Thermophilus Isolates Research Articles

Genetic diversity and phylogenetic relationships of Streptococcus thermophilus isolates from traditional Turkish yogurt: multilocus sequence typing (MLST).

Yogurt, a globally consumed fermented dairy product, is recognized for its taste and potential health benefits attributed to probiotic bacteria, particularly Streptococcus thermophilus. In this study, we employed Multilocus Sequence Typing (MLST) to investigate the genetic diversity and phylogenetic relationships of 13 S. thermophilus isolates from traditional Turkish yogurt samples. We also assessed potential correlations between genetic traits and geographic origins. The isolates were identified as S. thermophilus using VITEK® MALDI-TOF MS, ribotyping, and 16S rRNA analysis methods. MLST analysis revealed 13 different sequence types (STs), with seven new STs for Turkey. The most prevalent STs were ST/83 (n = 3), ST/135 (n = 2), and ST/134 (n = 2). eBURST analysis showed that these isolates mainly were singletons (n = 7) defined as sequence types (STs) that cannot be assigned to any group and differ at two or more alleles from every other ST in the sample. This information suggests that the isolates under study were genetically distinct from the other isolates in the dataset, highlighting their unique genetic profiles within the population. Genetic diversity analysis of ten housekeeping genes revealed polymorphism, with some genes showing higher allelic variation than others. Tajima's D values suggested that selection pressures differed among these genes, with some being more conserved, likely due to their vital functions. Phylogenetic analysis revealed distinct genetic diversity between Turkish isolates and European and Asian counterparts. These findings demonstrate the genetic diversity of S. thermophilus isolates in Turkish yogurt and highlight their unique evolutionary patterns. This research contributes to our understanding of local microbial diversity associated with yogurt production in Turkey and holds the potential for identifyic strains with enhanced functional attributes.

Genetic and technological diversity of Streptococcus thermophilus isolated from the Saint-Nectaire PDO cheese-producing area.

Streptococcus thermophilus is of major importance for cheese manufacturing to ensure rapid acidification; however, studies indicate that intensive use of commercial strains leads to the loss of typical characteristics of the products. To strengthen the link between the product and its geographical area and improve the sensory qualities of cheeses, cheese-producing protected designations of origin (PDO) are increasingly interested in the development of specific autochthonous starter cultures. The present study is therefore investigating the genetic and functional diversity of S. thermophilus strains isolated from a local cheese-producing PDO area. Putative S. thermophilus isolates were isolated and identified from milk collected in the Saint-Nectaire cheese-producing PDO area and from commercial starters. Whole genomes of isolates were sequenced, and a comparative analysis based on their pan-genome was carried out. Important functional properties were studied, including acidifying and proteolytic activities. Twenty-two isolates representative of the diversity of the geographical area and four commercial strains were selected for comparison. The resulting phylogenetic trees do not correspond to the geographical distribution of isolates. The clustering based on the pan-genome analysis indicates that isolates are divided into five distinct groups. A Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation of the accessory genes indicates that the accessory gene contents of isolates are involved in different functional categories. High variability in acidifying activities and less diversity in proteolytic activities were also observed. These results indicate that high genetic and functional variabilities of the species S. thermophilus may arise from a small (1,800 km2) geographical area and may be exploited to meet demand for use as autochthonous starters.

Multidimensional evaluation of techno-functional properties of yoghurt bacteria

Microbial diversity in traditional foods is decreasing due to widespread industrial production, using a limited number of strains, and industrial products as starter cultures in homemade production. Yoghurt is a fermented product, and this decrease is most common. This study investigated the microbiological and physicochemical properties of 82 traditional and 37 industrial yoghurt samples. Four hundred thirty-nine lactic acid bacteria isolated were molecularly identified. Of the isolates, 223 were Lactobacillus bulgaricus, and 216 were Streptococcus thermophilus. Characteristics of the isolates such as acidification, arginine hydrolysis and gas production from glucose, H2O2 production, growth at different temperatures, pH and NaCl concentrations, exopolysaccharide production, proteolytic and lipolytic activity, l (+) lactic acid production, cholesterol assimilation, haemolytic activity, DNase activity, antibiotic resistance, and virulence gene were investigated. As a result, 4 L. bulgaricus and 6 S. thermophilus isolates were the best starter cultures for yoghurt production.

