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1.
S. G. Botina N. V. Koroban K. M. Klimina A. A. Glazova N. V. Zakharevich V. V. Zinchenko V. N. Danilenko 《Russian Journal of Genetics》2010,46(12):1399-1406
The species and strain genetic diversity of bacterial cultures belonging to the genus Lactobacillus, which were isolated from the gastrointestinal microbiome of the human population living in the former Soviet Union in the
years 1960–1980, was studied. The bacteria demonstrated probiotic characteristics. Phylogenetic analysis of sequences of the
gene coding for 16S rRNA detected earlier by us, showed that the gene found in bacteria isolated from the intestinal content
of healthy adultsand represented by species L. plantarum, L. helveticus, L. casei/paracasei, L. rhamnosus, and L. fermentum has high homology (97–100%) with this gene in type representatives of the species. The genotypic and strain diversity of
cultures was studied using RAPD-PCR and nonspecific primers. This method with the use of the ERIC-1 primer gave reliable and
reproducible results as compared that using with M13 and MSP primers and allowed the identification of examined bacteria belonging
to the genus Lactobacillus at the level of species and certification at the strain level. 相似文献
2.
A high-throughput, medium-discrimination method for preliminary typing and selecting non-identical isolates of lactic acid
bacteria in cheeses was developed. RAMP, a PCR with one microsatellite-targeted and one random primer, was used for preliminary
typing of 1119 isolates of lactic acid bacteria from Slovak Bryndza cheese. A total of 59 genotypes were identified based
on RAMP profiles consisting of 12–23 DNA fragments of 150–3000 bp. For example, 18, 17, 13 and 7 different RAMP-types were
identified in Lactobacillus brevis, L. plantarum, L. paracasei and L. fermentum, respectively. The method facilitated well reproducible, medium-discrimination typing of Lactobacillus spp. and Pediococcus spp. at a subspecies level and proved to be suitable for preliminary typing of lactic acid bacteria isolated from cheese. 相似文献
3.
Frank Schmidt Thomas Fiege Hanne K. Hustoft Susanne Kneist Bernd Thiede Dr. 《Proteomics》2009,9(7):1994-2003
A taxonomical study of 90 isolates of lactobacilli isolated from soft and hard carious dentine of 70 deciduous molars is presented. The Lactobacillus strains were determined by shotgun mass mapping (SMM). This method based on MALDI‐MS analysis of Lactobacillus isolates treated with trypsin followed by database comparison against a library of mass spectra derived from 20 reference strains. The SMM method allowed to discriminate different Lactobacillus subspecies. The method was used to analyse Lactobacillus isolates of unknown identity derived from carious dentine. Application of the SMM method to isolates from hard carious dentine revealed a nearly similar distribution of L. paracasei ss paracasei (29%), L. paracasei ss tolerans (32%) and L. casei ss rhamnosus (23%) as dominant subspecies. On the other hand, samples derived from soft carious dentine showed a clear bias only to L. paracasei ss paracasei (60%), whereas L. paracasei ss tolerans (14%) and L. casei ss rhamnosus (12%) were clear minorities. Compared to existent methods, SMM has unique potential for the analysis of Lactobacillus strains on subspecies level. 相似文献
4.
S. T. Danova G. Stoyancheva Y. Manasiev V. Miteva 《World journal of microbiology & biotechnology》2005,21(6-7):835-842
Summary Nine Lactobacillus-specific and non-isotopically (digoxygenin) labelled probes were developed on the basis of Lactobacillus total chromosomal DNA. Their specificity and applicability for Lactobacillus discrimination was proven by DNA–DNA hybridization to reference strains from the American Type Culture Collection (ATCC).
The DNA probes were divided into three groups depending on the ability to hybridize to DNA from the same and/or from a group
of related Lactobacillus strains. They were assayed in the species-specific detection of vaginal strains from the genus Lactobacillus. Six DNA probes were successfully applied for characterization of 21 newly isolated vaginal Lactobacilli. The species affliation
of some isolates was determined. The developed DNA probes were evaluated for usage as a qualitative hybridization test for
detection of Lactobacillus species in mixed cultures, obtained directly from vaginal samples without strain isolation. 相似文献
5.
