ABP-118, a small heat-stable bacteriocin produced by Lactobacillus salivarius subsp. salivarius UCC118, a strain isolated from the ileal-caecal region of the human gastrointestinal tract, was purified to homogeneity. Using reverse genetics, a DNA fragment specifying part of ABP-118 was identified on a 10769 bp chromosomal region. Analysis of this region revealed that ABP-118 was a Class IIb two-peptide bacteriocin composed of Abp118alpha, which exhibited the antimicrobial activity, and Abp118beta, which enhanced the antimicrobial activity. The gene conferring strain UCC118 immunity to the action of ABP-118, abpIM, was identified downstream of the abp118beta gene. Located further downstream of abp118beta, several ORFs were identified whose deduced proteins resembled those of proteins involved in bacteriocin regulation and secretion. Heterologous expression of ABP-118 was achieved in Lactobacillus plantarum, Lactococcus lactis and Bacillus cereus. In addition, the abp118 locus encoded an inducing peptide, AbpIP, which was shown to play a role in the regulation of ABP-118 production. This novel bacteriocin is, to the authors' knowledge, the first to be isolated from a known human probiotic bacterium and to be characterized at the genetic level.

The transfer via the food chain from animals to humans of microbes that are resistant to antimicrobial agents is of increasing concern. To determine the contributions of nonpathogenic microflora to the occurrence and spread of antibiotic resistance (AR) genes in the food chain, 123 lactic acid bacteria were isolated from 29 samples of raw and processed pork and chicken meat products that had previously tested positive for one or more AR genes that encode clinically relevant ARs: tet(M), tet(O), tet(K), erm(A), erm(B), erm(C), aac (6')-Ie aph (2")-Ia, mecA, and blaZ. All of the isolates were initially tested for their AR gene profiles by PCR. The 59 isolates carrying a tet, erm, or blaZ gene were taken through molecular identification, analyzed by determination of the MIC, and subjected to genetic fingerprinting. Lactococcus garvieae was the predominant species (28 isolates), followed by Lactobacillus plantarum (11 isolates) and L. salivarius (6 isolates), whereas Lactococcus lactis subsp. lactis, Lactobacillus johnsonii, L. reuteri, L. crispatus, and L. brevis were identified at lower frequencies. The tet(M) and erm(B) genes were the most frequently detected. Assessment of multiple resistances in 18 tet positive (tet+) isolates revealed that tet(M) plus erm(B) and tet(K) plus erm(B) were the most frequent AR gene patterns. Partial sequencing of the tet(M) open reading frame of three selected strains showed high sequence similarities (> 99%) with tet(M) genes previously found in human pathogens (Listeria monocytogenes and Neisseria meningitidis). Southern hybridization with plasmid profiles revealed these strains contained tet(M)-carrying plasmids.

The antibiotic resistances of 45 lactic acid bacteria strains belonging to the genera Lactobacillus, Streptococcus, Lactococcus, Pediococcus, and Leuconostoc were investigated. The objective was to determine antibiotic resistances and to verify these at the genetic level, as is currently suggested by the European "qualified presumption of safety" safety evaluation system for industrial starter strains. In addition, we sought to pinpoint possible problems in resistance determinations. Primers were used to PCR amplify genes involved in beta-lactam antibiotic, chloramphenicol, tetracycline, and erythromycin resistance. The presence of ribosomal protection protein genes and the ermB gene was also determined by using a gene probe. Generally, the incidences of erythromycin, chloramphenicol, tetracycline, or beta-lactam resistances in this study were low (<7%). In contrast, aminoglycoside (gentamicin and streptomycin) and ciprofloxacin resistances were higher than 70%, indicating that these may constitute intrinsic resistances. The genetic basis for ciprofloxacin resistance could not be verified, since no mutations typical of quinolone resistances were detected in the quinolone determining regions of the parC and gyrA genes. Some starter strains showed low-level ampicillin, penicillin, chloramphenicol, and tetracycline resistances, but no known resistance genes could be detected. Although some strains possessed the cat gene, none of these were phenotypically resistant to chloramphenicol. Using reverse transcription-PCR, these cat genes were shown to be silent under both inducing and noninducing conditions. Only Lactobacillus salivarius BFE 7441 possessed an ermB gene, which was encoded on the chromosome and which could not be transferred in filter-mating experiments. This study clearly demonstrates problems encountered with resistance testing, in that the breakpoint values are often inadequately identified, resistance genes may be present but silent, and the genetic basis and associated resistance mechanisms toward some antibiotics are still unknown.

