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Identification of Lactobacillus isolates from the gastrointestinal tract, silage, and yoghurt by 16S-23S rRNA gene intergenic spacer region sequence comparisons.

Lactobacillus isolates were identified by PCR amplification and sequencing of the region between the 16S and 23S rRNA genes (spacer region). The sequences obtained from the isolates were compared to those of reference strains held in GenBank. A similarity of 97.5% or greater was considered to provide identification. To check the reliability of the method, the V2-V3 region of the 16S rRNA gene was amplified and sequenced in the case of isolates whose spacer region sequences were less than 99% similar to that of a reference strain. Confirmation of identity was obtained in all instances. Spacer region sequencing provided rapid and accurate identification of Lactobacillus isolates obtained from gastrointestinal, yoghurt, and silage samples. It had an advantage over 16S V2-V3 sequence comparisons because it distinguished between isolates of Lactobacillus casei and Lactobacillus rhamnosus.

Bacterial Typing Techniques↗

Anaerobic conversion of lactic acid to acetic acid and 1, 2-propanediol by Lactobacillus buchneri.

The degradation of lactic acid under anoxic conditions was studied in several strains of Lactobacillus buchneri and in close relatives such as Lactobacillus parabuchneri, Lactobacillus kefir, and Lactobacillus hilgardii. Of these lactobacilli, L. buchneri and L. parabuchneri were able to degrade lactic acid under anoxic conditions, without requiring an external electron acceptor. Each mole of lactic acid was converted into approximately 0.5 mol of acetic acid, 0.5 mol of 1,2-propanediol, and traces of ethanol. Based on stoichiometry studies and the high levels of NAD-linked 1, 2-propanediol-dependent oxidoreductase (530 to 790 nmol min(-1) mg of protein(-1)), a novel pathway for anaerobic lactic acid degradation is proposed. The anaerobic degradation of lactic acid by L. buchneri does not support cell growth and is pH dependent. Acidic conditions are needed to induce the lactic-acid-degrading capacity of the cells and to maintain the lactic-acid-degrading activity. At a pH above 5.8 hardly any lactic acid degradation was observed. The exact function of anaerobic lactic acid degradation by L. buchneri is not certain, but some results indicate that it plays a role in maintaining cell viability.

Acetic Acid↗

Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella species in human feces by using group-specific PCR primers and denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments generated by PCR with 16S ribosomal DNA-targeted group-specific primers was used to detect lactic acid bacteria (LAB) of the genera Lactobacillus, Pediococcus, Leuconostoc, and Weissella in human feces. Analysis of fecal samples of four subjects revealed individual profiles of DNA fragments originating not only from species that have been described as intestinal inhabitants but also from characteristically food-associated bacteria such as Lactobacillus sakei, Lactobacillus curvatus, Leuconostoc mesenteroides, and Pediococcus pentosaceus. Comparison of PCR-DGGE results with those of bacteriological culture showed that the food-associated species could not be cultured from the fecal samples by plating on Rogosa agar. On the other hand, all of the LAB species cultured from feces were detected in the DGGE profile. We also detected changes in the types of LAB present in human feces during consumption of a milk product containing the probiotic strain Lactobacillus rhamnosus DR20. The analysis of fecal samples from two subjects taken before, during, and after administration of the probiotic revealed that L. rhamnosus was detectable by PCR-DGGE during the test period in the feces of both subjects, whereas it was detectable by culture in only one of the subjects.

Adult↗

Surface display of foreign epitopes on the Lactobacillus brevis S-layer.