Ruíz FO Gerbaldo G García MJ Giordano W Pascual L Barberis IL 《Current microbiology》2012,64(4):349-356
Group B streptococci (GBS) are bacterial species that colonize the vagina in pregnant women and as such may cause serious
infections in neonates that passed through the birth channel. The objective of this work was to study the inhibitory activities
produced by each bacteriocin-like inhibitory substance (BLIS) of Lactobacillus rhamnosus L60 and Lactobacillus fermentum L23, and the effects of the combined BLIS-es of these lactobacilli on GBS. The interactions between the BLIS-es were assessed
by qualitative and quantitative methods on agar plates. The minimum inhibitory concentrations (MICs) and fractional inhibitory
concentrations (FICs) were determined by a modification of the broth microdilution and checkerboard methods, respectively.
Antibiotic susceptibilities of all S. agalactiae strains were assayed and the results of these tests were evaluated for statistical significance. A 7.5% of GBS isolates were
recovered from 760 pregnant women and 91% of those strains were susceptible to each BLIS produced by L. fermentum,
L. rhamnosus, and also to a mixture of them. The comparisons among the BLIS-es showed statistically significant differences, with a combination
of the BLIS-es from the two Lactobacillus species being better than the BLIS of each one alone (P < 0.05) as GBS growth inhibitors. Synergistic activities between the BLIS-es were found on 100% of susceptible GBS strains,
MICs ranges of BLIS of L23 and L60 were 80–160 and 160–320 UA ml−1, respectively. By the checkerboard method, the BLIS-es combination showed synergistic effect on all sensitive strains tested,
with values of FICs ranging from 0.131 to 0.218. The BLIS-es produced by these lactobacilli of vaginal origin were able to
inhibit S. agalactiae isolates. The results indicate that these strains may have probiotic potential for the control of GBS in women and may consequently
prevent GBS infections in newborns. 相似文献
6.
J. Bania A. Żarczyńska J. Molenda A. Dąbrowska K. Kosek-Paszkowska M. Więckowska-Szakiel B. Różalska 《Folia microbiologica》2009,54(1):17-24
Analysis of actA gene sequence polymorphism has been shown to be an effective and relatively inexpensive method for subtyping Listeria monocytogenes isolates, allowing the division of the population of this species into two deeply separate lineages. This sequence-based
method as well as PCR-mediated fingerprinting were applied here for the differentiation of 49 isolates of food and clinical
origin. Correlation between these two typing approaches was high. Both methods divided the isolates into two lineages, designated
I (33 isolates) and II (16 isolates). All the 33 lineage I isolates were assigned to the same, or closely related, six clusters
by both typing methods. For the lineage II isolates, PCR fingerprinting was found to be more discriminatory. The isolates
were characterized by cell invasion assay. All highly invasive isolates were assigned to lineage I, which constituted a heterogeneous
group also containing low-invasive isolates. High-invasive isolates were not found in the genetically determined lineage II.
A particular actA cluster, designated Ha, contained all the isolates showing the lowest invasiveness. A common trait of the isolates belonging
to this cluster was the presence of a threonine-441 of the deduced ActA sequence instead of the alanine-441 present in the
remaining isolates. Thirteen human isolates were classified to lineage I and five to lineage II. A PCR-based method can therefore
differentiate L. monocytogenes isolates in accordance with the current phylogenetic model of the evolution of this species. 相似文献
7.
