Development of a group-specific PCR combined with ARDRA for the identification of Bacillus species of environmental significance

A group-specific primer pair was designed to amplify the 16S rRNA gene of representative reference strains from environmentally sourced, mesophilic aerobic spore-forming Bacillus taxa. The PCR generated a 1114 bp amplicon but did not do so with DNA extracted from 16 other Eubacterial species. When amplicons were digested with restriction enzymes AluI or TaqI, different profiles containing between 2 and 5 fragments ranging in size from 76 to 804 base pairs were seen with different Bacillus species. This procedure, known otherwise as amplified ribosomal DNA restriction analysis or ARDRA, produced unique and distinguishable patterns to differentiate between 15 ATCC reference strains (10 Bacillus, 3 Paenibacillus and 2 Brevibacillus member species) as well as 3 misidentified Bacillus probiotic strains in a commercial collection. Our simplified PCR-ARDRA protocol provides a facile method for the identification of most environmentally important species of Bacillus.     

Inventory of non starter lactic acid bacteria from ripened Parmigiano Reggiano cheese as assessed by a culture dependent multiphasic approach

The objective of this study was to investigate microbial species diversity and strain complexity of the cultivable non starter lactic acid bacteria (NSLAB) occurring in 31 ripened Parmigiano Reggiano (PR) cheeses. Dereplication of 127 lactobacilli isolates by (GTG)(5)-PCR fingerprinting yielded a total of 51 genotypes. Phylogenetic relatedness of all the genotypes with known Lactobacillus species was determined by a novel combined amplified 16S rDNA restriction analysis (16S-ARDRA), species-specific PCR assays and 16S rRNA gene sequencing. The species Lactobacillus rhamnosus and Lactobacillus paracasei comprise the largest portions of the cultivable NSLAB community in PR cheese, with an inter-individual diversity ranging from one to four dominant genotypes per sample. Lactobacillus casei, Lactobacillus harbinensis and Lactobacillus fermentum species were also detected at low frequency. The data showed differences in cultivable NSLAB population, with an overall decrease in diversity and complexity from early to advanced stages of ripening. Finally the de-replicated collection of genotypes resulting from this work is the bases for further functional screening.     

Identification of Carnobacterium, Lactobacillus, Leuconostoc and Pediococcus by rDNA-based techniques

Ribosomal DNA-based techniques including the analysis of profiles generated by ISR amplification, ISR restriction and ARDRA have been evaluated as molecular tools for identifying Carnobacterium, Lactobacillus, Leuconostoc and Pediococcus. They have been applied for the molecular characterization of 91 strains with the following identities: eight Carnobacterium including the eight type species of the genus; 61 Lactobacillus including 40 type strains out of 45 species, 13 Leuconostoc, out of them 11 are type strains and three are subspecies of Lc. mesenteroides; and nine strains representing the six species of genus Pediococcus. The genetic relationship displayed between these species by rrn-based profiles is sustained by their phylogenetic relationships and can therefore be considered useful for taxonomic purposes. Profiles obtained by ISR amplification allowed identification at genus level of Carnobacterium and Leuconostoc, and even at species level in genus Carnobacterium. Genera Lactobacillus and Pediococcus could not be distinguished from each other by applying this technique. The Lactobacillus species analysed here (45) were differentiated using ARDRA-DdeI and ISR-DdeI profiles, sequentially, and Pediococcus species by ISR-DdeI profiles. It was necessary to combine profiles generated by restriction of ISR-DdeI, ARDRA-DdeI and ARDRA-HaeIII in order to complete the identification of Leuconostoc species.     

Bacterial population in traditional sourdough evaluated by molecular methods

AIMS: To study the microbial communities in artisanal sourdoughs, manufactured by traditional procedure in different areas of Sicily, and to evaluate the lactic acid bacteria (LAB) population by classical and culture-independent approaches. METHODS AND RESULTS: Forty-five LAB isolates were identified both by phenotypic and molecular methods. The restriction fragment length polymorphism and 16S ribosomal DNA gene sequencing gave evidence of a variety of species with the dominance of Lactobacillus sanfranciscensis and Lactobacillus pentosus, in all sourdoughs tested. Culture-independent method, such as denaturing gradient gel electrophoresis (DGGE) of the V6-V8 regions of the 16S rDNA, was applied for microbial community fingerprint. The DGGE profiles revealed the dominance of L. sanfranciscensis species. In addition, Lactobacillus-specific primers were used to amplify the V1-V3 regions of the 16S rDNA. DGGE profiles flourished the dominance of L. sanfranciscensis and Lactobacillus fermentum in the traditional sourdoughs, and revealed that the closely related species Lactobacillus kimchii and Lactobacillus alimentarius were not discriminated. CONCLUSIONS: Lactobacillus-specific PCR-DGGE analysis is a rapid tool for rapid detection of Lactobacillus species in artisanal sourdough. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports a characterization of Lactobacillus isolates from artisanal sourdoughs and highlights the value of DGGE approach to detect uncultivable Lactobacillus species.     

