS gene analysis, but not with those of MALDI-TOF MS analysis, the type strains of the subspecies plantarum and argentoratensis of La. plantarum, which showed ANIb and dDDH values typical of different species, 94.9% and 62.9%, respectively, should be considered different species, making it necessary to reclassify the subspecies argentoratensis in a novel species. In agreement with the results of the pheS gene analysis, but not with those of MALDI-TOF MS analysis, the type strains of the subspecies paracasei and tolerans of La. paracasei showed ANIb and dDDH values typical of the same species, 97.9% and 84.9%, respectively (Table 2).

4. Discussion

There is a growing interest in the identification of LAB present in artisanal cheeses elaborated with raw milk in Europe, with those elaborated with cow or/and goat raw milks being more analysed [6,7,8,10,11,13] compared to those elaborated with ewe’s raw milk [5,9].

In Spain, one of the most appreciated cheeses is the named type “Torta”, elaborated with ewe’s raw milk in Caceres province, and therefore it is also interesting to know the species of LAB present in these cheeses. In a work published in the last century, the lactic bacteria present in the “Torta del Casar” cheese were identified on the basis of phenotypic traits [4]. More recently, by 16S rRNA gene analysis, the species Lactobacillus sakei, Lactobacillus casei, Lactobacillus helveticus and Lc. lactis subsp. cremoris have been identified in this cheese [5]. These two works were only carried out in the “Torta del Casar” cheese and using techniques that have limitations for species and particularly for subspecies differentiation. For this reason, in this study we compared the results obtained in two cheeses type “Torta” elaborated in the same region by using more recent methodologies. From the species previously identified in the “Torta del Casar” cheese [5], in the present work only L. lactis has been identified in the “Torta de Trujillo” cheese. Nevertheless, the species identified in this study have been found in some of the European artisanal cheeses elaborated with raw milk [7,8,9,10,11,12,13].

From the mentioned cheeses, only the LAB present in the French cheese Maroilles were identified by MALDI-TOF MS [13]. The authors showed that this methodology is very useful to identify LAB belonging to different genera and species, but they do not demonstrate its usefulness in differentiating among subspecies. Considering that many species of LAB contain several subspecies, this is an essential issue to be discussed by comparison with other molecular techniques, particularly genomic ones.

In this study, we identified four species which contain several subspecies, La. plantarum, La. paracasei, Le. mesenteroides and Lc. lactis (http://www.bacterio.net/) of which the most common inhabitants in milk-related sources are present in the database Biotyper 3.0. In addition to the type strain, several strains are included in this database for Le. mesenteroides subsp. mesenteroides, Lc. lactis subsp. lactis, Lc. lactis subsp. cremoris, La. paracasei subsp. paracasei, La. paracasei subsp. tolerans and La. plantarum subsp. plantarum. The presence of most than one strain for a taxon in a database is a priori positive, but this can be an important disadvantage if some strains are not correctly assigned to a taxon, as seems to occur for several strains from the subspecies of Le. mesenteroides, Lc. lactis, La. paracasei and La. Plantarum, present in the Biotyper 3.0 database. For example, the non-type strain of La. plantarum subsp. plantarum DSM 20205 is more distant from the type strain of this subspecies than the type strain of La. plantarum subsp. argentoratensis (Figure 4A). In the case of La. paracasei, there is a greater distance among strains of the same subspecies than among strains of different species (Figure 4A). In the case of Le. mesenteroides, the non-type strain Le. mesenteroides subsp. mesenteroides DSM 2040 is more distant from the type strain of this subspecies than the strain Le. mesenteroides subsp. dextranicum DSM 20187 (Figure 4B). Several strains assigned to Lc. lactis subsp. lactis are more distant from the type strain of this subspecies than to that of Lc. lactis subsp. cremoris (Figure 4B). These results indicate that several non-type strains held in DSMZ culture collection which are included in the Biotyper 3.0 database are not correctly classified at species or subspecies levels, but, as no gene sequences are available for these strains, we cannot know their correct taxonomic name and this could lead to errors in the identification of any tested strain. For this reason, we always referred to a type strain in the identification of our strains, although the score values were lower than those found for non-type strains (Table 1).

Figure 4

Cluster analysis of MALDI-TOF MS spectra of the strains belonging to the species identified in this study which are included in the biotyper 3.0 database within genera Lactobacillus (A) and Leuconostoc and Lactococcus (B). Distance is displayed in relative units.

