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- Springer Journals
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- Copyright © 2017 by Springer-Verlag Berlin Heidelberg and the University of Milan
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- Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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- 1590-4261
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- 1869-2044
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- 10.1007/s13213-017-1258-2
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Abstract
Ann Microbiol (2017) 67:289–301 DOI 10.1007/s13213-017-1258-2 ORIGINAL ARTICLE In vitro assessment of safety and probiotic potential characteristics of Lactobacillus strains isolated from water buffalo mozzarella cheese 1,2 3 4 Sabrina Neves Casarotti & Bruno Moreira Carneiro & Svetoslav Dimitrov Todorov & 4 5 1 Luis Augusto Nero & Paula Rahal & Ana Lúcia Barretto Penna Received: 10 August 2016 /Accepted: 7 February 2017 /Published online: 28 February 2017 Springer-Verlag Berlin Heidelberg and the University of Milan 2017 Abstract The aim of this study was to evaluate the safety and presented resistance to several antibiotics, the resistance was probiotic potential characteristics of ten Lactobacillus spp. limited to antibiotics to which the strains had intrinsic resis- strains (Lactobacillus fermentum SJRP30, Lactobacillus casei tance. Furthermore, the strains presented a limited spread of SJRP37, SJRP66, SJRP141, SJRP145, SJRP146, and genes encoding virulence factors and resistance to antibiotics, SJRP169, and Lactobacillus delbrueckii subsp. bulgaricus and none of the strains presented hemolytic or mucin degra- SJRP50, SJRP76, and SJRP149) that had previously been dation activity. The L. delbrueckii subsp. bulgaricus strains isolated from water buffalo mozzarella cheese. The safety of showed the lowest survival rate after exposure to simulated GI the strains was analyzed based on mucin degradation, hemo- tract conditions, whereas all of the L. casei and L. fermentum lytic activity, resistance to antibiotics and the presence of strains showed good survivability. None of the tested lactobacilli genes encoding virulence factors. The in vitro tests concerning strains presented bile salt hydrolase (BSH) activity, and only probiotic potential included survival under simulated gastro- L. casei SJRP145 did not produce the β-galactosidase enzyme. intestinal (GI) tract conditions, intestinal epithelial cell adhe- The strains showed varied levels of adhesion to Caco-2 cells. sion, the presence of genes encoding adhesion, aggregation None of the cell-free supernatants inhibited the growth of path- and colonization factors, antimicrobial activity, and the pro- ogenic target microorganisms. Overall, L. fermentum SJRP30 duction of the β-galactosidase enzyme. Although all strains and L. casei SJRP145 and SJRP146 were revealed to be safe and to possess similar or superior probiotic characteristics com- pared to the reference strain L. rhamnosus GG (ATCC 53103). * Sabrina Neves Casarotti . . . sabrinacasarotti@yahoo.com.br Keywords Lactic acid bacteria Dairy Safety Antibiotic . . resistance Beneficial properties Gastrointestinal tract * Ana Lúcia Barretto Penna analucia@ibilce.unesp.br survival Departamento de Engenharia e Tecnologia de Alimentos, Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Introduction Paulista (UNESP), R. Cristovão Colombo, 2265, 15054-000 São José do Rio Preto, São Paulo, Brazil Lactobacillus spp. belong to the group of lactic acid bacteria Departamento de Alimentos e Nutrição, Faculdade de Nutrição, (LAB) and have a long history of use in the production of Universidade Federal de Mato Grosso (UFMT), dairy products due to their ability to convert lactose into lactic 78060-900 Cuiabá, MT, Brazil acid (Tulumoğlu et al. 2014). In addition to their use as tech- Instituto de Ciências Exatas e Naturais, Universidade Federal de nological agents in the food industry, some Lactobacillus spe- Mato Grosso (UFMT), 78735-901 Rondonópolis, MT, Brazil 4 cies can confer health benefits to the host when they are ad- Departamento de Veterinária, Universidade Federal de Viçosa ministered adequately as probiotics. Probiotics are currently (UFV), 36570-000 Viçosa, MG, Brazil 5 defined Bas live microorganisms that, when administered in Departamento de Biologia, Instituto de Biociências, Letras e Ciências adequate amounts, confer health benefit on the host^ (Hill Exatas, Universidade Estadual Paulista (UNESP), Rua Cristóvão Colombo, 2265, 15054-000 São José do Rio Preto, SP, Brazil et al. 2014). Although probiotics have been extensively 290 Ann Microbiol (2017) 67:289–301 studied and commercialized, and are the subject of national Materials and methods and international regulations, there is no agreement concerning the amount of probiotic bacteria necessary to pro- Bacterial strains duce their beneficial effects. Generally, probiotic food prod- ucts must contain 10 CFU/mL or CFU/g (Shah 2000). Ten Lactobacillus strains previously isolated and identified Nevertheless, some authors state that