Identification of Lactobacillus sakei and Lactobacillus curvatus by multiplex PCR-based restriction enzyme analysis

Two closely related lactic acid bacteria, Lactobacillus sakei and Lactobacillus curvatus, are very difficult to be rapidly differentiated. Here we report multiplex polymerase chain reaction (PCR)-based restriction enzyme analysis that is useful for rapid and reliable identification of these two species. This method employs both polymerase chain reaction (PCR) and restriction enzyme analysis (REA). First, multiplex-PCR using three primers that were designed from 16S rDNA sequence produces two bands, a 433-bp and a 623-bp band. A 433-bp band represents only L. sakei and L. curvatus among lactobacilli and genetically related bacteria, and a 623-bp band is used for further identification by restriction analysis. Second, restriction analysis of 623-bp band using Hind III restriction enzyme discriminates L. sakei from L. curvatus. This method could identify 28 strains as L. sakei or L. curvatus, which were frequently isolated from kimchi, a traditional fermented cabbage product in South Korea. Therefore, these results suggest that this method is simple, rapid, and reliable for the identification of L. sakei and L. curvatus species.     

A Lactobacillus helveticus plasmid detects restriction fragment length polymorphism in different bacterial species

A small cryptic Lactobacillus helveticus plasmid, pLBL4, was able to reveal restriction fragment length polymorphism in different bacterial species including Lactobacillus species, Bacillus species, and Escherichia coli when used as a DNA probe. The observed polymorphism was a result of the combined hybridization of several microsatellite sequences. The 6-bp sequence (TTGTTT) was repeated 12 times, seven of which were concentrated within the region between 1791 and 1997 bp of the plasmid sequence. The polymorphic patterns generated with pLBL4 differed from those obtained with M13 DNA in the larger number of bands observed. The results presented here open the possibility of using pLBL4 as a new broad-spectrum polymorphic DNA probe for fingerprint analysis.     

Differentiation of Lactobacillus plantarum, L. pentosus and L. paraplantarum species by RAPD-PCR and AFLP

Two high-resolution genotypic techniques (RAPD-PCR and AFLP) were evaluated for their possibility to discriminate the species Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus paraplantarum and to type these taxa at the infra-species level. In total 23 strains of L. plantarum, three strains of L. pentosus, two strains of L. paraplantarum and two related strains for which the species assignment was not clear, were studied. For RAPD-PCR, suitable oligonucleotides and amplification conditions were selected and tested. For AFLP, a double digest of total genomic DNA was used and a subset of restriction fragments was selectively amplified and visualised using different primer combinations. Both methodologies generated, species-specific electrophoretic profiles. Moreover, the presence of distinct subgroups was revealed within the species L. plantarum.     

Molecular identification of vaginal lactobacilli isolated from Bulgarian women

Lactobacilli play an important role in maintaining the vaginal health of women. The development of suitable bacterial replacement therapies for the treatment of vaginosis requires knowledge of the vaginal lactobacilli species representation. The aim of this study was to identify at the species level vaginal Lactobacillus isolates obtained from Bulgarian women in childbearing age by using different molecular methods. Twenty-two strains of lactobacilli isolated from vaginal samples were identified and grouped according to their genetic relatedness. A combined approach, which included amplified ribosomal DNA restriction analysis (ARDRA), ribotyping and polymerase chain reaction (PCR) with species-specific oligonucleotide primers was applied. All vaginal isolates were grouped into 5 clusters in␣comparison with a set of 21 reference strains based␣on the initial ARDRA results, which was then confirmed by ribotyping. Finally, the strains were subjected to PCR analysis with eight different species-specific primer pairs, which allowed most of␣them to be classified as belonging to one of␣the␣following species: Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus and Lactobacillus plantarum. In conclusion, this study suggests that the most straightforward identification strategy for vaginal lactobacilli would be grouping by ARDRA or ribotyping, followed by PCR specific primers identification at species level.     

Construction of a new shuttle vector for Lactobacillus.