Antibiotic Resistance Profile of Indigenous Streptococcus thermophilus and Lactobacillus bulgaricus Strains Isolated from Traditional Yogurt

Antibiotic resistance signifies a safety hazard to public health. Lactic acid bacteria, particularly, Streptococcus thermophilus and Lactobacillus bulgaricus, are useful organisms responsible for dairy fermentation. In keeping with this, they may transfer antibiotic resistance to human population. We conducted a study exanimated the antibiotic resistance pattern and distribution of antibiotic resistance genes of the S. thermophilus and L. bulgaricus strains isolated from traditional yogurt samples. Fifty-five traditional yogurt samples were collected, and S. thermophilus and L. bulgaricus strains were isolated using the microbial culture. The disk diffusion method in agar wells was utilized to assess the antibiotic resistance pattern of isolates against 13 antibiotic agents. The distribution of antibiotic resistance genes was assessed using conventional PCR. Lactobacillus bulgaricus and S. thermophilus were identified in 94.54% (52/55) and 87.27% (48/55) of yogurt samples, respectively. Both L. bulgaricus and S. thermophilus bacteria were simultaneously identified in 72.72% of samples. Streptococcus thermophilus isolates harbored the highest antibiotic resistance rate against tetracycline (31.25%), ampicillin (31.25%), gentamicin (25%), erythromycin (25%), penicillin (12%), and ciprofloxacin (20.83%). Lactobacillus bulgaricus isolates harbored the highest resistance rate against tetracycline (9.61%), ampicillin (9.61%), gentamicin (9.61%), and erythromycin (7.69%). Streptococcus thermophilus (33.33%) isolates harbored the higher distribution of multidrug resistance than L. bulgaricus (11.53%). aacA-D (20.83%), tetK (16.66%), ermA (14.58%), blaZ (14.58%), and gyrA (12.50%) were the most commonly detected antibiotic resistance genes in S. thermophilus. AacA-D (3.84%) and tet K (3.84%) were the most commonly detected antibiotic resistance genes in L. bulgaricus. Lactobacillus bulgaricus isolates did not harbor tetM, cat1, rpoB, vanA, linA, and strA-strB antibiotic resistance genes. Streptococcus thermophilus and L. bulgaricus strains used in traditional yogurt production can harbor antibiotic resistance genes and subsequently disseminate the resistance to human beings. Monitoring antibiotic resistance in fermented foods should be a common inspection for food quality.

Screening of mixed-species starter cultures for increasing flavour during fermentation of milk

Flavour is an important factor determining the quality of fermented dairy products. Three L.delbrueckii subsp. bulgaricus isolates and five Streptococcus thermophilus isolates with good fermentation characteristics were selected for evaluation as starter cultures when used in ten different combinations, and in comparison with a commercial starter. Using solid phase micro extraction-gas chromatography-mass spectrometry (SPME-GC-MS) analysis, a total of 55 volatile compounds and nine odour-active compounds were detected in milk fermented by mixed-species starter cultures C5 (L. delbrueckii subsp. bulgaricus IMAU62081: S. thermophilus IMAU40133). The results from partial least squares-discriminant analysis (PLS-DA), electronic nose (E-nose) analysis and sensory evaluation indicated that flavor composition and content of fermentation samples from the C5 mixture were closer to the commercial control than the other mixtures. These results help understand how volatile flavour compounds in yoghurt are affected by the starter culture; this will be useful in the development of value-added fermented dairy products.