So far, the inability to establish viable Lactobacillus surface layer (S-layer) null mutants has hampered the biotechnological applications of Lactobacillus S-layers. In this study, we demonstrate the utilization of Lactobacillus brevis S-layer subunits (SlpA) for the surface display of foreign antigenic epitopes. With an inducible expression system, L. brevis strains producing chimeric S-layers were obtained after testing of four insertion sites in the slpA gene for poliovirus epitope VP1, that comprises 10 amino acids. The epitope insertion site allowing the best surface expression was used for the construction of an integration vector carrying the gene region encoding the c-Myc epitopes from the human c-myc proto-oncogene, which is composed of 11 amino acids. A gene replacement system was optimized for L. brevis and used for the replacement of the wild-type slpA gene with the slpA-c-myc construct. A uniform S-layer, displaying on its surface the desired antigen in all of the S-layer protein subunits, was obtained. The success of the gene replacement and expression of the uniform SlpA-c-Myc recombinant S-layer was confirmed by PCR, Southern blotting MALDI-TOF mass spectrometry, whole-cell enzyme-linked immunosorbent assay, and immunofluorescence microscopy. Furthermore, the integrity of the recombinant S-layer was studied by electron microscopy, which indicated that the S-layer lattice structure was not affected by the presence of c-Myc epitopes. To our knowledge, this is the first successful expression of foreign epitopes in every S-layer subunit of a Lactobacillus S-layer while still maintaining the S-layer lattice structure.

Bacterial Proteins↗

In situ determination of the intracellular pH of Lactococcus lactis and Lactobacillus plantarum during pressure treatment.

Hydrostatic pressure may affect the intracellular pH of microorganisms by (i) enhancing the dissociation of weak organic acids and (ii) increasing the permeability of the cytoplasmic membrane and inactivation of enzymes required for pH homeostasis. The internal pHs of Lactococcus lactis and Lactobacillus plantarum during and after pressure treatment at 200 and 300 MPa and at pH values ranging from 4.0 to 6.5 were determined. Pressure treatment at 200 MPa for up to 20 min did not reduce the viability of either strain at pH 6.5. Pressure treatment at pH 6.5 and 300 MPa reduced viable cell counts of Lactococcus lactis and Lactobacillus plantarum by 5 log after 20 and 120 min, respectively. Pressure inactivation was faster at pH 5 or 4. At ambient pressure, both strains maintained a transmembrane pH gradient of 1 pH unit at neutral pH and about 2 pH units at pH 4.0. During pressure treatment at 200 and 300 MPa, the internal pH of L. lactis was decreased to the value of the extracellular pH during compression. The same result was observed during treatment of Lactobacillus plantarum at 300 MPa. Lactobacillus plantarum was unable to restore the internal pH after a compression-decompression cycle at 300 MPa and pH 6.5. Lactococcus lactis lost the ability to restore its internal pH after 20 and 4 min of pressure treatment at 200 and 300 MPa, respectively. As a consequence, pressure-mediated stress reactions and cell death may be considered secondary effects promoted by pH and other environmental conditions.

Animals↗

Analysis, characterization, and loci of the tuf genes in lactobacillus and bifidobacterium species and their direct application for species identification.

We analyzed the tuf gene, encoding elongation factor Tu, from 33 strains representing 17 Lactobacillus species and 8 Bifidobacterium species. The tuf sequences were aligned and used to infer phylogenesis among species of lactobacilli and bifidobacteria. We demonstrated that the synonymous substitution affecting this gene renders elongation factor Tu a reliable molecular clock for investigating evolutionary distances of lactobacilli and bifidobacteria. In fact, the phylogeny generated by these tuf sequences is consistent with that derived from 16S rRNA analysis. The investigation of a multiple alignment of tuf sequences revealed regions conserved among strains belonging to the same species but distinct from those of other species. PCR primers complementary to these regions allowed species-specific identification of closely related species, such as Lactobacillus casei group members. These tuf gene-based assays developed in this study provide an alternative to present methods for the identification for lactic acid bacterial species. Since a variable number of tuf genes have been described for bacteria, the presence of multiple genes was examined. Southern analysis revealed one tuf gene in the genomes of lactobacilli and bifidobacteria, but the tuf gene was arranged differently in the genomes of these two taxa. Our results revealed that the tuf gene in bifidobacteria is flanked by the same gene constellation as the str operon, as originally reported for Escherichia coli. In contrast, bioinformatic and transcriptional analyses of the DNA region flanking the tuf gene in four Lactobacillus species indicated the same four-gene unit and suggested a novel tuf operon specific for the genus Lactobacillus.

Adult↗

Characterization of a theta-type plasmid from Lactobacillus sakei: a potential basis for low-copy-number vectors in lactobacilli.