Lactobacillus species are usually used as starters for the production of fermented products, and some strains are capable of producing
antimicrobial substances, such as bacteriocins. Because these characteristics are highly desirable, research are continually
being performed for novel Lactobacillus strains with bacteriocinogenic potential for use by food industries. The aim of this study was to characterise the bacteriocinogenic
potential and activity of Lactobacillus isolates. From a lactic acid bacteria culture collection obtained from raw milk and cheese, 27 isolates were identified by
16S rDNA as Lactobacillus spp. and selected for the detection of lantibiotics biosynthesis genes, bacteriocin production, antimicrobial spectra, and
ideal incubation conditions for bacteriocin production. Based on the obtained results, 21 isolates presented at least one
of the three lantibiotics biosynthesis genes (lanB, lanC or lamM), and 23 isolates also produced antimicrobial substances with sensitivity to at least one proteinase, indicating their bacteriocinogenic
activity. In general, the isolates had broad inhibitory activity, mainly against Listeria spp. and Staphylococcus spp. strains, and the best antimicrobial performance of the isolates occurred when they were cultivated at 25 °C for 24 or
48 h or at 35 °C for 12 h. The present study identified the bacteriocinogenic potential of Lactobacillus isolates obtained from raw milk and cheese, suggesting their potential use as biopreservatives in foods. 相似文献
8.
A group of 69 lactobacilli was isolated from caries lesions and root canals of early childhood caries (ECC) affected children
treated in the Department of Pedodontics (Children’s Teaching Hospital, Brno, Czech Republic). Biochemical and physiological properties of all strains were characterized by API 50 CH kit and conventional
tube tests. The rep-PCR fingerprinting with the (GTG)5 primer was used for genotypic grouping of the isolates. The (GTG)5-PCR fingerprinting grouped all analyzed strains into a few clusters in nearly full agreement with phenotype identification
results and clarified the taxonomic position of 13 biochemically unidentified strains. In total, 20 strains of Lactobacillus fermentum, 17 L. rhamnosus, 14 L. casei/paracasei, 7 L. gasseri, 7 L. salivarius and 4 L. plantarum were identified. Mixtures of two or even three Lactobacillus spp. were isolated from a few root canal content samples. Results obtained by biotyping and (GTG)5-PCR were generally comparable except for L. gasseri strains that were not biochemically identified. The (GTG)5-PCR fingerprinting was shown to be quicker, easier to perform and more reliable than biotyping. Our results imply this molecular
method as a good tool for screening and identification of Lactobacillus spp. inhabiting dental plaque. 相似文献
9.
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. 相似文献
10.
Zhechko Panayotov Dimitrov Svetlana Minkova Michaela Michaylova 《World journal of microbiology & biotechnology》2008,24(8):1305-1312
We isolated a total of 49 strains of lactic acid bacteria from the faeces of healthy donors. The species in that group were
determined as L. plantarum (11 strains), L. casei (11 strains), L. rhamnosus (seven strains), L. fermentum (seven strains), L. gasseri (six strains), L. delbrueckii ssp. lactis (four strains), L. salivarius (two strains), and L. acidophilus (one strain). Genotyping at strain level was performed using random amplification of polymorphic DNA (RAPD), pulsed field
gel electrophoresis (PFGE) with endonucleases ApaI and XhoI and amplified fragment length polymorphism (AFLP) with enzymes XhoI and TaqI. The main objective was the comparison of three molecular typing techniques: AFLP, PFGE and RAPD in their applicability
to determine the genetic diversity among the isolates. RAPD was the easiest, comparatively rapid and fairly strain discriminative
tool. PFGE was the most laborious method but producing the most stable profiles with satisfactory discriminatory power. AFLP
proved to be the most discriminative approach for typing of the strains. AFLP could differentiate strains with the same PFGE
profiles. Therefore, AFLP successfully could replace the labor consuming PFGE. The specially developed AFLP and PFGE proved
very high potential to evaluate the strain diversity of Lactobacillus spp. with human origin. 相似文献
11.
Min Zhang Xiaomin Hang Xiaobing Fan Daotang Li Hong Yang 《World journal of microbiology & biotechnology》2008,24(1):7-14
Fourteen Lactobacillus strains of six species were investigated with their characteristics of bile salt tolerance, deconjugation of sodium taurocholate
and cholesterol removal in the spent broth. Meanwhile, a co-precipitation curve of cholesterol with cholic acid at concentrations
ranged 0.0–6.0 μM/ml was involved in the evaluation of cholesterol removal. Results demonstrated that both co-precipitation
and assimilation effects contributed to cholesterol removal during the incubation of these Lactobacillus strains. It was also indicated that the supplementation of bile salts influenced the cholesterol removal, not only as an
essential factor related to co-precipitation but also a critical condition for cholesterol assimilation. Out of all strains
tested, four L. plantarum strains LS12, LS31, Lp501 and Lp529 exhibited a high ability of cholesterol assimilation (maximum 20.76 μg/ml), deconjugation
of sodium taurocholate (maximum 5.00 μM/ml) and bile tolerance. They could be further studied and used as potential probiotics
strains to reduce serum cholesterol in humans 相似文献
12.