Isolation of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus from nature: Technological characterisation and antibiotic resistance

Yoghurt fermenting bacteria were isolated from natural sources including plants, dew, and rain samples (total of 300 samples) by the same methods nomadic peoples used for several centuries in Turkey. Inoculation into the reconstituted skim milk followed by planting on specific media and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis allowed for the identification of 18 Lactobacillus delbrueckii subsp. and 26 Streptococcus thermophilus. A multiplex polymerase chain reaction (PCR) assay applied to lactobacilli enabled the identification of 5 isolates as L. delbrueckii subsp. bulgaricus. The isolates showed a varying range of acidification rates and proteolytic activity in reconstituted skimmed milk (RSM). S. thermophilus isolates showed a broader range of resistance and the most frequent resistance was observed for streptomycin (69.2%), gentamycin (65.3%), clindamycin (61.5%), ampicillin (61.5%), kanamycin (53.8%), and erythromycin (50%). For L. delbrueckii subsp. the highest resistance was determined for vancomycin (38.8%), ciprofloxacin (33.3%), and penicillin (27.8%). The frequency of multiple resistance was tested on 14 different antimicrobials determining that 19 S. thermophilus (73%) and 3 L. delbrueckii subsp. (16.7%) demonstrated resistance to more than three different antibiotics. In contrast to this wide-ranging resistance, five isolates from each genus were found to be susceptible to all tested antibiotics. The present study indicates that lactic acid bacteria (LAB) isolated from nature may have broad-range of resistance to antibiotics and could be a source for the transfer of resistance.

Microbial exopolysaccharide production of Streptococcus thermophilus and its antiquorum sensing activity.

Interest in the production of exopolysaccharides by microorganisms has increased in the recent years. Using low-cost product is the main step of microbial production to reduce cost and compete with chemical production. In this work, EPS production of Streptococcus thermophilus isolates from yogurt (S2), kefir (S3), and S. thermophilus ATCC 19258 (S1) isolate which was used as control strains were investigated by using different fruit pulps. S. thermophilus isolates were identified by morphological and 16S sequence analysis. The amount of EPS obtained was measured spectrophotometrically using glucose as standard with phenol sulfuric acid method. All three isolates produced higher amounts of EPS on M17 medium than Nutrient medium. When the fruit pulp was added to the medium, EPS production increased in all three isolates. When different nitrogen sources were added together with fruit pulp juice, EPS production increased. The highest amount of EPS produced by ATCC 19258 strain (21.570mg/L) and S3 isolate (29.131mg/L) is the medium where mixed fruit pulp juice and nitrogen source is tryptophan. It has been shown that EPS production is increased by adding fruit pulps to the prepared media. It is thought that apricot pulp can be a good alternative in EPS production especially in the evaluation of wastes. Also, antiquorum sensing activity of the highest amount EPS was determined by using Chromobacterium violaceum CV026 strain and found effective on violacein pigment inhibition and C6-AHL production of biosensor strain.

A survey of the relationship between functional genes and acetaldehyde production characteristics in Streptococcus thermophilus by multilocus sequence typing

Streptococcus thermophilus is an important bacterium used in the production of fermented dairy products. Yogurt with good flavor is preferred by consumers; thus, variation in flavor-formation characteristics among isolates is attracting attention. Here, acetaldehyde production characteristics of 30 isolates were evaluated in parallel with genotyping and multilocus sequence typing of key functional genes involved in acetaldehyde production. The results showed that isolates could be divided into 3 phenotypically distinct groups: high-acetaldehyde-yielding isolates (>10 mg/L), medium-acetaldehyde-yielding isolates (5-10 mg/L) and low-acetaldehyde-yielding (<5 mg/L) based on evaluation of acetaldehyde production during yogurt storage. These groups, distinguishable by phenotypic characteristics, were clustered in corresponding groups based on functional gene multilocus sequence typing analysis. Combining functional gene sequence analysis of 30 Strep. thermophilus isolates with phenotypic evaluation of their flavor-related characteristics (specifically acetaldehyde production) demonstrated that groups of isolates established using genotype data analysis corresponded with groups identified based on their phenotypic traits. Interestingly, the 30 isolates of Strep. thermophilus showed significant phylogenetic clustering in acetaldehyde content by functional gene and acetaldehyde content analysis. A corresponding relationship exists between functional gene phylogenetic clustering and acetaldehyde content variation.