The complete nucleotide sequence of the 13-kb plasmid pRV500, isolated from Lactobacillus sakei RV332, was determined. Sequence analysis enabled the identification of genes coding for a putative type I restriction-modification system, two genes coding for putative recombinases of the integrase family, and a region likely involved in replication. The structural features of this region, comprising a putative ori segment containing 11- and 22-bp repeats and a repA gene coding for a putative initiator protein, indicated that pRV500 belongs to the pUCL287 subfamily of theta-type replicons. A 3.7-kb fragment encompassing this region was fused to an Escherichia coli replicon to produce the shuttle vector pRV566 and was observed to be functional in L. sakei for plasmid replication. The L. sakei replicon alone could not support replication in E. coli. Plasmid pRV500 and its derivative pRV566 were determined to be at very low copy numbers in L. sakei. pRV566 was maintained at a reasonable rate over 20 generations in several lactobacilli, such as Lactobacillus curvatus, Lactobacillus casei, and Lactobacillus plantarum, in addition to L. sakei, making it an interesting basis for developing vectors. Sequence relationships with other plasmids are described and discussed.

Amino Acid Sequence↗

Relative ability of orally administered Lactobacillus murinus to predominate and persist in the porcine gastrointestinal tract.

Five porcine-derived Lactobacillus or Pediococcus isolates administered to pigs (n = 4), either singly or as a combination at approximately 10(10) CFU per day varied with respect to intestinal survival and persistence. Two Lactobacillus murinus strains survived best and were excreted at approximately 10(7) to 10(8) CFU/g of feces. In contrast, Pediococcus pentosaceus DPC6006 had the lowest fecal count at approximately 10(5) CFU/g and was excreted at a significantly lower level than both L. murinus strains. Fecal L. murinus DPC6003 counts were also significantly higher than both Lactobacillus salivarius DPC6005 and Lactobacillus pentosus DPC6004 ( approximately 10(6) CFU/g). The L. murinus strains persisted for at least 9 days postadministration in both the feces and the cecum. Animals fed a combination of all five strains excreted approximately 10(7) CFU of the administered strains/g, with L. murinus predominating, as determined by randomly amplified polymorphic DNA PCR. Postadministration, variation was observed between animals fed the strain combination, but in general, L. murinus DPC6002 and DPC6003 and L. pentosus DPC6004 predominated in the feces and the cecum while P. pentosaceus DPC6006 was detected only in the cecum. Fifteen days after the start of culture administration, mean fecal Enterobacteriaceae counts were significantly lower in some of the treatment groups. In addition, when mean preadministration counts were compared with those obtained after 21 days of culture administration, Enterobacteriaceae counts were reduced by approximately 87 to 98% in pigs fed L. salivarius DPC6005, P. pentosaceus DPC6006, L. pentosus DPC6004, and the culture mix. In conclusion, the porcine intestinal isolates have potential as probiotic feed additives for pigs, with differences in strain performance highlighting the advantages of using culture combinations.

Administration, Oral↗

Detection, characterization, and in vitro and in vivo expression of genes encoding S-proteins in Lactobacillus gallinarum strains isolated from chicken crops.

Thirty-eight isolates of Lactobacillus gallinarum cultured from the crops of broiler chickens were screened for the presence of genes encoding S-layer proteins. All of the isolates had two S-protein genes, which were designated Lactobacillus gallinarum S-protein (lgs) genes. One gene in each isolate was either lgsA or lgsB. The Lactobacillus isolates were further characterized by pulsed-field gel electrophoresis of DNA digests, which grouped the isolates into 17 genotypes (strains). The second gene in each of eight representative strains was sequenced and shown to differ among strains (lgsC, lgsD, lgsE, lgsF, lgsG, lgsH, and lgsI). The genome of each strain thus encoded a common S-protein (encoded by either lgsA or lgsB) and a strain-specific S-protein. The extraction of cell surface proteins from cultures of the eight strains showed that each strain produced a single S-protein that was always encoded by the strain-specific lgs gene. Two of the strains were used to inoculate chickens maintained in a protected environment which were Lactobacillus-free prior to inoculation. DNAs and RNAs extracted from the digesta of the chickens were used for PCR and reverse transcription-PCR, respectively, to demonstrate the presence and transcription of lgs genes in vivo. In both cases, only the strain-specific gene was transcribed. Both of the strains adhered to the crop epithelium, consistent with published data predicting that S-proteins of lactobacilli are adhesins. The results of this study provide a basis for the investigation of gene duplication and sequence variation as mechanisms by which bacterial strains of the same species can share the same habitat.