Lactobacillus salivarius Rogosa et al. 1953 was described as a homofermentative lactobacillus with two varieties: salivarius, typified inter alia by the ability to ferment rhamnose, and salicinius, characterized by the ability to ferment the glucoside salicin. These varieties have become accepted as subspecies divisions. We have examined the relatedness of 32 L. salivarius strains by a polyphasic approach. Carbohydrate fermentation profile analysis did not support clear distinction of the two subspecies. L. salivarius UCC118 was shown to be facultatively heterofermentative, confirming in silico genome analysis. 16S rRNA gene sequences and 16S-23S rRNA intergenic spacer region sequences provided no discrimination between any of the strains or subspecies. Broad subdivisions were distinguishable by pulsed-field gel genomic digest patterns, but they did not allow subspecific or phenotypic distinctions. A phylogeny based upon groEL gene sequences was discordant with rhamnose or salicin fermentation data for many taxa, and no reliable phenotypic correlations could be established. In the absence of meaningful taxonomic criteria, we therefore propose that Lactobacillus salivarius comprises a single species with no infraspecific taxa. Based on the present study and literature data, an emended description of the species Lactobacillus salivarius is provided.

The relatedness among 91 Enterococcus strains representing all validly described species was investigated by comparing a 1,102-bp fragment of atpA, the gene encoding the alpha subunit of ATP synthase. The relationships observed were in agreement with the phylogeny inferred from 16S rRNA gene sequence analysis. However, atpA gene sequences were much more discriminatory than 16S rRNA for species differentiation. All species were differentiated on the basis of atpA sequences with, at a maximum, 92% similarity. Six members of the Enterococcus faecium species group (E. faecium, E. hirae, E. durans, E. villorum, E. mundtii, and E. ratti) showed > 99% 16S rRNA gene sequence similarity, but the highest value of atpA gene sequence similarity was only 89.9%. The intraspecies atpA sequence similarities for all species except E. faecium strains varied from 98.6 to 100%; the E. faecium strains had a lower atpA sequence similarity of 96.3%. Our data clearly show that atpA provides an alternative tool for the phylogenetic study and identification of enterococci.

Salivaricin CRL 1328 is a heat-stable bacteriocin produced by Lactobacillus salivarius CRL 1328, a strain isolated from healthy human vagina, with potential applications for preventing urogenital infections. The objective of this study was to characterize the locus responsible for salivaricin CRL 1328 production and its mechanism of action against Enterococcus faecalis MP97 as the sensitive strain. Oligonucleotides were designed based on sequences of antimicrobial peptides previously described in the literature. The salivaricin CRL 1328 cluster was identified, sequenced and analyzed. This cluster was similar to the previously described ABP118 which codified for a two-peptide bacteriocin. The putative mature peptides of salivaricin CRL 1328, Salalpha and Salbeta were chemically synthesized. These peptides did not show bacteriocin activity when assayed individually. Both peptides exhibited optimal antimicrobial activity at an equimolar ratio. Spectroscopic fluorescence assays were carried out using the synthetic peptides to study the effect of salivaricin on proton motive force. This bacteriocin was shown to dissipate membrane potential and the transmembrane proton gradient, both components of proton motive force. E. faecalis MP97 cells treated with salivaricin CRL 1328 peptides were observed in transmission electron microscopy which revealed ultrastructural modifications of the cell wall.