To clone the malolactic enzyme gene from Lactobacillus sp. 89, construction of a shuttle vector able to express itself in Lactobacillus sp. 89 and Escherichia coli was undertaken. The shuttle plasmid pLE16 resulted from the union of pBR328 and of the pLB10 plasmid extracted from Lactobacillus bulgaricus 10. The bacterial transformation in Lactobacillus sp. 89 was performed by electroporation, and the clones were selected on MRS medium with 30 micrograms.mL-1 chloramphenicol added. Fifty percent of the clones from Lactobacillus sp. 89 lost their resistance to chloramphenicol following 28 generations when the selection pressure was not maintained. The restriction map of pLE16 (7600 bp) was established using several restriction enzymes.     

The genetic diversity of lactic acid producing bacteria in the equine gastrointestinal tract

Seventy-two lactic acid producing bacterial isolates (excluding streptococci) were cultured from the gastrointestinal tract of six horses. Two of the horses were orally dosed with raftilose to induce lactic acidosis and laminitis while the remaining four were maintained on a roughage diet. Near complete 16S rDNA was amplified by PCR from the genomic DNA of each isolate. Following RFLP analysis with the restriction enzymes MboI, HhaI and HinfI, the PCR products from the 18 isolates that produced L- and/or D-lactate were subsequently cloned and sequenced. DNA sequence analysis indicated that the majority of the isolates were closely related to species within the genus Lactobacillus, including Lactobacillus salivarius, Lactobacillus mucosae and Lactobacillus delbrueckii. Four isolates were closely related to Mitsuokella jalaludinii. Lactic acid producing bacteria (LAB) from the equine gastrointestinal tract was dominated by representatives from the genus Lactobacillus, but also included D-lactate-producing bacteria closely related to M. jalaludinii. Identification and characterization of LAB from the equine gastrointestinal tract should contribute to our understanding and management of fermentative acidosis, ulceration of the stomach and laminitis.     

Estimation of 16S rRNA gene copy number in several probiotic Lactobacillus strains isolated from the gastrointestinal tract of chicken

The copy numbers of 16S rRNA genes in 12 probiotic Lactobacillus strains of poultry origin were analyzed. Genomic DNA of the strains was digested with restriction endonucleases that do not cut within the 16S rRNA gene of the strains. This was followed by Southern hybridization with a biotinylated probe complementary to the 16S rRNA gene. The copy number of the 16S rRNA gene within a Lactobacillus species was found to be conserved. From the hybridization results, Lactobacillus salivarius I 24 was estimated to have seven copies of the 16S rRNA gene, Lactobacillus panis C 17 to have five copies and Lactobacillus gallinarum strains I 16 and I 26 four copies. The 16S rRNA gene copy numbers of L. gallinarum and L. panis reported in the present study are the first record. Lactobacillus brevis strains I 12, I 23, I 25, I 211, I 218 and Lactobacillus reuteri strains C 1, C 10, C 16 were estimated to have at least four copies of the 16S rRNA gene. In addition, distinct rRNA restriction patterns which could discriminate the strains of L. reuteri and L. gallinarum were also detected. Information on 16S rRNA gene copy number is important for physiological, evolutionary and population studies of the bacteria.     

Identification and differentiation of Lactobacillus, Streptococcus and Bifidobacterium species in fermented milk products with bifidobacteria

The aim of this study was to identify and discriminate bacteria contained in commercial fermented milks with bifidobacteria by the use of amplified ribosomal DNA restriction analysis (ARDRA) and randomly amplified polymorphic DNA (RAPD) techniques. ARDRA of the 16S rDNA gene and RAPD were performed on 13 Lactobacillus strains, 13 Streptococcus and 13 Bifidobacterium strains isolated from commercial fermented milk. Lactobacillus delbrueckii, Streptococcus thermophilus and Bifidobacterium animalis isolates were identified by genus- and species-PCR and also, they were differentiated at genus and species level by ARDRA using MwoI restriction enzyme. The ARDRA technique allowed for the discrimination among these three related genus with the use of only one restriction enzyme, since distinctive profiles were obtained for each genus. Therefore it can be a simple, rapid and useful method for routine identification. Also, RAPD technique allowed the discrimination of all bacteria contained in dairy products, at genus- and strain-level by the performance of one PCR reaction.     

Septicemia from Lactobacillus rhamnosus GG,from a Probiotic Enriched Yogurt,in a Patient with Autologous Stem Cell Transplantation

Probiotics and Antimicrobial Proteins - Probiotic-rich foods are consumed without much restriction. We report here, a case of septic shock caused by yogurt derived Lactobacillus species in a...     