beneficial effects can through 16S rRNA gene sequencing by our group (Silva be achieved even when bacteria lose their viability (Adams et al. 2015; Silva 2015)as Lactobacillus fermentum 2010). (SJRP30), Lactobacillus casei (SJRP37, SJRP66, SJRP141, Some of the health effects attributed to probiotic consump- SJRP145, SJRP146, and SJRP169), and Lactobacillus tion include the regulation of gastrointestinal (GI) functions, delbrueckii subsp. bulgaricus (SJRP50, SJRP76 and relief of lactose intolerance, prevention of different types of SJRP149) were screened for their safety and probiotic poten- diarrhea besides urogenital infections, reduction in cholesterol tial. Lactobacillus rhamnosus GG (ATCC 53103) was used as levels, reduction in atopic and food allergies, and modulation a probiotic reference strain. The strains were maintained at of the immune system. Furthermore, in vitro studies have −80 °C in MRS broth (Difco, Becton Dickinson, Sparks, shown that probiotic bacteria reduce the number of pathogens MD) supplemented with 25% (v/v) glycerol (Vetec, Duque and their metabolic activities in the human intestine and com- de Caxias, RJ, Brazil). Each culture was sub-cultured at least pete with these microorganisms for attachment sites to intes- twice in MRS broth before use in the assays. tinal epithelial cells and nutrients (Guarner and Malagelada 2003;Mishra et al. 2015). Assessment of safety characteristics Although a large number of probiotic strains are available for commercial use worldwide, the isolation and characteriza- Hemolytic activity tion of new strains from different species is desirable; thus, many studies in this field have been published in recent years Fresh lactobacilli broth cultures (8.0–9.0 log CFU/mL) were (Jeronymo-Ceneviva et al. 2014; Peres et al. 2014; de Paula streaked in triplicate on Columbia agar plates containing 5% et al. 2015; Oh and Jung 2015). Probiotics targeted for human (w/v) sheep blood (NewProv, Pinhais, PR, Brazil). After 48 h consumption are usually isolated from humans or animals of incubation at 37 °C, the plates were examined for hemolytic because strains from these origins can better adapt to the con- reactions. The Lactobacillus rhamnosus GG (ATCC 53103) ditions encountered in the human/animal GI tract, which en- and Staphylococcus aureus ATCC 6538 strains were used as ables more successful gut colonization (Argyri et al. 2013). the negative and positive controls, respectively (Pieniz et al. However, certain food-associated Lactobacillus strains have 2014). The assay was repeated on three independent occasions probiotic characteristics even though they do not belong to the in triplicate. gut microbiota (Solieri et al. 2014; Tulumoğlu et al. 2014). According to the FAO/WHO (2002), a bacterial strain Mucin degradation should fulfill a number of requirements to be considered pro- biotic; these requirements must be verified by in vitro and Mucin degradation was determined according to Zhou et al. in vivo tests. In vitro tests are useful for the selection of strains (2001). Salmonella enterica subsp. enterica serovar that have greater probiotic potential; these tests increase Typhimurium ATCC 14028 and Lactobacillus rhamnosus knowledge regarding the strain as well as the mechanisms GG (ATCC 53103) were used as the positive and negative underlying the beneficial effects. Although LAB, particularly controls, respectively. The assay was repeated on three inde- Lactobacillus, are generally recognized as safe (GRAS), ad- pendent occasions in triplicate. ditional tests should be performed to check the safety of these strains because some cases recently associated systemic infec- Presence of genes encoding virulence factors, antibiotic tion with the consumption of probiotics (Liong 2008; Sharma resistance and biogenic amines and Devi 2014). Thus, evaluating their safety, assessing their resistance to antibiotics, investigating the presence of viru- The Lactobacillus strains were tested for the presence of vir- lence genes, and determining hemolytic activity are important ulence, antibiotic resistance and amino acid decarboxylase (Jeronymo-Ceneviva et al. 2014; Vijayakumar et al. 2015). genes (Table 1). DNA was extracted using the QIAgen Given these points, the aim of this study was to characterize DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany), the safety features and probiotic potential attributes of autoch- followed by DNA concentration estimation using the thonous Lactobacillus spp. isolated from water buffalo moz- NanoDrop2000 spectrophotometer (Thermo Scientific, zarella cheese using in vitro tests. Candidates that met the Waltham, MA). PCRs were performed according to the refer- established criteria may be used in the production of ences listed in Table 1, and the amplified products were sep- fermented products to promote their probiotic characteristics. arated by electrophoresis in 0.8 to 2.0% (w/v) agarose gels in Ann Microbiol (2017) 67:289–301 291 Table 1 Presence of genes implicated in virulence factors, antibiotic resistance and biogenic amine production in Lactobacillus spp. strains Gene Encoded factor L. fermentum