Imrich Turcovský Kristína Kuniková Hana Drahovská Eva Kaclíková 《Antonie van Leeuwenhoek》2011,99(2):257-269
The aim of this study was to identify and characterize Cronobacter spp. isolated from a range of foods. A total of 71 Cronobacter strains were isolated from 602 foods in our laboratory. The highest contamination was observed in foods of plant origin,
e.g. spices, teas, chocolate, nuts, pastries and vegetables. On the basis of genus and species identification performed using
genus-specific PCR, 16S rRNA sequencing and AFLP genotyping, most of the strains belonged to Cronobacter sakazakii. Biochemical profiling by the tests included in API 20E, complemented with relevant additional tests, classified the strains
into 13 biogroups. AFLP genotyping facilitated discrimination of six main groups at the 70% similarity level and strain grouping
correlated clearly with species identification. Our results indicate that molecular typing by AFLP may be applied as a useful
tool not only for direct comparison of Cronobacter isolates, providing traceability, but also for the reliable species classification. Moreover, tracing of these bacteria in
a wider variety of foods should be important to enhance the knowledge of their transmission. 相似文献
13.
Identification and characterization of the dominant lactic acid bacteria isolated from traditional fermented milk in Mongolia 总被引:1,自引:0,他引:1
Z. H. Sun W. J. Liu J. C. Zhang J. Yu W. Gao M. Jiri B. Menghe T. S. Sun H. P. Zhang 《Folia microbiologica》2010,55(3):270-276
Five samples of Airag and 20 of Tarag (both in Mongolia) were collected from scattered households. One hundred strains of
lactic acid bacteria (LAB) were isolated and identified from these samples according to phenotypic characterization and 16S
rRNA gene sequence analysis. Eighty-five isolates belonged to the genus Lactobacillus, 15 being classified as coccoid LAB. All isolates belonged to 5 genera and 11 to different species and subspecies. Lactobacillus (Lb.) helveticus was predominant population in Airag samples, Lb. fermentum and Lb. helveticus were the major LAB microflora in Tarag. 相似文献
14.
Chrobak D Kizerwetter-Świda M Rzewuska M Moodley A Guardabassi L Binek M 《Folia microbiologica》2011,56(5):415-422
The aim of this study was to determine the species distribution among 44 randomly selected clinical isolates (30 mecA-positive and 14 mecA-negative) of animal origin previously identified as Staphylococcus intermedius by phenotypic tests and species-specific PCR amplification of the 16S rRNA gene. For this purpose, we used a multiplex PCR
for the detection of the nuc gene and restriction fragment length polymorphism analysis of pta gene amplified by PCR. Both methods allow discrimination of Staphylococcus pseudintermedius from the other closely related members of the S. intermedius group and other coagulase-positive staphylococci isolated from animals. Genetic diversity of S. pseudintermedius strains was analyzed by staphylococcal protein A-encoding gene (spa) typing. Multiplex PCR method was used to identify staphylococcal cassette chromosome mec (SCCmec) type in mecA-positive strains. All isolates previously identified as S. intermedius were shown to belong to S. pseudintermedius. According to PCR-based SCCmec typing, SCCmecIII was the most prevalent type (n = 23), and solely seven isolates were designated as non-typeable. Furthermore, the assessment of spa-typing results revealed that the majority of all strains (n = 27) harbored spa type t02, and 17 strains were classified as non-typeable. 相似文献
15.
