Introduction

Beneficial bacteria are responsible for a healthy environment in the gut ecosystem when they are present in adequate concentrations [1]. Evaluation of the beneficial potential and safety properties of such strains is important, as is their use in commercial dairy products [2]. The genus Corynebacterium consists of numerous species, some of which are increasingly recognized as important pathogens related to human and animal diseases [3]. However, some of the strains of this genus are safe and can even be considered beneficial. Corynebacterium vitaeruminis has already been shown to be safe and non-pathogenic [4, 5], but it has not been studied in greater detail. In the evaluation process of newly isolated strains with beneficial potential, the bacteria as well as their virulence potential need to be identified to make sure that these strains do not present risks to consumers. Thus, this communication aimed to present select beneficial properties and safety characteristics of C. vitaeruminis MRU4 isolated from the cow rumen.

Materials and Methods

Corynebacterium vitaeruminis MRU4

The strain C. vitaeruminis MRU4 was isolated from a cow rumen sample after plating on de Man Rogosa Sharpe (MRS) agar (Oxoid Ltd., Basingstoke, England) and incubating at 37 °C for 48 h. This isolate was characterized by Gram staining (positive) and the catalase test (positive), and it was subjected to phenotypical tests to assess its resistance to gastric pH (2.0, 2.5, and 3.0; control 7.2) and bile (0.5 and 3%; control 0%) by plating and optical density, according to Argyri et al. [6]; results were compared by ANOVA and Tukey’s test (p < 0.05) using XLSTAT 2016.01.26192 (AddinSoft, New York, NY, USA). The isolate was then differentiated from other isolated bacteria by rep-PCR and random amplification of polymorphic DNA-PCR (RAPD-PCR) [7]. Taxonomical identification was confirmed by sequencing the PCR-amplified 16S rRNA gene (Center for Human Genome Studies, Institute of Biomedical Sciences, University of São Paulo, Brazil). The resulting sequences were compared to known sequences in GenBank using the Basic Local Alignment Search Tool (BLAST) [8]. In addition, selected enzymatic activities were detected by the API ZYM Kit (bioMérieux, Basingstoke, Hants) according to the manufacturer’s instructions.

Beneficial Properties

Resistance to Simulated Gastric and Intestinal Conditions

The resistance of C. vitaeruminis MRU4 to gastric and intestinal conditions was confirmed using an in vitro model according to dos Santos et al. [9]. Mean counts of log C. vitaeruminis MRU4 populations were compared by ANOVA and Tukey’s test (p < 0.05) using XLSTAT 2016.01.26192 (AddinSoft).

Aggregation Properties

Auto-aggregation and co-aggregation abilities of C. vitaeruminis MRU4 with co-aggregation partners L. monocytogenes Scott A, Enterococcus faecalis ATCC 19443 and Lactobacillus sakei ATCC 15521 were assessed according to dos Santos et al. [9].

Cell Surface Hydrophobicity

The test for bacterial cell surface hydrophobicity, related to adhesion of the studied strain to hydrocarbons, was performed according to dos Santos et al. [9], with 37 °C as the incubation temperature.

Evidence for the Presence of Genes Related to Beneficial Properties

DNA from C. vitaeruminis MRU4, cultured in MRS for 24 h at 37 °C, was isolated by the ZR Fungal/Bacterial DNA Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions, and the concentration was determined by spectrophotometry (NanoDrop, Thermo Scientific, Whaltam, MA, USA). DNA obtained from C. vitaeruminis MRU4 was subjected to PCR analysis for the presence of genes related to the bacterial adhesion characteristics. The target genes used were EF1249 (fibrinogen binding protein), EF2380 (membrane-associated zinc metalloprotease), EF2662 (choline binding protein), prgB (surface protein), EFTu (adhesion-like factor), and map and mub (mucus adhesion genes) [10].

Bile Salt Deconjugation

The strain’s ability to perform bile salt deconjugation was evaluated according to the method described by dos Santos et al. [9].

Lactose Assimilation

The ability of the selected strain to metabolize lactose was tested according to Pelinescu et al. [11], using glucose as control. Mean counts of log C. vitaeruminis MRU4 populations were compared by ANOVA (p < 0.05) using XLSTAT 2016.01.26192 (AddinSoft).

Statistical Analysis

All experiments were conducted in duplicate with three repetitions. Populations were compared by ANOVA (p < 0.05) using XLSTAT 2016.01.26192 (AddinSoft, New York, NY, USA).

Safety Characteristics

Phenotypical Evidence for Virulence

C. vitaeruminis MRU4 was subjected to phenotypical tests to identify its hemolytic, DNase, gelatinase, and lipase activities, according to Barbosa et al. [12]. All tests were performed at 25 °C and 37 °C.

Biogenic Amine Production

The production of biogenic amines was evaluated according to Bover-Cid, Holzapfel [13] at 25 °C and 37 °C.