L. casei L. delbrueckii subsp. bulgaricus Reference SJRP30 SJRP37 SJRP66 SJRP141 SJRP145 SJRP146 SJRP169 SJRP50 SJRP76 SJRP149 Virulence gelE Gelatinase – – + –– –– – + + Vankerckhoven et al. (2004) hyl Hyaluronidase –– – – – – – ++ – Vankerckhoven et al. (2004) asa1 Aggregation substance –– – – – – – + –– Vankerckhoven et al. (2004) esp Enterococcal surface protein – ++ + –– + –– – Vankerckhoven et al. (2004) cylA Cytolysin –– – – – – + –– – Vankerckhoven et al. (2004) efaA Endocarditis antigen – – – – –– –– – – Martín-Platero et al. (2009) ace Adhesion of collagen – – – – –– –– – – Martín-Platero et al. (2009) fsrA Gelatinase – ++ + + – + –– – Lopes et al. (2006) fsrB Gelatinase – ++ + –– + –– – Lopes et al. (2006) fsrC Gelatinase –– – – – – – ++ – Lopes et al. (2006) sprE Serine protease –– – – + –– – – + Lopes et al. (2006) ccf Sex pheromones, chemotactic for – + – ++ + – + –– Eaton and Gasson (2001) human leukocytes; facilitate conjugation cob Sex pheromones, chemotactic for –– – – – – – + – + Eaton and Gasson (2001) human leukocytes; facilitate conjugation cpd Sex pheromones, chemotactic for – + – + – ++ + –– Eaton and Gasson (2001) human leukocytes; facilitate conjugation Biogenic amine hdc1 Histidine decarboxylase – + –– – – + –– +deLasRivasetal.(2005) hdc2 Histidine decarboxylase – – – – –– –– – – de Las Rivas et al. (2005) tdc Tyrosine decarboxylase –– – – – – – ++ – de Las Rivas et al. (2005) odc Ornithine decarboxylase – – – – –– –– – – de Las Rivas et al. (2005) Antibiotic resitance van A Vancomycin resistance – ++ + –– + –– – Martín-Platero et al. (2009) van B Vancomycin resistance – ++ + – ++ –– – Martín-Platero et al. (2009) vanC1 Vancomycin resistance –– – + –– – + + + Dutka-Malen et al. (1995) vanC-2,vanC-3 Vancomycin resistance + –– – – – + –– – Dutka-Malen et al. (1995) vanC1 Vancomycin resistance + –– – – – + –– – Miele et al. (1995) tet(M) Tetracycline resistance – – – – –– –– + – Aarestrup et al. (2000b) tet(L) Tetracycline resistance –– – + –– – ++ – Aarestrup et al. (2000b) 292 Ann Microbiol (2017) 67:289–301 Table 1 (continued) Gene Encoded factor L. fermentum L. casei L. delbrueckii subsp. bulgaricus Reference SJRP30 SJRP37 SJRP66 SJRP141 SJRP145 SJRP146 SJRP169 SJRP50 SJRP76 SJRP149 tet(K) Tetracycline resistance – + –– – – – + – + Aarestrup et al. (2000b) tet(O) Tetracycline resistance – + + ++ ++ –– – Aarestrup et al. (2000b) tet(S) Tetracycline – – – – –– –– – – Aarestrup et al. (2000a) bcr(B) Bacitracin resistance –– + – ++ + –– – Manson et al. (2004) bcr(D) Bacitracin resistance – – – – –– –– – – Manson et al. (2004) bcr(R) Bacitracin resistance –– – – – + –– – – Manson et al. (2004) erm(A) Erythromycin resistance – – – – –– –– – – Sutcliffe et al. (1996) erm(B) Erythromycin resistance – – – – –– –– – – Sutcliffe et al. (1996) erm(C) Erythromycin resistance – + – ++ ++ – + – Sutcliffe et al. (1996) erm(B) Erythromycin resistance – – – – –– –– – – Gevers et al. (2003) ant(4′)-Ia Aminoglycoside resistance + + + + + – + –– – Fortina et al. (2008) aph(3′)-III-a Aminoglycoside resistance – + –– – – + + + + Fortina et al. (2008) aph(2″)-Ib Aminoglycoside resistance ++ – ++ + –– – – Fortina et al. (2008) aph(2″)-Ic Aminoglycoside resistance – ++ + + – + –– – Fortina et al. (2008) aph(2″)-Id Aminoglycoside resistance –– – ++ –– + + + Fortina et al. (2008) Aminoglycoside resistance + + + – + –– + – + Fortina et al. (2008) aac(6′)-Ie-ap- h(2″)-Ia aac(6′)-Ii Aminoglycoside resistance + + + + + – ++ + – Costa et al. (1993) catA(PIP501) Chloramphenicol resistance – ++ –– –– – – – Aarestrup et al. (2000a) int-Tn Tetracycline resistance –– + –– –– – – + Fortina et al. (2008) int Transposon integrase gene + + + + – + – ++ + Geversetal.(2003) + Indicates the presence and – absence of genes Ann Microbiol (2017) 67:289–301 293 0.5× TAE buffer. The gels were stained in 0.5× TAE buffer Bile salt hydrolase activity containing 0.5 μg/mL of ethidium bromide (Sigma-Aldrich, St. Louis, MO). Fresh bacterial cultures of the studied lactobacilli (8.0–9.0 log CFU/mL) were screened for bile salt hydrolase (BSH) activity as previously described by de Paula et al. (2014) using MRS Antibiotic susceptibility plates supplemented with taurodeoxycholic acid sodium salt (TDCA) or taurocholic acid sodium salt hydrate (TC); MRS The disc diffusion assay was applied to determine the antibi- plates without TDCA and TC were used as negative controls, otic susceptibility of the strains. Diluted culture (100 μL; 6.0 whereas L. mesenteroides SJRP 55 was used as a positive log CFU/mL) was spread onto MRS agar media (Difco), and control. The plates were incubated anaerobically at 37 °C for antibiotic discs (Oxoid, Basingstoke, UK) containing (per 48 h. The presence of precipitated bile acid around the spots disc) ampicillin (10 μg), vancomycin (30 μg), gentamicin was considered a positive result (Rodríguez et al. 2012). The (10 μg), kanamycin (30 μg), streptomycin (300 μg), tetracy- assay was repeated on three independent occasions in cline (30 μg), chloramphenicol (30 μg), erythromycin triplicate. (15 μg), and clindamycin (2 μg) were placed manually on the surface of the