Commensal lactobacilli frequently produce bile salt hydrolase (Bsh) enzymes whose roles in intestinal survival are unclear. Twenty-six Lactobacillus salivarius strains from different sources all harbored a bsh1 allele on their respective megaplasmids. This allele was related to the plasmid-borne bsh1 gene of the probiotic strain UCC118. A second locus (bsh2) was found in the chromosomes of two strains that had higher bile resistance levels. Four Bsh1-encoding allele groups were identified, defined by truncations or deletions involving a conserved residue. In vitro analyses showed that this allelic variation was correlated with widely varying bile deconjugation phenotypes. Despite very low activity of the UCC118 Bsh1 enzyme, a mutant lacking this protein had significantly lower bile resistance, both in vitro and during intestinal transit in mice. However, the overall bile resistance phenotype of this and other strains was independent of the bsh1 allele type. Analysis of the L. salivarius transcriptome upon exposure to bile and cholate identified a multiplicity of stress response proteins and putative efflux proteins that appear to broadly compensate for, or mask, the effects of allelic variation of bsh genes. Bsh enzymes with different bile-degrading kinetics, though apparently not the primary determinants of bile resistance in L. salivarius, may have additional biological importance because of varying effects upon bile as a signaling molecule in the host.

Twenty-one homofermentative lactic acid bacteria were isolated from fermented cummingcordia (pobuzihi), a traditional food in Taiwan. The isolates had identical 16S rRNA gene sequences that were distinct from those of other lactobacilli, and their closest neighbours in the 16S rRNA gene sequence phylogenetic tree were strains of Lactobacillus acidipiscis. Levels of DNA-DNA relatedness between representative pobuzihi isolates and strains of L. acidipiscis were 17% and below. Furthermore, the new isolates could be differentiated clearly from L. acidipiscis NBRC 102163T and NBRC 102164 in terms of acid production from L-arabinose, rhamnose, mannitol, lactose and 5-ketogluconate. It was concluded that the new isolates represent a single novel species of the genus Lactobacillus, for which the name Lactobacillus pobuzihii sp. nov. is proposed. The type strain is E100301T (=RIFY 6501T =NBRC 103219T =KCTC 13174T).

The aim of this study was to evaluate the use of the phenylalanyl-tRNA synthase alpha subunit (pheS) and the RNA polymerase alpha subunit (rpoA) partial gene sequences for species identification of members of the genus Lactobacillus. Two hundred and one strains representing the 98 species and 17 subspecies were examined. The pheS gene sequence analysis provided an interspecies gap, which in most cases exceeded 10 % divergence, and an intraspecies variation of up to 3 %. The rpoA gene sequences revealed a somewhat lower resolution, with an interspecies gap normally exceeding 5 % and an intraspecies variation of up to 2 %. The combined use of pheS and rpoA gene sequences offers a reliable identification system for nearly all species of the genus Lactobacillus. The pheS and rpoA gene sequences provide a powerful tool for the detection of potential novel Lactobacillus species and synonymous taxa. In conclusion, the pheS and rpoA gene sequences can be used as alternative genomic markers to 16S rRNA gene sequences and have a higher discriminatory power for reliable identification of species of the genus Lactobacillus.

A phylogenetic tree showing diversities among 116 partial (499-bp) Lactobacillus hsp60 (groEL, encoding a 60-kDa heat shock protein) nucleotide sequences was obtained and compared to those previously described for 16S rRNA and tuf gene sequences. The topology of the tree produced in this study showed a Lactobacillus species distribution similar, but not identical, to those previously reported. However, according to the most recent systematic studies, a clear differentiation of 43 single-species clusters was detected/identified among the sequences analyzed. The slightly higher variability of the hsp60 nucleotide sequences than of the 16S rRNA sequences offers better opportunities to design or develop molecular assays allowing identification and differentiation of either distant or very closely related Lactobacillus species. Therefore, our results suggest that hsp60 can be considered an excellent molecular marker for inferring the taxonomy and phylogeny of members of the genus Lactobacillus and that the chosen primers can be used in a simple PCR procedure allowing the direct sequencing of the hsp60 fragments. Moreover, in this study we performed a computer-aided restriction endonuclease analysis of all 499-bp hsp60 partial sequences and we showed that the PCR-restriction fragment length polymorphism (RFLP) patterns obtainable by using both endonucleases AluI and TacI (in separate reactions) can allow identification and differentiation of all 43 Lactobacillus species considered, with the exception of the pair L. plantarum/L. pentosus. However, the latter species can be differentiated by further analysis with Sau3AI or MseI. The hsp60 PCR-RFLP approach was efficiently applied to identify and to differentiate a total of 110 wild Lactobacillus strains (including closely related species, such as L. casei and L. rhamnosus or L. plantarum and L. pentosus) isolated from cheese and dry-fermented sausages.