Determination of some Antibiotic Resistances genes by Polymerase Chain Reaction of Lactic Acid Bacteria Isolates from Local Dairy Products

Background: This study sheds light on the current resistance situation in local dairy product associated microorganisms Like Lactic Acid Bacteria (LAB) and will provide a basis for further experiments concerning gene transferability.Objective: Isolation of Lactic Acid Bacteria (LAB) from local dairy products that have resistance phenotypes to antibiotics, and detection by PCR presence of certain known antibiotic resistance genes.Materials and methods: This study was conducted during the period from 1st of September 2016 to the end of January 2017. Two hundred samples from local dairy products were collected as 50 samples from each raw milk, sour-milk, yogurt, cream and soft cheese. All isolates were identified according to biochemical characters, antimicrobial sensitivity test and MIC of the isolates was done by the Kirby–Bauer disc diffusion and micro dilution methods respectively. PCR amplification genes associated with resistance to β-lactam antibiotics (bla Z) gene, erythromycin (ermB), genes, and tetracycline ribosomal protection proteins (tet M), were done by PCR.Results: The results showed that the most common bacteria presents in all dairy source samples were Lactobacillus as followed:75(31.9%), 40(17%), 30(12.7%) and 90(38.2%) in yogurt, white cheese, cream and sour milk respectively. Lactococcus isolates was appeared as 20(36.3%) and 15(27.27%) in yogurt and creams respectively and 10(18.1%) in each of white cheese and sour milk. Streptococcus was found as 45(40.9%) in white cheese and 30(27.2%) in yogurt, while in cream and sour milk was found as 27(24.5%) and 8(7.2%) respectively, whereas the total number of Lactococcus and Pediococcus were 55(12.6%) and 36(8.25%) respectively. The physiological characters and phenotypic identification was found that out of the 436 LAB isolates; Lactobacillus isolates were the dominant genus appeared followed by Streptococcus species. Isolates of LAB demonstrated different profiles of antibiotic resistance, all Lb. delbrukii, Lb. plantarum and Lb. fermentum were resistance to penicillin G, while showed a variable susceptibility rates to other antibiotics. The detection of tet M and erm B and bla Z resistance genes in LAB isolates showed that some isolates harbor tet M and/or erm B and bla Z genes. Twenty isolates of Lactobacillus delbrukii showed the presence of tet M and erm B and 17 isolates harbor bla Z genes corresponding to their resistance phenotypes. As well as 10 isolates of Lb. plantarum showed these gens in some of them as follow; five of the ten possessed the resistance tet M gen and 4 isolates have erm B while 8 isolates showed the penicillin G resistance gen. From the ten isolates of Lb. fermentum; only 4 isolates harbor tet M gene and 5 isolates possessed erm B gens while 9 isolates had the resistance gens bla Z. The isolates Pedicoccus sp. and Lc. lactis showed only presence of tet M gens in 8 and 7 isolates respectively. On the other hand Strep. thermophilus (20 isolates); the band of the detected gens appeared in 18 isolates for tet M and 15 for the erm B, but a β-lactamase gene detected in only 4 of these resistant isolates. Leu. mesntroids (10 isolates) showed 6 and 4 isolates possessed bla Z and tet M gen respectively but only one isolates showed to possessing the gene erm B.Conclusion: This study had established that wide varieties of LAB the most common bacteria presents in all local dairy source samples were lactobacillus species which show high resistance properties to amoxicillin and ampicillin. Lactococcus lactis isolates showed resistance to tetracycline and all Leuconostoc cremoris isolates showed sensitivity to all tested antibiotics except, some isolates resisted to tetracycline and chloramphenicol. While Leuconostoc mesntroids had variable resistance to tetracycline, chloramphenicol, erythromycin, vancomycin, amoxicillin, ampicillin and penicillin G. All Streptococcus thermophilus isolates were resisting to tetracycline. The genes tet M, erm B and bla Z were detected in Lactic acid bacterial isolates.

Antibiotic Resistance of Lactobacillus spp. and Streptococcus thermophilus Isolated from Chinese Fermented Milk Products.