Moreover, we found some surprising score values for the type strains of the subspecies from La. paracasei and Lc. lactis because the subspecies are infraspecific taxa and score values higher than 2.3 among these subspecies are expected after MALDI-TOF MS analysis (Table 2). In the case of the type strains of the subspecies paracasei and tolerans of La. paracasei, the score value was clearly lower than 2.0, indicating that these strains do not belong to the same species (Table 2). This contrasts with the high similarity value of the pheS gene sequences and the high ANIb and dDDH values calculated from their genomes (Table 2). These two values, which are clearly higher than those proposed for species differentiation [28], confirmed that the type strains of paracasei and tolerans belong to the same species, therefore the type strains of these subspecies held in DSMZ culture collection and in the Biotyper 3.0 database should be revised. In addition, they showed that the dDDH values are higher than those proposed for subspecies differentiation [29], therefore the taxonomic status of these subspecies should be revised.

In the case of the type strains of the subspecies lactis and cremoris of Lc. lactis, the low values found in the pheS gene analysis agree with the calculated ANIb and dDDH values, which were lower than those proposed for species differentiation, confirming that they belong to different species (Table 2). Concerning to the other two subspecies, hordniae and tructae, not included in Biotyper database, the pheS gene analysis showed that their type strains are phylogenetically related to the subspecies lactis and cremoris, respectively (Table 2). The calculated ANIb and dDDH values confirmed that Lc. lactis really contains two different species with two subspecies each, although the dDDH values were near to or slightly lower than those proposed for subspecies differentiation in both cases (Table 2). These results clearly indicate that the taxonomic status of the subspecies currently included within Lc. lactis should be revised in order to separate the subspecies cremoris as a novel species and to evaluate whether the subspecies hordniae and tructae can maintain their current taxonomic status.

Conversely, the score value found between the type strains of the subspecies plantarum and argentoratensis of La. plantarum was surprisingly high (2.424) considering the low similarity value found between their pheS genes (90.5%), and that the calculated ANIb and dDDH values were lower than those proposed for bacterial species differentiation (Table 2). These results indicate that the type strain of the subspecies argentoratensis held in DSMZ culture collection and in the Biotyper 3.0 database should be revised and that it should be reclassified as a novel species.

In the case of the subspecies mesenteroides and cremoris of Le. mesenteroides score, values higher than 2.3 were expected, as this corresponds to strains from the same species (Table 2). In agreement, they showed high similarity in their pheS genes and the calculated ANIb and dDDH values were higher than those proposed for bacterial species differentiation (Table 2). In the case of the subspecies dextranicum and jonggajibkimchii, absent in the Biotyper 3.0 database, the pheS gene analysis also showed high similarity values in agreement with those of the ANIb and dDDH, which were also higher than those proposed for bacterial species’ differentiation. Moreover, the dDDH values among the type strains of all these subspecies considerably exceed the upper limit proposed for subspecies differentiation, and therefore their taxonomic status should be revised.

Therefore, the application of the currently accepted ANIb and dDDH cut-off values for species differentiation [28] will lead to the promotion of some subspecies to the taxonomic status of species. Conversely, the application of the dDDH threshold value for subspecies differentiation [29] will lead to the loss of taxonomic status for some subspecies, as recently occurred with the subspecies sakuensis of Serratia marcescens [30]. Although Chun et al. [28] considered that currently there is not enough information to establish general guidelines for species differentiation on the basis of genome data, we face the dilemma of whether to increase the dDDH threshold value for subspecies differentiation, maintain the existing ones, or reject many of the existing subspecies in several species of LAB. Before trying to solve this, we should take into account that the increase necessary to maintain the current subspecies would cause a dramatic increase in the number of these taxa, since the pheS gene similarity cut-off values would be above 99% and only in this study several strains cannot be assigned to any of the described subspecies because they fall within these limits and could be considered as novel subspecies. We should also consider that applying the dDDH thresholds values proposed by Meier-Kolthoff et al. [29] would mean that none of the subspecies from the species identified in this study can maintain their taxonomic status, except perhaps the subspecies hordniae of Lc. lactis. This second option better agrees with the results of the MALDI-TOF MS analysis that clearly allows the identification of the strains isolated at species level, compared to identification at subspecies level. In any case, this situation should be clarified, since it affects several LABs from different genera and species, and currently there is an increasing interest in the identification of these bacteria, particularly in fermented foods.

5. Conclusions

The LAB present in the two cheeses of type “Torta” analysed in this study were identified as La. curvatus, La. diolivorans, La. paracasei, La. plantarum, La. rhamnosus, Lc. lactis and Le. mesenteroides through MALDI-TOF MS and pheS gene analyses. These results confirmed that MALDI-TOF MS is a reliable method for the identification of LAB comparable to pheS gene sequence analysis and presents important advantages over gene sequencing in terms of rapidity and cost per sample. The analysis of pheS gene showed low similarity values for some subspecies of Lc. lactis and for the two subspecies of La. plantarum and values near to 100% for the subspecies of Le. mesenteroides and La. paracasei. These results were confirmed by the calculated ANIb and dDDH values of their whole genomes, showing the need for a revision of the taxonomic status of these species and their subspecies, which should be based on additional criteria.