Detection and identification of Lactobacillus species in crops of broilers of different ages by using PCR-denaturing gradient gel electrophoresis and amplified ribosomal DNA restriction analysis

The microflora of the crop was investigated throughout the broiler production period (0 to 42 days) using PCR combined with denaturing gradient gel electrophoresis (PCR-DGGE) and selective bacteriological culture of lactobacilli followed by amplified ribosomal DNA restriction analysis (ARDRA). The birds were raised under conditions similar to those used in commercial broiler production. Lactobacilli predominated and attained populations of 10(8) to 10(9) CFU per gram of crop contents. Many of the lactobacilli present in the crop (61.9% of isolates) belonged to species of the Lactobacillus acidophilus group and could not be differentiated by PCR-DGGE. A rapid and simple ARDRA method was developed to distinguish between the members of the L. acidophilus group. HaeIII-ARDRA was used for preliminary identification of isolates in the L. acidophilus group and to identify Lactobacillus reuteri and Lactobacillus salivarius. MseI-ARDRA generated unique patterns for all species of the L. acidophilus group, identifying Lactobacillus crispatus, Lactobacillus johnsonii, and Lactobacillus gallinarum among crop isolates. The results of our study provide comprehensive knowledge of the Lactobacillus microflora in the crops of birds of different ages using nucleic acid-based methods of detection and identification based on current taxonomic criteria.     

Ribotyping ofLactobacillus casei group strains isolated from dairy products

A series of lactobacilli isolated from dairy products were characterized using biotyping and ribotyping with EcoRI and HindIII restriction enzymes. Biotyping assigned 14 strains as Lactobacillus casei, 6 strains as Lactobacillus paracasei subsp. paracasei and 12 as Lactobacillus rhamnosus. The obtained ribotype patterns separated all analyzed strains into two clearly distinguished groups corresponding to L. rhamnosus and L. casei/L. paracasei subsp. paracasei. The HindIII ribotypes of individual strains representing these two groups were visually very similar. In contrast, EcoRI ribotyping revealed high intraspecies variability. All ribotypes of L. casei and L. paracasei subsp. paracasei dairy strains were very close and some strains even shared identical ribotype profiles. The type strains L. casei CCM 7088T (= ATCC 393T) and Lactobacillus zeae CCM 7069T revealing similar ribopatterns formed a separate subcluster using both restriction enzymes. In contrast, the ribotype profile of L. casei CCM 7089 (= ATCC 334) was very close to ribopatterns obtained from the dairy strains. These results support synonymy of L. casei and L. paracasei species revealed by other studies as well as reclassification of the type strain L. casei CCM 7088T as L. zeae and designation of L. casei CCM 7089 as the neotype strain.     

Characterization of a gram-positive broad-host-range plasmid isolated from Lactobacillus hilgardii

Two plasmids, pLAB1000 and pLAB2000 (3.3 and 9.1 kb, respectively), have been isolated from a grass silage strain of Lactobacillus hilgardii. Both plasmids were cloned in Escherichia coli and characterized through restriction mapping. A 1.6-kb XbaI-SacI fragment of pLAB1000 appeared to be sufficient for autonomous replication in Lactobacillus plantarum and in Bacillus subtilis. Different shuttle vectors for E. coli and gram-positive bacteria were developed using the pLAB1000 plasmid. These could stably be maintained in Lactobacillus, Enterococcus, and Bacillus under selective conditions. Plasmids sharing DNA homologies with pLAB1000 have been observed in different strains of the related species L. plantarum.     

属特异性T-RFLP技术用于乳酸杆菌的群落分析

【目的】设计乳酸杆菌属特异性T-RFLP技术(末端限制性片段长度多态性分析)对14株乳酸杆菌进行分型。【方法】采用源于16S-23S rRNA基因间隔区序列的乳酸杆菌属特异性引物LAB-rev,乳酸杆菌的属特异性引物,6-FAM荧光标记后结合16S上游通用引物7f用于乳酸杆菌的PCR扩增。【结果】选取HaeⅢ和HhaⅠ进行限制性酶切,最后对酶切后的产物末端测序得到T-RFLP峰谱图,该图谱能够快速准确地对不同种的乳酸杆菌进行定性、定量的分析。【结论】实验成功搭建T-RFLP技术用于微生态环境中乳酸杆菌检测的平台,对于在功能性食品、乳酸饮料和药物对肠道微生态的影响及菌种鉴定等领域有重大意义。     