M. Tamburro G. Ripabelli I. Fanelli G. Maria Grasso M. Lucia Sammarco 《Journal of applied microbiology》2010,108(5):1602-1611
Aims: In this study, 105 Listeria monocytogenes strains isolated from humans, foods and environmental samples were characterized using several typing methods. Moreover, serotyping procedure was evaluated, and a cost‐effective methodological approach based on preliminary PCRs screening was proposed. Methods and Results: The isolates were analysed by conventional serotyping, multiplex‐PCRs for serogroup and lineage identification and PCR–RFLP of inlA gene to identify potentially noninvasive L. monocytogenes. Among the strains, only the serotypes 1/2a, 1/2c, 1/2b, 4b and 3a were identified. The isolates were classified into serogroups I (58·10%), II (22·85%), III (12·38%) and IV (6·67%). Among clinical strains, lineage I was more represented (68·75%) than lineage II; whereas, lineage II was more associated with food (90·24%) and environmental (85·72%) isolates. Most of food (89·02%) and environmental (85·71%) isolates were classified into truncated InlA profiles, whereas the 93·75% of clinical strains were associated with a complete form of the protein. Conclusion: Molecular techniques were sensitive and specific for classifying strains into serogroup and lineage and in agreement with the serotyping. Moreover, a preliminary PCRs‐based screening was proposed to select only the necessary antisera by a flow chart; this methodological approach allows cost saving up to 42%. Our results further suggest the role of InlA protein in human listeriosis, particularly in immunocompetent individuals, and a correlation between truncated protein and serotype. Significance and Impact of the Study: This study further validates molecular methods for L. monocytogenes analysis and proposed a new cost‐effective approach for serotyping. It could help to improve a national surveillance network for L. monocytogenes infections in Italy. 相似文献
16.
Abstract
Intestinal microbiota comprise a complex ecosystem whose equilibrium is crucial for the health of animal species. For humans,
data exist on the microbiota composition in adult subjects, but few studies have addressed the microbiota composition in infants.
In particular, data on the presence and species distribution of members of the genus Lactobacillus in newborns (less than one week old) are lacking. In the present work, the feces of healthy newborns were sampled to determine
the taxonomic composition of Lactobacillus in the intestinal microbiota in a group of 16 neonates. In total, 1640 colony-forming units (CFU) were isolated, of which
420 grouped in the Lactobacillus genus by means of primary phenotypic characterization. The 420 isolates were further grouped into 125 strains on the basis
of identical plasmid profiles. Of these 125 strains, 21 turned out to be permanent, i.e., they were identified in the feces of the same subject on several consecutive days. Sugar fermentation, DNA/DNA hybridization,
and S-layer protein determination enabled us to classify 52 of the 125 strains as follows: L. paracasei (40 strains), L. delbrueckii sp. (1 strain), and L. acidophilus (sensu stricto) (11 strains). Based on the same criteria, the remaining 73 strains were tentatively allotted to the Johnson subgroup B,
although hybridization experiments with probes specific for L. gasseri and L. johnsonii species were not performed. The presence of new species among these 73 strains cannot be excluded. Surprisingly, the obligately
heterofermentative lactobacilli, L. reuteri in particular, were entirely absent from the feces of healthy newborns.
Received: 26 March 1997; Accepted: 24 July 1997 相似文献
17.
Rajesh Kumar Sunita Grover Virender Kumar Batish 《Probiotics and antimicrobial proteins》2011,3(3-4):186-193
In the present scenario, it is now well documented that probiotics confer health benefits to the host and the purported probiotic effects are highly strain specific. Hence, accurate genotypic identification is extremely important to link the strain to the specific health effect. With this aim, specific primed-PCR assays were developed and explored for the molecular identification and typing of a putative indigenous probiotic isolate Lp91 of human faecal origin. PCR with specific primers targeting 23S rRNA gene of genus Lactobacillus and 16S rRNA gene of species L. plantarum resulted positive for Lp91. In addition, BLAST analysis of 16S rRNA gene sequence of Lp91 and multiple sequence alignment of 16S rRNA gene variable (V2-V3) regions along with the reference sequences revealed it as L. plantarum with a sequence identity of more than 99%. Furthermore, resolution of 16S rRNA gene sequences was sufficient to infer a phylogenetic relationship amongst Lactobacillus species. In order to determine strain-level variations, randomly amplified polymorphic DNA (RAPD) banding profiles of Lp91 obtained with OPAA-01, OPAP-01 and OPBB-01 primers were compared with those of reference strains of Lactobacillus spp., and Lp91 could be delineated as a distinct strain. Apart from this, presence of probiotic markers viz. bile salt hydrolase (bsh) and collagen-binding protein (cbp) encoding genes in Lp91 genome could be attributed to its exploitation as a potential probiotic adjunct in the development of indigenous functional foods. Lactobacillus isolates/or strains from the gastrointestinal system, fermented products and other environmental niches could be identified and characterized by employing the PCR methods developed in this study; they are rapid, reproducible and more accurate than the conventional methods based on the fermentation profiles. 相似文献
18.