The aim of the present study was to investigate the phenotypic and genotypic antimicrobial resistance and the transferability of resistance markers in 87 lactic acid bacterial strains recovered from fermented milk products obtained from different areas of China. The isolates were identified as 21 Lactobacillus bulgaricus, 8 Lactobacillus casei, 6 Lactobacillus rhamnosus, 3 Lactobacillus paracasei, 2 Lactobacillus acidophilus, and 47 Streptococcus thermophilus strains. High levels of intrinsic resistance were revealed among the tested species. The following resistance genes were detected in strains isolated from fermented milk products: tet(M) in two L. bulgaricus and two S. thermophilus isolates, strA and strB in nine and seven S. thermophilus isolates, respectively; sul1 in six L. bulgaricus and seven S. thermophilus isolates, sul2 in one S. thermophilus isolate, aac(6')-aph(2″) in two L. bulgaricus isolates, and aph(3″)-II and aph(3″)-III in one S. thermophilus and two L. bulgaricus isolates, respectively. Transfer of the monitored antibiotic resistance genes was not observed in the filter mating assays of this study. To our knowledge, the strA, strB, sul1, sul2, and aph(3″)-II genes in S. thermophilus, and the sul1 and aac(6')-aph(2″) genes in L. bulgaricus were identified for the first time. These results indicate the potential risks posed by lactic acid bacteria (LAB) in fermented milk products in expanding the antibiotic resistance gene reservoir and transferring antibiotic resistance genes among bacteria. Further investigations are required to identify the potential sources of contamination and the dissemination routes of antibiotic resistance genes among LAB in fermented milk products.

Exact identification of six starter‐strain candidates of Streptococcus thermophilus by analysing genotypic and industrial properties

Six strains of Streptococcus thermophilus isolated as starter candidates were precisely identified by analysing their genotypic and industrial properties. The combination of three methods, multilocus sequence typing, CRISPR1 and epsCD with 16S‐23S rRNA internal transcribed spacer, differentiated the six isolates into five groups. Only strains s60 and s79 could not be distinguished from each other. The industrial properties of the six tested strains were more diverse, especially in their firmness, viscosity and exopolysaccharide production. The six S. thermophilus isolates were identified and typed at the strain level by analysing genotypic and industrial properties.

Comparison of RAPD-PCR and PFGE analysis for the typing of Streptococcus thermophilus strains isolated from traditional Turkish yogurts

The dairy industry is constantly developing new starters with specific traits. Therefore, strains from natural sources have huge potential for exploration and characterization. The aim of this work was to identify the most efficient, useful, and reliable option for typing 55 Streptococcus thermophilus isolates from traditional Turkish yogurts and four industrial strains. Then, we statistically evaluated whether our conclusions could be extended to the true population. For this purpose, the results of the rapid amplified polymorphic DNA polymerase chain reaction (RAPD-PCR; using the OPI-02 MOD, M13, and XD9 primers) and pulsed-field gel electrophoresis (PFGE; using the SmaI and ApaI enzymes) typing methods were compared. The discriminatory power, typeability, and reproducibility were analyzed. Additionally, the congruence between typing methods was quantified using the adjusted Rand index (AR), Wallace index (W), and expected Wallace coefficient. Both methods revealed high genetic diversity of the S. thermophilus strains, even in the same yogurt sample. The numerical combination of results for these primers or restriction enzymes increased the congruence between the methods and provided more complete information on the strains. The comparison of these two options showed that using SmaI with ApaI was more advisable and useful than using OPI-02 MOD with M13. Additionally, the first combination represented the best tool to discriminate S. thermophilus strains (Simpson’s index of diversity [DI] of 0.999 [0.997–1.000]). This finding was statistically supported. The RAPD (OPI-02 MOD) typing result showed an ability to confidently predict the PFGE (SmaI, ApaI) type (AR = 0.782 [0.618–0.949], W = 0.946 [0.865–1.000]). This result had some statistical support and might represent an important application in the dairy industry for screening strains.

Identification of Coccoidal Bacteria in Traditional Fermented Milk Products from Mongolia, and the Fermentation Properties of the Predominant Species, Streptococcus thermophilus.