inoculated plates using sterile forceps. Adhesion to Caco-2 cells These antibiotics were chosen according to the list proposed by the European Food Safety Authority (EFSA 2012). The The Caco-2 cell line BCRJ 0059 (Rio de Janeiro Cell Bank, plates were incubated at 37 °C under anaerobic conditions, Rio de Janeiro, Brazil) was cultured (passages 29–31) in and the diameters of the inhibition zones were evaluated Dulbecco’s modified Eagle’s minimum (DMEM, Sigma- 24 h after incubation. The susceptibility of the isolates was Aldrich) supplemented with 10% heat-inactivated fetal bovine scored as resistant, moderately susceptible, or susceptible ac- serum (Cultilab, Campinas, Brazil), a mixture of penicillin cording to the cut-off values proposed by Charteris et al. (100 UI/mL) and streptomycin (100 μg/mL) (Sigma- (1998). The assay was repeated on three independent occa- Aldrich), and 1% non-essential amino acid solution (Sigma- sions in triplicate. Aldrich) at 37 °C in a 5% CO atmosphere. The adhesion assay was performed as described by Argyri et al. (2013). All bacterial cultures were grown for 18 h in Assessment of probiotic potential characteristics MRS at 37 °C before the assays, harvested by centrifugation (7000 g, 7 min, 5 ° C), washed twice with phosphate-buffered Tolerance to simulated GI tract conditions saline (PBS) and re-suspended in DMEM without any serum or antibiotics. The commercial probiotic L. rhamnosus GG The tolerance to simulated GI tract conditions test was per- (ATCC 53103) was used as a reference strain. Subsequently, formed by successively exposing the strains to gastric and 1 mL containing approximately 8.0–9.0 log CFU bacterial enteric simulated juices as described by Botta et al. (2014). cells was added to each well, and each strain was evaluated The lactobacilli strains were grown for 18 h at 37 °C in MRS for adherence in duplicate wells in each experiment. After broth, and 1 mL of each culture (8.0–9.0 CFU/mL) was dis- incubation for 2 h at 37 °C, the cells were washed three times tributed into four sterile flasks (two for the gastric phase and with sterile PBS to remove non-adherent bacteria, and then two for the enteric phase). The solutions simulating the gastric detached from each well by the addition of 1 mLTriton X-100 and enteric juices were prepared according to the method of (0.5% v/v) (Sigma-Aldrich). Following incubation for 5 min Bautista-Gallego et al. (2013). The pH values used in the at 37 °C, the cell lysates were serially diluted and plated on gastric and enteric phases were 2.5 and 8.0, respectively. All MRS agar. Bacterial adhesion (%) was calculated by the ratio enzyme solutions were prepared and filter-sterilized using a of adhered bacteria to the total number of added bacteria. The 0.22-μm membrane filter (Merck Millipore, Cork, Ireland) on experiment was performed on three independent occasions. the day of analysis. The cells were counted at the beginning (T )and the end of the gastric phase (T ) and after the enteric phase (T ). Presence of genes encoding adhesion, aggregation 120 360 The cell count was performed by serial dilution and plating and colonization factors in MRS agar (Difco). The plates were incubated at 37 °C for 48 h under anaerobic conditions (Anaerobac, Probac, The investigated Lactobacillus strains were tested for the pres- São Paulo, Brazil). The commercial probiotic L. rhamnosus ence of adhesion, aggregation and colonization genes GG (ATCC 53103) was used as a reference strain. The (Table 2) as described in the section BPresence of genes assay was repeated on three independent occasions in encoding virulence factors, antibiotic resistance and biogenic duplicate. amines^. 294 Ann Microbiol (2017) 67:289–301 Antimicrobial activity All lactobacilli strains were tested for antimicrobial activity against Escherichia coli ATCC 25922, E. coli ATCC 8739, Listeria innocua ATCC 33090, Listeria monocytogenes ATCC 15313, Klebsiella pneumoniae subsp. pneumoniae ATCC 10031, Staphylococcus aureus subsp. aureus ATCC 25923, Salmonella enterica subsp. enterica serovar Typhimurium ATCC 14028 and Shigella sonnei ATCC 25931 according to the method described by de Paula et al. (2014). The antibiotic ciprofloxacin (5 μg) was used as a positive control, whereas MRS broth adjusted to pH 6.5 and filtered was used as a negative control. The assay was repeated on three independent occasions in triplicate. β-Galactosidase activity The β-galactosidase activity of the Lactobacillus spp. strains was assessed by employing sterile filter paper discs impreg- nated with o-nitrophenyl-β-D-galactopyranose (ONPG Discs, Fluka, Buchs, Switzerland) according to the manufacturer’s instructions. The test was performed in three independent ex- periments in duplicate. S. enterica subsp. enterica serovar Typhimurium ATCC 14028 and E. coli ATCC 25922 were used as the negative and positive controls, respectively. Statistical analysis The statistical analysis was performed using the Statistica 7.0 software (StatSoft, Inc., 2004, Tulsa, OK). One-way ANOVA followed by Tukey’s test was applied to detect significant differences (P ≤ 0.05) in the data