The increasing interest in probiotic lactobacilli implicates the requirement of techniques that allow a rapid and reliable identification of these organisms. In this study, group-specific PCR and RAPD-PCR analyses were used to identify strains of the Lactobacillus casei and Lactobacillus acidophilus groups most commonly used in probiotic yogurts. Group-specific PCR with primers for the L. casei and L. acidophilus groups, as well as L. gasseri/johnsonii, could differentiate between 20 Lactobacillus strains isolated from probiotic yogurts and assign these into the corresponding groups. For identification of these strains to species or strain level, RAPD profiles of the 20 Lactobacillus strains were compared with 11 reference strains of the L. acidophilus and L. casei group. All except one strain could be attributed unambigously to the species L. acidophilus, L. johnsonii, L. crispatus, L. casei, and L. paracasei. DNA reassociation analysis confirmed the classification resulting from the RAPD-PCR. 相似文献
19.
Apetroaie C Andersson MA Spröer C Tsitko I Shaheen R Jääskeläinen EL Wijnands LM Heikkilä R Salkinoja-Salonen MS 《Archives of microbiology》2005,184(3):141-151
Producers of cereulide, the emetic toxin of Bacillus cereus, are known to constitute a specific subset within this species. We investigated physiological and genetic properties of 24
strains of B. cereus including two high cereulide producers (600–1,800 ng cereulide mg−1 wet weight biomass), seven average producers (180–600 ng cereulide mg−1 wet weight biomass), four low cereulide producers (20–160 ng cereulide mg−1 wet weight biomass) and 11 non-producers representing isolates from food, food poisoning, human gut and environment. The
13 cereulide producers possessed 16S rRNA gene sequences identical to each other and identical to that of B. anthracis strains Ames, Sterne from GenBank and strain NC 08234–02, but showed diversity in the adk gene (two sequence types), in ribopatterns obtained with EcoRI and PvuII (three types of patterns), in tyrosin decomposition, haemolysis and lecithin hydrolysis (two phenotypes). The cereulide-producing
isolates from the human gut represented two ribopatterns of which one was novel to cereulide-producing B. cereus and two phenotypes. We conclude that the cereulide-producing B. cereus are genetically and biochemically more diverse than hitherto thought. 相似文献
20.
It is hard to accurately identify specific species of the Lactobacillus casei group using phenotypic techniques alone. Some strains of this species group are considered to be probiotic and are widely
applied in the food industry. In this study, we compared the use of two phylogenetic markers, the 16S rRNA and dnaK genes, for species discrimination of the members of the L. casei group using sequencing and RFLP. The results showed that L. casei, Lactobacillus
paracasei, Lactobacillus
zeae and Lactobacillus
rhamnosus could be clearly distinguished based on the dnaK gene. The average sequence similarity for the dnaK gene (87.8%) among type strains was significantly less than that of the 16S rRNA sequence (99.1%). Therefore, the dnaK gene can be proposed as an additional molecular phylogenetic marker for L. casei that provides higher resolution than 16S rRNA. Species-specific RFLP profiles of the Lactobacillus strains were obtained with the enzyme ApoI. Our data indicate that the phylogenetic relationships between these strains are easily resolved using sequencing of the
dnaK gene or RFLP assays. 相似文献