The objective of this study was to identify the coccoidal bacteria present in 188 samples of fermented yaks’, mares’ and cows’ milk products collected from 12 different regions in Mongolia. Furthermore, we evaluated the fermentation properties of ten selected isolates of the predominant species, Streptococcus (S.) thermophiles, during the process of milk fermentation and subsequent storage of the resulting yoghurt at 4℃. Overall, 159 isolates were obtained from 188 samples using M17 agar. These isolates were presumed to be lactic acid bacteria based on their gram-positive and catalase-negative properties, and were identified to species level using 16S rRNA gene sequence analysis. These coccoid isolates were distributed in four genera and six species: Enterococcus (E.) durans, Enterococcus (E.) faecalis, Lactococcus (Lac.) subsp. lactis, Leuconostoc (Leuc.) lactis, Leuconostoc (Leuc.) mesenteroides. subsp. mesenteroides and S. thermophilus. Among these S. thermophilus was the most common species in most samples. From evaluation of the fermentation characteristics (viable counts, pH, titratable acidity [TA]) of ten selected S. thermophilus isolates we could identify four isolates (IMAU 20246, IMAU20764, IMAU20729 and IMAU20738) that were fast acid producers. IMAU20246 produced the highest concentrations of lactic acid and formic acid. These isolates have potential as starter cultures for yoghurt production.

Preparation of low galactose yogurt using cultures of Gal(+) Streptococcus thermophilus in combination with Lactobacillus delbrueckii ssp. bulgaricus.

Streptococcus thermophilus is an important lactic starter used in the production of yogurt. Most strains of S. thermophilus are galactose negative (Gal(-)) and are able to metabolize only glucose portion of lactose and expel galactose into the medium. This metabolic defect leads to the accumulation of free galactose in yogurt, resulting in galactosemia among consumers. Hence there is an absolute need to develop low galactose yogurt. Therefore, in this study, three galactose positive (Gal(+)) S. thermophilus strains from National Collection of Dairy Cultures (NCDC) viz. NCDC 659 (AJM), NCDC 660 (JM1), NCDC 661 (KM3) and a reference galactose negative (Gal(-)) S. thermophilus NCDC 218 were used for preparation of low galactose yogurt. In milk fermented using S. thermophilus isolates alone, NCDC 659 released less galactose (0.27%) followed by NCDC 661 (0.3%) and NCDC 660 (0.45%) after 10 h at 42°C. Milk was fermented in combination with Gal(-) L. delbrueckii subsp. bulgaricus NCDC 04, in which NCDC 659 released least galactose upto 0.49% followed by NCDC 661 (0.51%) and NCDC 660 (0.60%) than reference Gal(-) NCDC 218(0.79%). Low galactose yogurt was prepared following standard procedure using Gal(+) S. thermophilus isolates and Gal(-) L. delbrueckii subsp. bulgaricus NCDC 04 in 1:1 ratio. Among which low galactose yogurt by NCDC 659 combination contained less galactose 0.37% followed by NCDC 661 (0.51%), NCDC 660 (0.65%) and reference Gal(-) NCDC 218 (0.98%) after 4h of fermentation. This study clearly reveals that Gal(+) S. thermophilus isolates can be paired with Gal(-) L. delbrueckii subsp. bulgaricus for developing low galactose yogurt.

Biogenic amines formation in Streptococcus thermophilus isolated from home-made natural yogurt

Twelve different biogenic amines formation in 58 isolates of Streptococcus thermophilus from home-made natural yogurt were investigated in histidine (HDB) and lysine decarboxylase broth (LDB). All S. thermophilus isolates had an ability to produce twelve different biogenic amines in HDB and LDB. Most of the S. thermophilus isolates formed low amounts of histamine (1–50mg/L) from histidine. Apart from one isolate, S. thermophilus produced tyramine at low (47 isolates) and medium (10 isolates) levels. The amount of each specific biogenic amine produced by S. thermophilus was generally lower than 100mgL−1. Also, the presence of hdcA gene was investigated using PCR technique and relation between gene and histamine production was conducted in S. thermophilus isolates. This study showed that most of the S. thermophilus isolates have the ability to form biogenic amines, especially histamine, and tyramine, which is an important consideration when selecting strains as starter cultures.