regarding tolerance to simu- lated GI tract conditions and in vitro adhesion to Caco-2 cells. Results Hemolytic activity None of the examined strains revealed β-hemolytic (i.e., red blood cell lysis) activity when grown in Columbia sheep blood agar. Most of the strains (L. fermentum SJRP30 and L. casei strains SJRP37, SJRP66, SJRP145, SJRP146, and SJRP169) were γ-hemolytic (i.e., no hemolysis), whereas four strains showed partial hemolysis (L. delbrueckii subsp. bulgaricus SJRP50, SJRP76, and SJRP149 and L. casei SJRP141). Staphylococcus aureus ATCC 6538 (positive con- trol) showed hemolytic activity. Mucin degradation Neither the Lactobacillus spp. nor the reference strain (nega- tive control) showed mucinolytic activity in either type of Table 2 Presence of genes implicated in adhesion, aggregation and colonization in Lactobacillus spp. strains Gene Encoded factor L. fermentum L. casei L. delbrueckii subsp. bulgaricus Reference SJRP30 SJRP37 SJRP66 SJRP141 SJRP145 SJRP146 SJRP169 SJRP50 SJRP76 SJRP149 Mub Adhesion proteins – – – –– –– + – + Ramiah et al. (2007) mapA Adhesion proteins –– – – + –– + – + Ramiah et al. (2007) EF-Tu Elongation factor – ++ + + + – + + + Ramiah et al. (2007) EF2662-cbp Choline binding protein – – – –– ––– – + Fortina et al. (2008) EF1249-fbp Fibrinogen binding protein –– – – ++ –– + Fortina et al. (2008) EF2380-maz Membrane-associated zinc metalloprotease–– ++ – + – + –– Fortina et al. (2008) prgB Surface protein –– – – – + – ––– Fortina et al. (2008) + Indicates the presence and – absence of genes Ann Microbiol (2017) 67:289–301 295 tested medium (with or without glucose). Conversely, Tolerance to simulated GI tract conditions S. enterica subsp. enterica serovar Typhimurium ATCC 14028 (positive control) was able to degrade mucin in vitro There was a significant decrease (P ≤ 0.05) in the popula- in a medium in which mucin was the only energy source. tions of all strains evaluated after consecutive exposure to the gastric and small intestine conditions (Fig. 1). The L. delbrueckii subsp. bulgaricus strains showed the lowest Presence of genes encoding virulence factors, antibiotic population at the end of the in vitro assay, with a cell count resistance and biogenic amines reduction of 3.38 log CFU/mL on average. In contrast, L. casei and L. fermentum showed good viability during None of the tested isolates presented a positive result for the the simulated GI digestion, with reductions of 0.85–2.48 efaA, ace, hdc2, odc, tet(S), erm(A),and erm(B) genes, which log units (Fig. 1). Two groups of tolerance were outlined were related to endocarditis antigen, collagen adhesion, tyro- after exposure to simulated gastric juice at pH 2.5; L. casei sine decarboxylase, ornithine decarboxylase, tetracycline re- SJRP37, SJRP66, SJRP141, SJRP145, SJRP146 and sistance and erythromycin resistance, respectively (Table 1). SJRP169, L. delbrueckii subsp. bulgaricus SJRP76 and L. casei SJRP169 had the highest frequency of genes L. rhamnosus GG maintained the same populations, encoding virulence factors, antibiotic resistance and biogenic whereas L. fermentum SJRP30 and L. delbrueckii subsp. amines (42.55%). Conversely, the L. fermentum SJRP30 bulgaricus SJRP50 and SJRP149 showed a significant strain showed the lowest frequency (14.89%) of positive (P ≤ 0.05) reduction in their populations. Tolerance to the results. enteric condition was variable among the strains. The L. fermentum SJRP30 and L. casei SJRP146 strains suf- fered a reduction of less than 1 log unit after exposure to Antibiotic susceptibility simulated enteric juice. The other L. casei strains and L. delbrueckii subsp. bulgaricus SJRP50 suffered a reduc- All of the strains were sensitive to ampicillin, tetracycline, tion between 1 and 2 log units, whereas L. delbrueckii chloramphenicol, erythromycin, and clindamycin, which are subsp. bulgaricus SJRP76 and SJRP149 revealed a reduc- frequently used to treat bacterial infections (Table 3). All of tion of 3.07 and 3.59 log CFU/mL, respectively. the L. delbrueckii subsp. bulgaricus strains were sensitive to vancomycin and gentamicin, whereas the other strains were BSH activity resistant. Most strains were susceptible to streptomycin, with the exception of L. fermentum SJRP30, which was classified All L. casei and L. fermentum strains were able to grow in as moderately susceptible. All of the tested strains were clas- MRS agar plates containing 0.5% (w/v) TDCA sodium sified as resistant to kanamycin. salts, whereas the growth of L. delbrueckii subsp. Table 3 Antibiotic susceptibility of Lactobacillus spp. strains. AMP Ampicillin, VA vancomycin, CN gentamicin, K kanamycin, S streptomycin, TE tetracycline, C chloramphenicol, E erythromycin, DA clindamycin Species Strains AMP VA CN K S TE C E DA (10 μg) (30 μg) (10 μg) (30 μg) (300 μg) (30 μg) (30 μg) (15 μg) (2 μg) L. fermentum SJRP30 29 S 0 R 9 R 0 R 14 MS 26 S 27 S 27 S 27 S L. casei SJRP37 29 S 0 R 9 R 0 R 19 S 30 S 29 S 30 S 27 S SJRP66 32 S 0 R 13 S 0 R 23 S 35 S 31 S 37 S 34 S SJRP141 33 S 0 R 12 R 0 R 26 S 36 S 29 S 37 S 33 S SJRP145 28 S 0 R 10 R 0 R 20 S 31 S 27 S 31 S 27 S SJRP146 33 S 0 R 13 S 