Animals↗

A high-molecular-mass surface protein (Lsp) and methionine sulfoxide reductase B (MsrB) contribute to the ecological performance of Lactobacillus reuteri in the murine gut.

Members of the genus Lactobacillus are common inhabitants of the gut, yet little is known about the traits that contribute to their ecological performance in gastrointestinal ecosystems. Lactobacillus reuteri 100-23 persists in the gut of the reconstituted Lactobacillus-free mouse after a single oral inoculation. Recently, three genes of this strain that were specifically induced (in vivo induced) in the murine gut were identified (38). We report here the detection of a gene of L. reuteri 100-23 that encodes a high-molecular-mass surface protein (Lsp) that shows homology to proteins involved in the adherence of other bacteria to epithelial cells and in biofilm formation. The three in vivo-induced genes and lsp of L. reuteri 100-23 were inactivated by insertional mutagenesis in order to study their biological importance in the murine gastrointestinal tract. Competition experiments showed that mutation of lsp and a gene encoding methionine sulfoxide reductase (MsrB) reduced ecological performance. Mutation of lsp impaired the adherence of the bacteria to the epithelium of the mouse forestomach and altered colonization dynamics. Homologues of lsp and msrB are present in the genomes of several strains of Lactobacillus and may play an important role in the maintenance of these bacteria in gut ecosystems.

Animals↗

Rapid differentiation and in situ detection of 16 sourdough lactobacillus species by multiplex PCR.

A two-step multiplex PCR-based method was designed for the rapid detection of 16 species of lactobacilli known to be commonly present in sourdough. The first step of multiplex PCR was developed with a mixture of group-specific primers, while the second step included three multiplex PCR assays with a mixture of species-specific primers. Primers were derived from sequences that specify the 16S rRNA, the 16S-23S rRNA intergenic spacer region, and part of the 23S rRNA gene. The primer pairs designed were shown to exclusively amplify the targeted rrn operon fragment of the corresponding species. Due to the reliability of simultaneously identifying Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum, a previously described multiplex PCR method employing recA gene-derived primers was included in the multiplex PCR system. The combination of a newly developed, quick bacterial DNA extraction method from sourdough and this multiplex PCR assay allows the rapid in situ detection of several sourdough-associated lactobacilli, including the recently described species Lactobacillus rossii, and thus represents a very useful alternative to culture-based methodologies.

Bacterial Typing Techniques↗

Some slime-forming heterofermentative species of the genus Lactobacillus.

Lactobacillus coprophilus subsp. confusus (NCDO 1586) and 18 other strains of slime-forming heterofermentative lactobacilli obtained from diverse sources are considered to form a new species on the basis of their physiological characteristics and similarities of their pyruvate reductases and lactate dehydrogenases. It is suggested that these strains should be named Lactobacillus confusus (Holzapfel & Kandler) comb. nov. (L. coprophilus subsp. confusus Holzapfel & Kandler), reasons being given. The type strain is NCDO 1586 (NCIB 9311, ATCC 10881). Strains of Lactobacillus vermiforme and Lactobacillus viridescens also form slime. However, L. vermiforme was readily separated from the other two by the criteria used. Although physiological characteristics separated L. viridescens from L. confusus, a relationship between these two species and also between them and the leuconostocs was indicated by the properties of the pyruvate reductases and lactate dehydrogenases. The slime produced by all species was found to be a glucan, probably a dextran, containing primarily alpha-1-6-glycosidic linkages.

Ammonia↗

Divergent patterns of colonization and immune response elicited from two intestinal Lactobacillus strains that display similar properties in vitro.