Antibiotic Resistance

The tested culture was subjected to phenotypical analysis of antibiotic resistance using antibiotic discs (Oxoid) and Etest® strips (bioMérieux SA, Marcy l’Etoile, France). The following antibiotics were used: oxacillin (1 μg/disc), sulfa/trimethoprim (25 μg/disc), tetracycline (30 μg/disc), imipenem (10 μg/disc), ampicillin (10 μg/disc), erythromycin (15 μg/disc), vancomycin (30 μg/disc), rifampicin (5 μg/disc), gentamicin (10 μg/disc), penicillin (10 U/disc), clindamycin (2 μg/disc), and chloramphenicol (30 μg/disc). The inhibition zones around the discs was measured and classified as presenting resistance (R) or sensitivity (S) according to the manufacturer’s instructions and the recommendations of the European Committee on Antimicrobial Susceptibility Testing [14]. The presence of intermediate resistance was considered as resistant. In addition, the minimum inhibitory concentration (MIC) of five antibiotics (vancomycin, gentamicin, chloramphenicol, ampicillin, and rifampicin), representative of the important antibiotic classes, were determined. Considering the recorded MIC (μg/mL) for each antibiotic against C. vitaeruminis MRU4, the studied strain was classified as presenting resistance (R) or sensitivity (S), according to the manufacturer’s instructions for rifampicin, and the recommendations of the European Committee on Antimicrobial Susceptibility Testing [14] for the other antibiotics tested.

Detection of Virulence and Resistance Genes

The presence of 50 virulence, antibiotic resistance and biogenic amine-related genes was investigated: vanA, vanB, vanC1, vanC-1, vanC2, and vanC2/C3 (vancomycin resistance); tet(K), tet(L), tet(M), tet(O), and tet(S) (tetracycline resistance); ermA, ermB, and ermC (erythromycin resistance); catA (chloramphenicol resistance); aph(2″)-lb., ant(4′)-la, aph(2″)-ld, aph(2″)-lcand aph(3′)-llla (aminoglycoside antibiotic family resistance); aac(6′)-le-aph(2″)-Ia (gentamicin and aminoglycoside resistance); vat(E) (streptogramin resistance); bcrB, bcrD, and bcrR (bacitracin resistance); ant(6)-la (streptomycin resistance); mur-2ed (specific for E. durans); aac(6′)-li (specific for E. faecium); mur-2 (specific for E. hirae); Ddl E. faecalis (specific for E. faecalis); ace (adhesion of collagen of E. faecalis); asa1 (aggregation substance); cyt2 (cytolysin and hemolytic endotoxins); esp. (enterococcal surface protein); efaA (endocarditis antigen); cob, cpd, and ccf (chemotactic for human leukocytes and facilitated conjugation); sprE (serine protease); fsrA, fsrB, and fsrC (gelE regulation); gelE (gelatinase production); int and int-Tn (transposon integrase gene); odc (ornithine decarboxylase); tdc (tyrosine decarboxylase); hdc1 and hdc2 (histidine decarboxylase); hyl (hyaluronidase) [15, 16].

Results and Discussion

Samples of raw milk, cow, and goat salivary and vaginal mucosa swabs; ruminal boluses; consumption water; and silage were screened for presence of beneficial bacteria in order to investigate their potential application as future probiotics and to ensure their safety. A collection of 500 isolates was built based on the preliminary screening, including survival at low pH and in the presence of bile. The rep-PCR and RAPD PCR were used as basic tools for the differentiation of isolated, potentially beneficial, strains. Based on the previous 16S rRNA sequencing, according to the BLAST database analysis, the isolated strain (encoded MRU4) presented 97% similarity to C. vitaeruminis strain DSM 20294 and was named C. vitaeruminis MRU4. To the best of our knowledge, this is the first report regarding the isolation of C. vitaeruminis from the cow rumen. As shown in Fig. 1, C. vitaeruminis MRU4 presented a high survival rate in the screening process for resistance to low pH and the presence of bile salts. Comparing the initial counts and after 3 h of different pH treatments, we observed that C. vitaeruminis MRU4 presented a slight decrease in the microbial population at pH 2.0, and the same behavior was observed in absorbance (A) at 650 nm (Fig. 1). In addition, the strain survived at the tested bile salts concentrations and exhibited good bile tolerance after 4 h of incubation (Fig. 1). Survival at different pH values and bile salts concentrations is mandatory for probiotic cultures, since this is related to survival of these bacteria in the passage through the gastrointestinal tract [17]. Based on its enzymatic profile (Table 1), C. vitaeruminis MRU4 generated positive results for the presence of esterase, esterase lipase, leucine arilamidase, α- chymotrypsin, acid phosphatase, naphthol phosphohydrolase and α- galactosidase. There was no activity for the 12 enzymes included in the API ZYM test of the total 19 present enzymes: alkaline phosphatase, lipase, valine arylamidase, cystine arylamidase, trypsin, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase.