0 R 22 S 38 S 32 S 37 S 32 S SJRP169 35 S 0 R 14 S 0 R 25 S 36 S 34 S 36 S 33 S L. delbrueckii subsp. SJRP50 28 S 21 S 10 R 11 R 24 S 31 S 29 S 32 S 30 S bulgaricus SJRP76 27 S 21 S 10 R 7 R 23 S 31 S 30 S 32 S 29 S SJRP149 38 S 23 S 10 R 0 R 25 S 31 S 30 S 33 S 31 S L. rhamnosus GG ATCC 30 S 0 R 10 R 0 R 22 S 33 S 31 S 32 S 28 S Inhibition zones were measured in millimeters, and the susceptibility of the isolates was scored as resistant (R), moderately susceptible (MS) and susceptible (S) according to the cut-off values proposed by Charteris et al. (1998) 296 Ann Microbiol (2017) 67:289–301 Fig. 1 Survival of Lactobacillus spp. strains before ( )and during during the same sampling period of the in vitro assay. Different lower exposure to in vitro simulated gastric conditions for 120 min ( , case letters denote significant differences (P ≤0.05) among sampling pH 2.5) and enteric conditions for 360 min ( , pH 8). Different periods for the same strain in the in vitro assay. The results are capital letters denote significant differences (P ≤ 0.05) among strains expressed as the mean ± SD. n= 3 bulgaricus was completely inhibited. Conversely, all Presence of genes encoding adhesion, aggregation strains grew in MRS agar plates with 0.5% (w/v) TC. and colonization factors Nevertheless, none of the tested lactobacilli strains showed BSH activity. L. delbrueckii subsp. bulgaricus SJRP149 showed positive results for all tested genes encoding adhesion, aggregation and colonization factors with the exception of the prgB gene. Adhesion to Caco-2 cells In contrast, strain L. fermentum SJRP30 did not harbor any of these genes. The other studied strains possessed at least one of All of the tested strains could adhere to Caco-2 cells, but they the genes (Table 2). did so to different degrees. The adhesion rates ranged from 2.59% to 18.58%. L. fermentum SJRP30, L. casei strains Antimicrobial activity SJRP37, SJRP145, and SJRP146 and L. delbrueckii subsp. bulgaricus SJRP76 showed similar adherence to the reference The growth of pathogenic target microorganisms was not strain after 2 h of incubation (P ≤ 0.05). L. casei SJRP141 was inhibited by any of the cell-free supernatants (CFS) (adjusted the most adhesive strain and presented adherent bacteria to pH 6.5) obtained from the tested LAB strains (data not counts higher than the positive control L. rhamnosus GG. shown). L. casei SJRP66 and L. delbrueckii subsp. bulgaricus SJRP149 had the lowest adhesion capacities. The results indi- β-galactosidase activity cate that adhesion properties are strain-specific because the strains do not show similar adhesion values even though they Strains L. casei SJRP146 and L. delbrueckii subsp. bulgaricus SJRP50 and SJRP76 displayed an intense yellow color in their are from the same species or genus (Fig. 2). Ann Microbiol (2017) 67:289–301 297 is not considered a desirable feature for probiotic strains be- cause it contributes to changes in the intestinal mucosal barrier in addition to favoring mucosal invasion by pathogens and other toxic agents (Monteagudo-Mera et al. 2012; Peres et al. 2014). Our results suggest that the evaluated probiotic candidates may not be able to invade the intestinal mucosa. These findings were in agreement with previous studies that investigated mucin degradation by several LAB species (Fernández et al. 2005; Abe et al. 2010; Rodríguez et al. 2012). The main concern regarding probiotic safety is the resis- tance to antibiotics because these strains may transfer anti- biotic resistance genes to pathogenic bacteria in the intesti- nal habitat, which can represent a serious risk for the treat- Fig. 2 Adhesion capacity of Lactobacillus spp. strains to Caco-2 cells. ment of infected patients. Antibiotic resistance is consid- The adhesion capacity is calculated using the ratio of the number of ered a negative characteristic for probiotics (Lee et al. bacterial cells that remained attached to the total number of bacterial cells 2014). The strains were found to be resistant to vancomycin, added initially to each well. Different lower case letters denote significant gentamicin, streptomycin, and kanamycin; however, this differences (P ≤ 0.05) among the adhesion capacities of the strains. The results are expressed as the mean ± SD. n= 3 resistance pattern is considered an intrinsic feature of LAB because it is chromosomally encoded and, thus, the corre- tests. The other strains, except for L. casei SJRP145, also sponding genes will not be transferred to pathogens (Tulini showed positive results; however, these strains produced a less et al. 2013;Botta etal. 2014;Sharmaetal. 