The genome of Lactobacillus salivarius UCC118 includes a 242-kb megaplasmid, pMP118. We now show that 33 strains of L. salivarius isolated from humans and animals all harbor a megaplasmid, which hybridized with the repA and repE replication origin probes of pMP118. Linear megaplasmids that did not hybridize with the pMP118 repA probe were also found in some strains of L. salivarius, showing for the first time that a lactic acid bacterium has multiple megaplasmids. Phylogenetic analysis of the repE and groEL sequences of 28 L. salivarius strains suggested similar evolutionary paths for the chromosome and megaplasmid. Although the replication origin of circular megaplasmids in L. salivarius was highly conserved, genotypic and phenotypic comparisons revealed significant variation between megaplasmid-encoded traits. Furthermore, megaplasmids of sizes ranging from 120 kb to 490 kb were present in seven strains belonging to six other Lactobacillus species from among 91 strains and 47 species tested. The discovery of the widespread presence of megaplasmids in L. salivarius, and restricted carriage by other Lactobacillus species, provides an opportunity to study the contribution of large extrachromosomal replicons to the biology of Lactobacillus.

Bile salt hydrolase (BSH) is a gut-bacterial enzyme that negatively influences host fat digestion and energy harvesting. The BSH enzyme activity functions as a gateway reaction in the small intestine by the deconjugation of glycine-conjugated or taurine-conjugated bile acids. Extensive gut-microbiota studies have suggested that BSH is a key mechanistic microbiome target for the development of novel non-antibiotic food additives to improve animal feed production and for the design of new measures to control obesity in humans. However, research on BSH is still in its infancy, particularly in terms of the structural basis of BSH function, which has hampered the development of BSH-based strategies for improving human and animal health. As an initial step towards the structure-function analysis of BSH, C-terminally His-tagged BSH from Lactobacillus salivarius NRRL B-30514 was crystallized in this study. The 1.90 ? resolution crystal structure of L. salivarius BSH was determined by molecular replacement using the structure of Clostridium perfringens BSH as a starting model. It revealed this BSH to be a member of the N-terminal nucleophile hydrolase superfamily. Crystals of apo BSH belonged to space group P21212, with unit-cell parameters a = 90.79, b = 87.35, c = 86.76 ? (PDB entry 5hke). Two BSH molecules packed perfectly as a dimer in one asymmetric unit. Comparative structural analysis of L. salivarius BSH also identified potential residues that contribute to catalysis and substrate specificity.

Lactobacillus salivarius strains are increasingly being exploited for their probiotic properties in humans and animals. Dissemination of antibiotic resistance genes among species with food or probiotic-association is undesirable and is often mediated by plasmids or integrative and conjugative elements. L. salivarius strains typically have multireplicon genomes including circular megaplasmids that encode strain-specific traits for intestinal survival and probiotic activity. Linear plasmids are less common in lactobacilli and show a very limited distribution in L. salivarius. Here we present experimental evidence that supports an unusually complex multireplicon genome structure in the porcine isolate L. salivarius JCM1046. JCM1046 harbours a 1.83 Mb chromosome, and four plasmids which constitute 20% of the genome. In addition to the known 219 kb repA-type megaplasmid pMP1046A, we identified and experimentally validated the topology of three additional replicons, the circular pMP1046B (129 kb), a linear plasmid pLMP1046 (101 kb) and pCTN1046 (33 kb) harbouring a conjugative transposon. pMP1046B harbours both plasmid-associated replication genes and paralogues of chromosomally encoded housekeeping and information-processing related genes, thus qualifying it as a putative chromid. pLMP1046 shares limited sequence homology or gene synteny with other L. salivarius plasmids, and its putative replication-associated protein is homologous to the RepA/E proteins found in the large circular megaplasmids of L. salivarius. Plasmid pCTN1046 harbours a single copy of an integrated conjugative transposon (Tn6224) which appears to be functionally intact and includes the tetracycline resistance gene tetM. Experimental validation of sequence assemblies and plasmid topology resolved the complex genome architecture of L. salivarius JCM1046. A high-coverage draft genome sequence would not have elucidated the genome complexity in this strain. Given the expanding use of L. salivarius as a probiotic, it is important to determine the genotypic and phenotypic organization of L. salivarius strains. The identification of Tn6224-like elements in this species has implications for strain selection for probiotic applications.