Lactobacilli derived from the endogenous flora of normal donors are being increasingly used as probiotics in functional foods and as vaccine carriers. However, a variety of studies done with distinct strains of lactobacilli has suggested heterogeneous and strain-specific effects. To dissect this heterogeneity at the immunological level, we selected two strains of lactobacilli that displayed similar properties in vitro and studied their impact on mucosal and systemic B-cell responses in monoxenic mice. Germfree mice were colonized with Lactobacillus johnsonii (NCC 533) or Lactobacillus paracasei (NCC 2461). Bacterial loads were monitored for 30 days in intestinal tissues, and mucosal and systemic B-cell responses were measured. Although both Lactobacillus strains displayed similar growth, survival, and adherence properties in vitro, they colonized the intestinal lumen and translocated into mucosal lymphoid organs at different densities. L. johnsonii colonized the intestine very efficiently at high levels, whereas the number of L. paracasei decreased rapidly and it colonized at low levels. We determined whether this difference in colonization correlated with an induction of different types of immune responses. We observed that colonization with either strain induced similar germinal center formation and immunoglobulin A-bearing lymphocytes in the mucosa, suggesting that both strains were able to activate mucosal B-cell responses. However, clear differences in patterns of immunoglobulins were observed between the two strains in the mucosa and in the periphery. Therefore, despite similar in vitro probiotic properties, distinct Lactobacillus strains may colonize the gut differently and generate divergent immune responses.

Animals↗

Probiotic Lactobacillus spp. diminish Helicobacter hepaticus-induced inflammatory bowel disease in interleukin-10-deficient mice.

Clinical and experimental evidence has demonstrated the potential role of probiotics in the prevention or treatment of inflammatory bowel disease. Probiotic clones with direct immunomodulatory activity may have anti-inflammatory effects in the intestine. We investigated the roles of tumor necrosis factor alpha (TNF-alpha)-inhibitory Lactobacillus clones with a pathogen-induced murine colitis model. Murine-derived probiotic lactobacilli were selected in vitro for their ability to inhibit TNF-alpha secretion by Helicobacter hepaticus-stimulated macrophages. Interleukin-10 (IL-10)-deficient mice were treated with probiotic Lactobacillus reuteri in combination with Lactobacillus paracasei and then challenged with H. hepaticus. Ten weeks postinoculation, the severity of typhlocolitis was assessed by histologic examination of the cecocolic region. Intestinal proinflammatory cytokine responses were evaluated by real-time quantitative reverse transcriptase PCR and immunoassays, and the quantities of intestinal H. hepaticus were evaluated by real-time PCR. Intestinal colonization by TNF-alpha-inhibitory lactobacilli reduced intestinal inflammation in H. hepaticus-challenged IL-10-deficient mice despite similar quantities of H. hepaticus in cocolonized animals. Proinflammatory colonic cytokine (TNF-alpha and IL-12) levels were lowered in Lactobacillus-treated animals. In this H. hepaticus-challenged IL-10-deficient murine colitis model, lactobacilli demonstrated probiotic effects by direct modulation of mucosal inflammatory responses.

Animals↗

Immunological relationships among lactic dehydrogenases in the genera Lactobacillus and Leuconostoc.

Antisera were prepared against pure nicotinamide adenine dinucleotide-dependent d-lactic dehydrogenases of Lactobacillus leichmannii, L. jensenii, and L. fermenti. When tested against these three antisera, crude extracts of almost all species of Lactobacillus containing a d-lactic dehydrogenase give cross-reactions. Extensive pairwise comparisons between cross-reacting crude extracts by double diffusion experiments permit the recognition of groups of identical antigenic specificity among the lactic dehydrogenases of the various nomenspecies of Lactobacillus. The same groups are revealed by each of the three antisera. By analyses of spur formation, the groups of identical antigenic specificity can be arranged in order of decreasing similarity to the homologous d-lactic dehydrogenase used as the reference point. From the combined results obtained with the three antisera, a map of the antigenic relationships among the d-lactic dehydrogenases of lactobacilli can be constructed. Microcomplement fixation experiments with two of the three anti-d-lactic dehydrogenases antisera support the conclusions drawn from double diffusion experiments and provide a quantitative estimation of the antigenic relationships among the various d-lactic dehydrogenases. An antiserum was also prepared against the pure l-lactic dehydrogenase of L. acidophilus group III. It cross-reacts with extracts of almost all lactobacilli containing an l-lactic dehydrogenase. With respect to species that contain both d- and l-lactic dehydrogenases, this antiserum reveals the same groups of identical antigenic specificity as do the antisera directed against d-lactic dehydrogenases. Other than the genus Lactobacillus, only extracts of Leuconostoc cross-react with anti-d-lactic dehydrogenase. No extrageneric cross-reactions were obtained with the anti-l-lactic dehydrogenase.