2015). Taking intense yellow color. The reference strain L. rhamnosus GG these reports into consideration, the resistance to vancomy- and L. casei SJRP145 did not present β-galactosidase activity. cin, kanamycin, streptomycin, and gentamicin found in the strains and in the reference strain L. rhamnosus GG can be considered acceptable. Discussion During the course of testing the strains for the presence of virulence genes, at least one of the genes responsible for In this study, we performed an in vitro analysis to determine gelatinase production (gelE, fsrA, fsrB and fsrC) was detected the safety and probiotic potential characteristics of ten in all strains except L. fermentum SJPR30. The gelE gene is responsible for the production of gelatinase, which is an en- Lactobacillus strains. The assays were chosen based on inter- national guidelines for evaluation of probiotic potential (FAO/ zyme that hydrolyzes gelatin and collagen. Moreover, gelE WHO 2002). Although a large number of studies has been expression is thought to be regulated in a cell density- published in this field in the past, the identification of new dependent manner by the products of fsrA, fsrB and fsrC. strains with probiotic potential is always desirable, mainly However, the presence of gelE does not seem to be sufficient because each strain shows different methods of action and for gelatinase activity, and a complete fsr operon may be man- several benefits to health. Nevertheless, before being used as datory for gelE expression (Lopes et al. 2006). In our study, a probiotic, the safety of the strains needs to be assessed to none of the strains contained the complete fsr operon. ensure that they will not represent a risk to consumer health. Only L. casei SJRP169 presented the cylA gene; howev- Recently, some cases relating infections to probiotic consump- er, four isolates (L. casei SJRP141 and L. delbrueckii subsp. tion have been reported (Kochan et al. 2011; Zbinden et al. bulgaricus SJRP50, SJRP76 and SJRP149) showed partial 2015). Therefore, determining whether a strain is safe is of hemolysis in the phenotypic test, whereas SJRP169 did not. great concern among researchers. Other lytic genes most likely cause this hemolytic reaction A lack of hemolytic activity is considered a safety require- in the phenotypic tests when the cylA gene is not expressed ment when selecting a probiotic strain (FAO/WHO 2002)be- (Perin et al. 2014). Four L. casei strains contained the esp cause such bacteria are not virulent, and the lack of hemolysin gene, which may be a result of horizontal transference by ensures that opportunistic virulence will not appear among the Enterococcus genus. Conversely, the adhesion proper- strains (Peres et al. 2014). Previous reports also revealed that ties conferred by the esp gene can be a significant charac- different LAB species did not show hemolysis (Argyri et al. teristic for potential probiotic bacteria (de Paula et al. 2014). 2013; Bautista-Gallego et al. 2013; Ryu and Chang 2013; Sex pheromone genes (ccf, cob and cpd)werepresent in Ilavenil et al. 2015). The production of enzymes capable of some of the evaluated strains. These genes are also consid- degrading mucin was proposed as a determinant factor of ered virulence factors because they might induce an inflam- virulence for some enteropathogens. Therefore, this property matory response. Moreover, these genes have shown 298 Ann Microbiol (2017) 67:289–301 in vitro chemotactic activity for human and rat polymorpho- histamine, tyramine and ornithine into biogenic amines, re- nuclear leukocytes, and elicited superoxide production and spectively. Low levels of biogenic amines in food are not the secretion of lysosomal enzymes (Eaton and Gasson considered a serious risk to the consumer; however, they can 2001). be toxic when present in high concentrations (50–100 mg) Genes encoding antibiotic resistance were also tested. (Jeronymo-Ceneviva et al. 2014). The presence of these genes The antibiotic resistance genes vanA and vanB were fre- has been reported for other LAB (Coton et al. 2010; quently present among the tested strains. Almost all of the Jeronymo-Ceneviva et al. 2014). L. casei strains showed positive results for both genes, ex- Concerning the probiotic potential characteristics, the eval- cept L. casei SJRP145 and L. casei SJRP146 in which the uated strains showed good resistance to simulated gastric vanA gene was absent. The vanC genes were harbored by juice. In response to stress caused by acid, LAB use various L. fermentum SJRP30, L. casei SJRP141 and SJRP169 and mechanisms to overcome the damage, including maintaining all of the L. delbrueckii subsp. bulgaricus strains. The vanA the intracellular pH and cell membrane functionality and in- phenotype is characterized by a higher resistance level to ducing stress-response proteins (Wu et al. 2014). These mech- vancomycin than the vanB phenotype and cross-resistance anisms vary both within species and according to exogenous to teicoplanin. The gene cluster for both vanA and vanB conditions, including the growth media and incubation condi- resistance is usually located on a plasmid that is transfer- tions (Madureira et al. 2011). The negative effect of bile on the able, and thus represents a major concern for safety due to viability of most lactobacilli was more accentuated than the the spread of antibiotic resistance via horizontal gene trans- effect of low pH. The bile concentration in the human body fer (Klein et al. 2000; Perin et al. 2014). Conversely, vanC is usually ranges from 0.3% to 0.5% (García-Ruiz et al. 2014); located in the bacterial chromosome (Martín-Platero