Lactic acid bacteria (LAB) resistant to erythromycin were isolated from different food samples on selective media. The isolates were identified as Enterococcus durans, Enterococcus faecium, Enterococcus lactis, Enterococcus casseliflavus, Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus fermentum, Pediococcus pentosaceus and Leuconostoc mesenteroides. Of the total 60 isolates, 88 % harbored the ermB gene. The efflux gene msrA was identified in E. faecium, E. durans, E. lactis, E. casseliflavus, P. pentosaceus and L. fermentum. Further analysis of the msrA gene by sequencing suggested its homology to msrC. Resistance to tetracycline due to the genes tetM, tetW, tetO, tetK and tetL, alone or in combination, were identified in Lactobacillus species. The tetracycline efflux genes tetK and tetL occurred in P. pentosaceus and Enterococcus species. Since it appeared that LAB had acquired these genes, fermented foods may be a source of antibiotic resistance.

Degenerate oligodeoxyribonucleotides complementary to sequences encoding conserved amino acid (aa) motifs in D-alanine:D-alanine ligases (Ddl) were used to amplify approx. 600-bp fragments from glycopeptide-resistant strains of Leuconostoc mesenteroides (Lm), Lactobacillus plantarum, La. salivarius and La. confusus, and from a susceptible strain of La. leichmannii. Comparison of the deduced aa sequences of the PCR products revealed that the Ddl-related enzymes of resistant Lm and Lactobacillus spp. are more akin to each other (47-63% aa identity) than to that of susceptible La. leichmannii (33-37% aa identity), indicating that the Ddl-related enzymes in these intrinsically resistant species of Gram+ bacteria exhibit structural differences with those in susceptible species. The Ddl-related enzymes, VanA and VanB, implicated in acquired resistance to glycopeptides in enterococci, were not closely related to their counterparts in Lm and Lactobacillus spp., as they displayed only 26-32% aa identity.

The aim of this study was to identify Lactobacillus isolates derived from turkeys from six Polish farms and to characterize their phenotypic and genotypic antibiotic resistance profiles. Among 62 isolates identified by MALDI-TOF mass spectrometry and restriction analysis of 16S rDNA, the dominant species was L. salivarius (35%), followed by L. crispatus (21%), L. ingluviei (14.5%) and L. johnsonii (10%). A high prevalence of resistance to tetracycline (68% resistant isolates), lincomycin (64.5%) and enrofloxacin (60%) among the lactobacilli tested was observed. Fewer than 50% isolates were resistant to ampicillin (47%), erythromycin (45%), streptomycin (31%), chloramphenicol (29%) and gentamicin (10%). As many as 64,5% of the isolates showed multidrug resistance. High MIC values for ampicillin (?64 �gg/ml) were usually accompanied by elevated MICs for cephalosporins (?16 �gg/ml) and high MICs for tiamulin, i.e. ?32 �gg/ml, were noted in most of the turkey lactobacilli (61%). The occurrence of resistance genes was associated with phenotypic resistance, with the exception of five phenotypically susceptible isolates that contained the tetM, tetL, ermC, ermB or cat genes. The most frequently identified were ermB (45% isolates), tetL (40%), tetW (37%) and tetM (29%), and the occurrence of lnuA (18%), cat (10%), ermC (6%), ant(6)-Ia (5%) and aadE (5%) was less frequent. The mechanism of ampicillin resistance has not been elucidated, but the results of nitrocefin test confirmed that it is not involved in the production of beta-lactamases. The high rate of antibiotic resistance observed in this study indicates the need to implement the principles of rational use of antibiotics in poultry. The presence of transmissible resistant genes in lactobacilli may contribute to the development of antibiotic resistant pathogenic strains that pose a threat to both poultry and consumers. The results of these studies may be useful for committees providing guidance on antibiotic susceptibility of microorganisms in order to revise and supplement current microbiological cut-offs values within the genus Lactobacillus.