Antibiotic resistance of lactic acid bacteria isolated from Chinese yogurts

The aim of this study was to evaluate the susceptibility of 43 strains of lactic acid bacteria, isolated from Chinese yogurts made in different geographical areas, to 11 antibiotics (ampicillin, penicillin G, roxithromycin, chloramphenicol, tetracycline, chlortetracycline, lincomycin, kanamycin, streptomycin, neomycin, and gentamycin). The 43 isolates (18 Lactobacillus bulgaricus and 25 Streptococcus thermophilus) were identified at species level and were typed by random amplified polymorphic DNA analysis. Thirty-five genotypically different strains were detected and their antimicrobial resistance to 11 antibiotics was determined using the agar dilution method. Widespread resistance to ampicillin, chloramphenicol, chlortetracycline, tetracyclines, lincomycin, streptomycin, neomycin, and gentamycin was found among the 35 strains tested. All of the Strep. thermophilus strains tested were susceptible to penicillin G and roxithromycin, whereas 23.5 and 64.7% of Lb. bulgaricus strains, respectively, were resistant. All of the Strep. thermophilus and Lb. bulgaricus strains were found to be resistant to kanamycin. The presence of the corresponding resistance genes in the resistant isolates was investigated through PCR, with the following genes detected: tet(M) in 1 Lb. bulgaricus and 2 Strep. thermophilus isolates, ant(6) in 2 Lb. bulgaricus and 2 Strep. thermophilus isolates, and aph(3′)-IIIa in 5 Lb. bulgaricus and 2 Strep. thermophilus isolates. The main threat associated with these bacteria is that they may transfer resistance genes to pathogenic bacteria, which has been a major cause of concern to human and animal health. To our knowledge, the aph(3′)-IIIa and ant(6) genes were found in Lb. bulgaricus and Strep. thermophilus for the first time. Further investigations are required to analyze whether the genes identified in Lb. bulgaricus and Strep. thermophilus isolates might be horizontally transferred to other species.

Persistence of wild Streptococcus thermophilus strains on wooden vat and during the manufacture of a traditional Caciocavallo type cheese

The present work was undertaken to evaluate the influence of the wooden dairy plant equipment on the microbiological characteristics of curd to be transformed into Caciocavallo Palermitano cheese. Traditional raw milk productions were performed concomitantly with standard cheese making trials carried out in stainless steel vat inoculated with a commercial starter. Milk from two different farms (A and B) was separately processed. The wooden vat was found to be a reservoir of lactic acid bacteria (LAB), while unwanted (spoilage and/or pathogenic) microorganisms were not hosted or were present at very low levels. All microbial groups were numerically different in bulk milks, showing higher levels for the farm B. LAB, especially thermophilic cocci, dominated the whole cheese making process of all productions. Undesired microorganisms decreased in number or disappeared during transformation, particularly after curd stretching. LAB were isolated from the wooden vat surface and from all dairy samples, subjected to phenotypic and genetic characterization and identification. Streptococcus thermophilus was the species found at the highest concentration in all samples analyzed and it also dominated the microbial community of the wooden vat. Fourteen other LAB species belonging to six genera (Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Streptococcus and Weissella) were also detected. All S. thermophilus isolates were genetically differentiated and a consortium of four strains persisted during the whole traditional production process. As confirmed by pH and the total acidity after the acidification step, indigenous S. thermophilus strains acted as a mixed starter culture.

Genotyping of Streptococcus thermophilus strains isolated from traditional Egyptian dairy products by sequence analysis of the phosphoserine phosphatase (serB) gene with phenotypic characterizations of the strains