Streptococcus alactolyticus is the dominating culturable lactic acid bacterium species in canine jejunum and feces of four fistulated dogs

Canine intestinal lactic acid bacterium (LAB) population in four fistulated dogs was cultured and enumerated using MRS agar. LAB levels ranging from 1.4x10(6) to 1.5x10(7) CFU ml(-1) were obtained in jejunal chyme. In the fecal samples 7.0x10(7) and 2.0x10(8) CFU g(-1) were detected. Thirty randomly selected isolates growing in the highest sample dilutions were identified to species level using numerical analysis of 16S and 23S rDNA restriction fragment length polymorphism patterns (ribotyping) and 16S rDNA sequence analysis. According to these results, Streptococcus alactolyticus was the dominant culturable LAB species in both feces and jejunal chyme. In addition, Lactobacillus murinus and Lactobacillus reuteri were detected.     

A survey of the microbial and chemical composition of seven semi-ripened Provola dei Nebrodi Sicilian cheeses

AIMS: The microbial and chemical composition of seven different semi-ripened (45 days) Provola dei Nebrodi Sicilian cheese samples were assessed in order to investigate the diversity of the microbial population in cheese made from different geographical areas throughout Sicily. METHODS AND RESULTS: The samples, which were obtained from seven different Provola dei Nebrodi manufacturers, were assessed using selective media. Interestingly, concentrations of presumptive lactobacilli represented over 90% of the total microbial population. In total, 105 presumptive Lactobacillus isolates were characterized to determine the relatedness of the isolates between the seven different cheeses. Randomly amplified polymorphic DNA polymerase chain reaction (RAPD PCR) analysis of the 105 presumptive lactobacilli indicated the presence of 22 distinct isolates. Further investigation of the isolates using pulsed field gel electrophoresis (PFGE) following restriction with the enzyme ApaI revealed the presence of 19 distinct macrorestriction patterns and the presence of between one and four distinct isolates per cheese sample (out of a total of 15 isolates per cheese randomly taken from Lactobacillus selective media plates). Analysis of the 16S rDNA sequence of each genetically distinct isolate demonstrated the dominance of the Lactobacillus casei species in all cheese samples assessed. Lactobacillus delbrueckii and Pediococcus pentosaceus species were also detected. The concentration of free amino acids, used to estimate the extent of proteolysis in each cheese, ranged from 59 to 433 mg 100 g(-1) cheese. CONCLUSIONS: Microbiological assessment of the cheeses demonstrated the dominance of Lactobacillus species after 45 days of ripening with levels ranging from 8.3 to 9.4 log CFU g(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new information on the diversity of lactobacilli within an artisanal Sicilian cheese, enabling the identification of 17 strains of Lact. casei, one strain of Lact. delbrueckii and Ped. pentosaceus through the combined use of RAPD PCR, PFGE and 16S rDNA sequencing.     

Effects of two probiotic Lactobacillus strains on jejunal and cecal microbiota of broiler chicken under acute heat stress condition as revealed by molecular analysis of 16S rRNA genes

We examined the effects of probiotic Lactobacillus strains of Lactobacillus agilis JCM 1048 and Lactobacillus salivarius subsp. salicinius JCM 1230 on jejunal and cecal microbiota of broiler chicken under heat stress condition using terminal restriction fragment length polymorphism (T-RFLP) analysis. The jejunal bacterial community was limited to a few bacterial groups, mostly Lactobacillus spp. A relatively abundant and higher prevalence of Lactobacillus spp. were observed in the jejunal and cecal microbiota of the probiotic chickens compared with those of the control chickens under heat stress condition. In general, the probiotic strains did not significantly affect the abundance of L. agilis and L. salivarius in chicken intestine but clearly contributed to increasing their prevalence in the probiotic chickens. The probiotic Lactobacillus strains enriched the diversity of Lactobacillus flora in chicken jejunum and cecum by increasing the abundance and prevalence of Lactobacillus spp. inhabiting the intestine. The richness of Lactobacillus species tended to be similar among the jejunal and cecal microbiota. The bacterial community of cecum was complex and age-dependent. The major components of the cecal microbiota were clostridia and lactobacilli. The Clostridium subcluster XIVa was the most predominant group in chicken cecum. Probiotic Lactobacillus strains restored the microbial balance and maintained the natural stability of indigenous bacterial microbiota following heat stress-induced changes.