Probiotic gut bacteria employ specific metabolic pathways to degrade dietary carbohydrates beyond the capabilities of their human host. Here, we report how individual commercial probiotic strains degrade prebiotic (inulin type) fructans. First, a structural analysis of commercial fructose oligosaccharide-inulin samples was performed. These �]-(2-1)-fructans differ in termination by either glucose (GF) or fructose (FF) residues, with a broad variation in the degrees of polymerization (DPs). The growth of individual probiotic bacteria on short-chain inulin (sc-inulin) (Frutafit CLR), a �]-(2-1)-fructan (DP 2 to DP 40), was studied. Lactobacillus salivarius W57 and other bacteria grew relatively poorly on sc-inulin, with only fractions of DP 3 and DP 5 utilized, reflecting uptake via specific transport systems followed by intracellular metabolism. Lactobacillus paracasei subsp. paracasei W20 completely used all sc-inulin components, employing an extracellular exo-inulinase enzyme (glycoside hydrolase family GH32 [LpGH32], also found in other strains of this species); the purified enzyme converted high-DP compounds into fructose, sucrose, 1-kestose, and F2 (inulobiose). The cocultivation of L. salivarius W57 and L. paracasei W20 on sc-inulin resulted in cross-feeding of the former by the latter, supported by this extracellular exo-inulinase. The extent of cross-feeding depended on the type of fructan, i.e., the GF type (clearly stimulating) versus the FF type (relatively low stimulus), and on fructan chain length, since relatively low-DP �]-(2-1)-fructans contain a relatively high content of GF-type molecules, thus resulting in higher concentrations of GF-type DP 2 to DP 3 degradation products. The results provide an example of how in vivo cross-feeding on prebiotic �]-(2-1)-fructans may occur among probiotic lactobacilli.IMPORTANCE The human gut microbial community is associated strongly with host physiology and human diseases. This observation has prompted research on pre- and probiotics, two concepts enabling specific changes in the composition of the human gut microbiome that result in beneficial effects for the host. Here, we show how fructooligosaccharide-inulin prebiotics are fermented by commercial probiotic bacterial strains involving specific sets of enzymes and transporters. Cross-feeding strains such as Lactobacillus paracasei W20 may thus act as keystone strains in the degradation of prebiotic inulin in the human gut, and this strain-exo-inulinase combination may be used in commercial Lactobacillus-inulin synbiotics.

In this report, we describe the draft genome sequence of a newly discovered probiotic strain, Lactobacillus salivarius L28. L. salivarius L28 demonstrates antagonistic effects against human foodborne pathogens, including Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes, in coculture experiments and food matrices.

Antibiotic growth promoters (AGPs) have been used as feed additives to improve average body weight gain and feed efficiency in food animals for more than 5 decades. However, there is a worldwide trend to limit AGP use to protect food safety and public health, which raises an urgent need to discover effective alternatives to AGPs. The growth-promoting effect of AGPs has been shown to be highly correlated with the decreased activity of intestinal bile salt hydrolase (BSH), an enzyme that is produced by various gut microflora and involved in host lipid metabolism. Thus, BSH inhibitors are likely promising feed additives to AGPs to improve animal growth performance. In this study, the genome of Lactobacillus salivarius NRRL B-30514, a BSH-producing strain isolated from chicken, was sequenced by a 454 GS FLX sequencer. A BSH gene identified by genome analysis was cloned and expressed in an Escherichia coli expression system for enzymatic analyses. The BSH displayed efficient hydrolysis activity for both glycoconjugated and tauroconjugated bile salts, with slightly higher catalytic efficiencies (k(cat)/K(m)) on glycoconjugated bile salts. The optimal pH and temperature for the BSH activity were 5.5 and 41�XC, respectively. Examination of a panel of dietary compounds using the purified BSH identified some potent BSH inhibitors, in which copper and zinc have been recently demonstrated to promote feed digestion and body weight gain in different food animals. In sum, this study identified and characterized a BSH with broad substrate specificity from a chicken L. salivarius strain and established a solid platform for us to discover novel BSH inhibitors, the promising feed additives to replace AGPs for enhancing the productivity and sustainability of food animals.