Animals↗

Quantitative analysis of diverse Lactobacillus species present in advanced dental caries.

Our previous analysis of 65 advanced dental caries lesions by traditional culture techniques indicated that lactobacilli were numerous in the advancing front of the progressive lesion. Production of organic acids by lactobacilli is considered to be important in causing decalcification of the dentinal matrix. The present study was undertaken to define more precisely the diversity of lactobacilli found in this environment and to quantify the major species and phylotypes relative to total load of lactobacilli by real-time PCR. Pooled DNA was amplified by PCR with Lactobacillus genus-specific primers for subsequent cloning, sequencing, and phylogenetic analysis. Based on 16S ribosomal DNA sequence comparisons, 18 different phylotypes of lactobacilli were detected, including strong representation of both novel and gastrointestinal phylotypes. Specific PCR primers were designed for nine prominent species, including Lactobacillus gasseri, L. ultunensis, L. salivarius, L. rhamnosus, L. casei, L. crispatus, L. delbrueckii, L. fermentum, and L. gallinarum. More than three different species were identified as being present in most of the dentine samples, confirming the widespread distribution and numerical importance of various Lactobacillus spp. in carious dentine. Quantification by real-time PCR revealed various proportions of the nine species colonizing carious dentine, with higher mean loads of L. gasseri and L. ultunensis than of the other prevalent species. The findings provide a basis for further characterization of the pathogenicity of Lactobacillus spp. in the context of extension of the carious lesion.

Colony Count, Microbial↗

Randomised trial of cranberry-lingonberry juice and Lactobacillus GG drink for the prevention of urinary tract infections in women.

OBJECTIVE: To determine whether recurrences of urinary tract infection can be prevented with cranberry-lingonberry juice or with Lactobacillus GG drink. DESIGN: Open, randomised controlled 12 month follow up trial. SETTING: Health centres for university students and staff of university hospital. PARTICIPANTS: 150 women with urinary tract infection caused by Escherichia coli randomly allocated into three groups. INTERVENTIONS: 50 ml of cranberry-lingonberry juice concentrate daily for six months or 100 ml of lactobacillus drink five days a week for one year, or no intervention. MAIN OUTCOME MEASURE: First recurrence of symptomatic urinary tract infection, defined as bacterial growth >/=10(5 )colony forming units/ml in a clean voided midstream urine specimen. RESULTS: The cumulative rate of first recurrence of urinary tract infection during the 12 month follow up differed significantly between the groups (P=0.048). At six months, eight (16%) women in the cranberry group, 19 (39%) in the lactobacillus group, and 18 (36%) in the control group had had at least one recurrence. This is a 20% reduction in absolute risk in the cranberry group compared with the control group (95% confidence interval 3% to 36%, P=0.023, number needed to treat=5, 95% confidence interval 3 to 34). CONCLUSION: Regular drinking of cranberry juice but not lactobacillus seems to reduce the recurrence of urinary tract infection.

Adult↗

Promotion of IgA immune response in patients with Crohn's disease by oral bacteriotherapy with Lactobacillus GG.

The effect of oral bacteriotherapy with human Lactobacillus casei strain GG (10(10) colony-forming units twice daily for 10 days) was investigated in Crohn's disease and in juvenile chronic arthritis which are chronic inflammatory diseases associated with impaired mucosal barrier function. During oral bacteriotherapy, the gut immune response was indirectly assessed by solid-phase enzyme-linked immunoassay in 14 children with Crohn's disease, in 9 with juvenile chronic arthritis, and in 7 controls. The immunostimulatory effect of Lactobacillus GG was specific for Crohn's disease, irrespective of its activity: the mean (95% confidence interval) number of specific antibody secreting cells in the IgA class to beta-lactoglobulin increased significantly from 0.2 (0.04-1.3) to 1.4 (0.3-6.0)/10(6) cells and to casein from 0.3 (0.1-1.4) to 1.0 (0.2-4.8)/10(6) cells. The results indicate that orally administered Lactobacillus GG has the potential to increase the gut IgA immune response and thereby to promote the gut immunological barrier. Consequently, Lactobacillus GG could provide an adjunct nutritional therapy for Crohn's disease.

Adolescent↗