et al. therefore, probiotic bacteria must to be able to withstand these 2009). Therefore, despite the observation of resistance dur- bile concentrations. Bile is a toxic component that damages ing the disc diffusion test, the resistance towards vancomy- the membrane by modifying its integrity and permeability. cin in L. fermentum SJRP30 and L. casei SJRP145 recorded Additionally, bile disturbs the stability of macromolecules, in our study was considered intrinsic, chromosomally including RNA, DNA and proteins, and may cause oxidative encoded, and not inducible or transferable (Tynkkynen stress (Begley et al. 2006). et al. 1998). The lack of genes encoding vancomycin resis- Generally, L. casei strains showed higher viability after tance has been reported (Casado Muñoz et al. 2014). successive exposures to gastric and intestinal conditions than However, other studies have demonstrated the presence of L. delbrueckii subsp. bulgaricus. The survival of the selected vanA and vanB genes in a variety of LAB (Jeronymo- strains, with the exception of L. delbrueckii subsp. bulgaricus Ceneviva et al. 2014; Perin et al. 2014). SJRP149, was greater than the survival observed for the com- mercial strain used as a reference; similar results were obtain- In this study, all strains contained at least one gene encoding aminoglycoside resistance, which could be associ- ed by Argyri et al. (2013). The good tolerance of ated with the intrinsic resistance towards this antibiotic class Lactobacillus strains to gastric juice and bile is in accordance among Lactobacillus spp. The most common tetracycline re- with the results reported in a previous study (Jensen et al. sistance gene among Lactobacillus spp. strains was tet(O), 2012). Nevertheless, no recovery in cell viability was ob- which was detected in all of the L. casei strains. However, served for any of the strains in the subsequent treatment with tet(S) was not recorded in any of the strains. Tetracycline enteric juice, which was in contrast to reports by other authors resistance genes have been found in other Lactobacillus spp. (Corsetti et al. 2008; Bautista-Gallego et al. 2013). This dis- strains isolated from fermented dry sausages, cheese and yo- crepancy most likely occurred because the strains did not have gurt (Zonenschain et al. 2009;Zhou et al. 2012). The int gene the ability to metabolize conjugated bile salts. The ability to was detected in eight strains, indicating that these strains hydrolyze bile salts is usually included as one of the criteria might harbor the transposon responsible for tet gene dissem- for the selection of strains with probiotic potential (Rodríguez ination. Although the int gene has not been found in et al. 2012). Nonetheless, BSH activity is rare among bacteria Lactobacillus strains to date, it has been identified in that have been isolated from environments with an absence of Enterococcus and Lactococcus strains with food origins bile, such as the strains used in the present study. This finding (Bulajić et al. 2015; Morandi et al. 2015; Jaimee and Halami is in agreement with other studies (Bautista-Gallego et al. 2016). Regarding erythromycin resistance, only the erm(C) 2013; Solieri et al. 2014). gene was found among the Lactobacillus sp. strains. This gene Adhesion to intestinal epithelial cells is commonly includ- was previously detected in a variety of Lactobacillus species ed as an in vitro test to select probiotic strains. Although (Kastner et al. 2006; Klare et al. 2007;Egervärn etal. 2009). in vitro adhesion assays are useful for providing information The hdc1 and tdc genes, which are related to biogenic on the differences among the strains being assessed, the results amine production, were present in five strains. The hdc1, obtained from adhesion tests are different from the reality hdc2, tdc and odc genes express enzymes that degrade in vivo. The human GI tract has defense systems, resident Ann Microbiol (2017) 67:289–301 299 Acknowledgements This research was financially supported by the flora, and bowel movements that may change the strain adhe- Sao Paulo Research Foundation (FAPESP, grants 2014/02131-8 and sion ability (Jensen et al. 2012). Moreover, different values for 2014/02132-4). lactobacillus adhesion to Caco-2 cells have been reported, ranging from less than 1% to more than 70% (Jensen et al. Compliance with ethical standards 2012; Nikolic et al. 2012; Ramos et al. 2013; Tulumoğlu et al. 2014), possibly due to variation in the conditions used during Conflict of interest The authors declare that they have no conflict of interest. This article does not contain any studies with human participants the assay, such as the type of cell line, incubation time and or animals performed by any of the authors. number of probiotic cells added. The presence of genes encoding adhesion, aggregation and colonization factors is desirable in LAB and can also indicate References that the bacteria are able to adhere to the mucus layer (Jeronymo-Ceneviva et al. 2014). 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Journal
Annals of Microbiology – Springer Journals
Published: Feb 28, 2017