Fig. 1
figure 1

Viability of C. vitaeruminus MRU4 at different pH values (a treatment of 3 h) and bile concentrations (b treatment of 4 h). Results obtained by plating (white and gray bars, indicating log CFU/mL counts at 0 h and 3 h/4 h, respectively) and optical density (lines, indicating recorded absorbances at 650 nm), with their respective standard deviations. Values with different uppercase or lowercase letters differs by ANOVA and Tukey’s test (p < 0.05)

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Table 1 Beneficial and safety properties of Corynebacterium vitaeruminis MRU4 isolated from the cow rumen
Full size table

The beneficial and safety characteristics related to C. vitaeruminis MRU4 are also summarized in Table 1. The confirmatory test for resistance at different pH values and bile salts concentrations was conducted considering the gastrointestinal tract characteristics. C. vitaeruminis MRU4 was able to survive in the gastric phase with a survival rate of 99.6%. In addition, the strain was able to survive and even multiply in the intestinal phase with a survival rate of 100.9%. Many studies have shown survival rates of more than 98% for potential probiotic strains [18, 19].

The aggregation (auto-aggregation and co-aggregation) ability is the capacity of the strain to adhere and form biofilms on various surfaces, allowing the beneficial strain to persist in the gastrointestinal environment, which facilitates the beneficial effects for the host. The results showed that C. vitaeruminis RU4 had a 53.2% auto-aggregation rate. C. vitaeruminis MRU4 showed the following results for co-aggregation: 42.4% with L. monocytogenes ScottA, 41.6% with E. faecalis ATCC 19443, and 10.0% with Lb. sakei ATCC 15521. Many studies have shown a large range for auto-aggregation and co-aggregation presented by probiotic bacteria, which is in agreement with our study [18].

C. vitaeruminis MRU4 showed 98.2% of cell surface hydrophobicity. Vinderola et al. [20] considered this feature to be a species-specific parameter. Moreover, some studies showed cell surface hydrophobicity rates of 5.4 to 79% for probiotic cultures [18, 21]. In addition, the selected strain generated positive results for the presence of two genes: EFTu and map. The first one is an adhesion-like factor gene that also aids in cell adhesion, and the second one is up-regulated in the presence of mucus [10].

The selected strain had a high ability to grow on MRS agar plates containing 0.5% (w/v) sodium salts of TC, TDC, GC, and GDC. This indicates a good capability to reduce cholesterol, and it is therefore desirable for use in probiotic products for human consumption [22]. In agreement with our study, many authors have reported the deconjugation capacity of probiotic cultures [18, 21].

Production of the β-galactosidase enzyme allows the probiotic culture to assimilate lactose and minimize lactose intolerance [19, 21]. C. vitaeruminis MRU4 showed no β-galactosidase activity in the API ZIM kit test. The ability of beneficial bacteria to assimilate lactose is a great advantage for use in probiotic foods targeted for lactose intolerant individuals. The results for the lactose assimilation test showed that C. vitaeruminis MRU4 presented better lactose assimilation (9.24 ± 0.02) than glucose (8.15 ± 0.01, p < 0.05) which was used as the control (Table 1).

C. vitaeruminis MRU4 did not express any virulence factors, such as for hemolytic activity, gelatinase production, lipase production, and deoxyribonuclease activity, in in vitro tests at both 25 °C and 37 °C. The same was verified for in vitro detection of biogenic amine production: C. vitaeruminis MRU4 was negative for lysine, tyrosine, histidine, and ornithine biogenic amines, as expected for safe strains [23]. Pisano et al. [24] highlighted the importance of the lack of these virulence factors for probiotic cultures.

Regarding antimicrobial resistance, C. vitaeruminis MRU4 was resistant to oxacillin, gentamicin, erythromycin, clindamycin, sulfa/trimethoprim, and rifampicin based on the disc diffusion method. Considering the Etest® strips, C. vitaeruminis MRU4 was resistant just to vancomycin. This vancomycin resistance can be due to an intrinsic characteristic of the studied bacteria. This is in agreement with the observation that based on the performed PCR, we could not detect the presence of genes related to vancomycin resistance. Studies of antimicrobial resistance in probiotic cultures have shown that resistance is species-specific and there is no pattern for resistance with the tested antibiotics [25].

Based on the performed PCR screening for the presence of virulence related genes, C. vitaeruminis MRU4 was negative for the 50 tested genes. The results obtained in this study agree with those obtained by other authors, and they show that these results are species and strain-specific [21]. The absence of antibiotic resistance or virulence genes suggests that there might be a new virulence mechanism that can occur by either acquiring genes or by mutation of endogenous genes [26].

In summary, C. vitaeruminis MRU4 demonstrated safety and potential beneficial functions in in vitro tests. However, it is necessary to emphasize the importance of additional studies regarding the safety of this strain, as well as confirmation of the benefits through in vivo testing in animal models and humans.