To develop a new, simplified genotyping method for examining the genetic diversity of Streptococcus thermophilus strains isolated from traditional Egyptian fermented dairy products and to characterize phenotypic traits of those strains related to their potential use in bioprocessing applications. A novel, simplified approach was developed for genotyping Strep. thermophilus involving the analysis of nucleotide sequence variations within a housekeeping gene encoding the phosphoserine phosphatase (SerB). Using this method, it was possible to identify ten genotypes involving diverse serB alleles among 54 Strep. thermophilus isolates cultured from Egyptian dairy products. These isolates harboured five de novo serB alleles that have not been detected in other Strep. thermophilus strains, deposited in a multilocus sequence typing (MLST) database. To assess distinct genotypes of the organism with phenotypic traits relevant to their potential use in industry, Strep. thermophilus strains were all subjected to a series of phenotypic characterizations. The strains were found to exhibit phenotypic diversity in terms of their ability to ferment lactose and galactose, express urease activity, produce exopolysaccharides and develop acidity. The analysis of nucleotide sequence variations within the serB gene could serve as a suitable tool for probing diverse genotypes of Strep. thermophilus. Streptococcus thermophilus isolates associated with traditional Egyptian dairy products show high degree of genetic and phenotypic diversity. This study presents a novel, simplified procedure based on serB nucleotide sequencing for genotyping Strep. thermophilus. It also provides a pool of phenotypically diverse Strep. thermophilus cultures, from which certain strains could be selected for use in bioprocessing applications including the preparation of fermented dairy products.

Occurrence, isolation and DNA identification of Streptococcus thermophilus involved in Algerian traditional butter ‘Smen’

Streptococcus thermophilus isolates from traditional butter 'Smen', a fermented product from cow's and ewe's milk in arid area was subjected to taxonomical investigations. The identification procedure included phenotypic approaches, molecular characterization by using genus polymerase chain reaction (PCR) amplifications for sodA gene encoding the manganese-dependant superoxide dismutase A, and species-specific primers from gene encoding glucose kinase (glcK), gene encoding DNA polymerase III (dnaE) and gene encoding threonyl-tRNA synthetase (thrS) housekeeping genes in order to distinguish among reference, and wild strains of S. thermophilus and for their differentiation from Enterococcus spp. A total of 12 strains were tested by DNA identification analysis and these indigenous isolates were unambiguously characterised by their housekeeping gene profiles. Finally, four genotypes were recognised. To our knowledge, this is the first report on the isolation and molecular characterization of S. thermophilus strains from fermented milk transformed into home-production butter provided from Southern West Algeria. These results are of particular interest as they favour the selection of future cultures starters as S. thermophilus from traditional dairy products. It is, therefore, of great importance to preserve the genetic pool of the wild strains.

Safety aspects, genetic diversity and technological characterisation of wild-type Streptococcus thermophilus strains isolated from north Italian traditional cheeses

Antibiotic susceptibility, antimicrobial activity, genotypic and technological properties of 52 Streptococcus thermophilus isolates, collected from four north Italian traditional cheeses, was investigated. RAPD-PCR, was used to study genetic variability and distinguish closely related strains; the results showed a high degree of heterogeneity among isolates. With regard to technological properties, after 6 h of incubation in milk 25% of the streptococcal strains could be defined as fast acid producers, while after 24 h the majority of isolates (79%) displayed only weak acidification activity. Reduction activity was generally low; in fact, none of these S. thermophilus strains showed a Eh < −102 mV). All the studied S. thermophilus were susceptible to ciprofloxacin, levofloxacin, penicillin G, ampicillin, mupirocin, nitrofurantoin, quinupristin/dalfopristin and rifampicin. Nine isolates were classified as resistant to tetracycline, 6 to streptomycin, 5 to oxacillin, 3 to erytromycin, 3 to vancomycin and only one to chloramphenicol. PCR-based detection did not identify any of the common genetic determinants for vancomycin (vanA, vanB, vanC1, vanC2, vanC3, vanD, vanE, vanG) or erythromycin (ermB and ermC). The genetic basis of the tetracycline resistance phenotype in these strains was linked to tetS-tetL genes (8 isolates) or the tetM gene (1 isolate), and the integrase element int of the Tn916/Tn1545 family of transposons was negative. Four strains were able to produce antimicrobial compounds against Clostridium tyrobutyricum. The study provides new evidence of the resistance of S. thermophilus to antimicrobial agents, confirming the importance of including an accurate safety assessment of phenotypic/biotechnological data when carrying out strain selection for dairy applications.