The genus Lactobacillus includes, among others, Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus, species that are collectively referred to as the Lactobacillus casei group. Many studies have shown that strains belonging to this group may decrease lactose intolerance, the effects of inflammatory bowel disease, diarrhea, constipation, food allergies and even colon cancer. Moreover, evidences exists of positive effects of these bacteria on mucosal immunity and blood cholesterol level. Because of their beneficial influence on human health, many of them are used as food additives and probiotic pharmaceuticals. It should be stressed that health-promoting properties are not attributed at the species level, but to specific strains. Therefore, procedures are necessary to allow specific identification at each phylogenetic level—genus, species and strain. In this paper we present a practical overview of molecular methods for the identification and differentiation of L. casei bacteria. The research included 30 bacterial strains belonging to three species: L.casei, L. paracasei and L. rhamnosus. Among the tested procedures were genus- and species-specific PCR, multiplex-PCR, Real-Time HRM analysis, RFLP-PCR, rep-PCR, RAPD-PCR, AFLP-PCR, and proteomic methods such as MALDI-TOF MS typing and SDS-PAGE fingerprinting. The obtained results showed that multiplex-PCR and MALDI-TOF MS turned out to be the most useful methods to identify the tested bacteria at the species level. At the strain level, the AFLP-PCR method showed the highest discriminatory power. We hope that the presented results will allow for the easy selection of an appropriate procedure, depending on the experiment conducted and the equipment capabilities of any given laboratory.

Lactobacillus rhamnosus Pen is a human endogenous strain with well-documented health promoting properties that is used for production of probiotics. It has a long safety history of application, and its effectiveness in the prevention of antibiotic-associated diarrhoea has also been confirmed in clinical trials. Here we present the complete genome sequence of L. rhamnosus Pen, which consists of a circular 2,884,4966-bp chromosome with a GC content of 46.8%. Within 2907 open reading frames (ORFs), genes involved with probiotic properties were identified. A CRISPR locus, consisting of a 1092-nt region with 16 spacers, was also detected. Finally, an intact prophage of ~ 40.7 kb, 57 ORFs, GC content 44.8% was identified. Genomic analysis confirmed the probiotic properties of L. rhamnosus Pen and may indicate new biotechnological applications of this industrially important strain.

The newly-isolated strain Enterobacter sp. LU1, which has previously been shown to be an effective producer of succinic acid on glycerol with the addition of lactose, was used for further intensive works aimed at improving the production parameters of the said process. The introduction of an initial stage of gentle culture aeration allowed almost 47 g/L of succinic acid to be obtained after 168 h of incubation, which is almost two times faster than the time previously taken to obtain this amount. Furthermore, the replacement of glycerol with crude glycerin and the replacement of lactose with whey permeate allowed the final concentration of succinic acid to be increased to 54 g/L. Considering the high content of yeast extract (YE) in the culture medium, tests were also performed with a reduced YE content via its partial substitution with urea. Although this substitution led to a deterioration of the kinetic parameters of the production process, using the fed-batch strategy, it allowed a succinic acid concentration of 69 g/L to be obtained in the culture medium, the highest concentration ever achieved using this process. Furthermore, the use of microaerophilic conditions meant that the addition of lactose to the medium was not required, with 37 g/L of succinic acid being produced on crude glycerol alone.

It is widely believed that microorganisms belonging toL. caseigroup can have positive effects on the human body. Therefore, these bacteria are used in many industrial processes, including the production of dietary supplements and probiotic preparations. When using live microorganisms in technological processes, it is important to use those without phage sequences within their genomes that can ultimately lead to lysis of the bacteria. It has been shown that many prophages have a benign nature, meaning that they don’t directly lead to lysis or inhibit microbial growth. Moreover, the presence of phage sequences in the genomes of these bacteria increases their genetic diversity, which may contribute to easier colonization of new ecological niches. In the 439 analyzed genomes of theL. caseigroup, 1509 sequences of prophage origin were detected. The average length of intact prophage sequences analyzed was just under 36 kb. GC content of tested sequences was similar for all analyzed species (44.6 ± 0.9%). Analyzing the protein coding sequences collectively, it was found that there was an average of 44 putative ORFs per genome, while the ORF density of all phage genomes varied from 0.5 to 2.1. The average nucleotide identity calculated on sequence alignments for analyzed sequences was 32.7%. Of the 56L. caseistrains used in the next part of the study, 32 did not show culture growth above the OD600 value of 0.5, even at a mitomycin C concentration of 0.25 μg/ml. Primers used for this study allowed for the detection of prophage sequences for over 90% of tested bacterial strains. Finally, prophages of selected strains were induced using mitomycin C, phage particles were isolated and then genomes of viruses obtained were sequenced and analyzed.

Lactobacillus rhamnosus Pen is a human endogenous strain used for the production of probiotic formula, which is effective in the prevention of antibiotic-associated diarrhoea. Our study showed that this probiotic strain releases bacteriophage BH1 without the addition of any inducing agent. Our research revealed that phage BH1 has a circular genome with a length of 40721 nt and a GC content of 44.8%. The genome of phage BH1 possesses 57 open reading frames which could be divided into functional modules associated with DNA packaging, morphogenesis, lysis, integration, genetic switch, and replication. In spite of similarity in morphology and genomic organization, comparative analysis revealed substantial genetic diversity and mosaic genomic architecture among phages described for the Lactobacillus casei group. Additionally, qPCR and ddPCR analysis confirmed earlier microscopic observations indicating that L. rhamnosus Pen liberates bacteriophage particles during growth. This occurs spontaneously, and is not a result of external inducing factors. For samples collected after 4 and 24 h of L. rhamnosus Pen culture, the number of attB and attP copies increased 2.5 and 12 times, respectively. This phenomenon, by introducing resistance to other phages or enhancing the biofilm-forming capabilities, may increase the survivability of microorganisms in their natural ecological niche. Conversely, spontaneous phage induction may be an important virulence factor for bacteria, posing a potential threat for the human host.

Lacticaseibacillus rhamnosus is widely recognized for its health-promoting properties, which have led to its broad application in the production of food and dietary supplements. Nevertheless, although rare and typically limited to patients with underlying conditions, adverse effects have also been reported. In this study, we sequenced and characterized the genomes of seven L. rhamnosus strains isolated from blood. Using a hybrid approach that combined Illumina and Oxford Nanopore technologies, we obtained complete genomes ranging from 2.96 to 3.13 Mb, with a GC content of 46.7–46.8%. Comparative analyses with publicly available L. rhamnosus genomes revealed that these isolates were genetically related to strains from highly diverse origins, including plants, dairy products, dietary supplements, the gastrointestinal and genitourinary tracts, as well as blood and other clinical samples from geographically distant regions. Importantly, neither core genome multilocus sequence typing (cgMLST) nor prophage and CRISPR module analyses indicated similarity to the widely used probiotic strain L. rhamnosus GG. Gene-based analysis identified determinants associated with bacteriocin production, adhesion, health-promoting traits, and potential pathogenicity of the strains. Notably, several genes linked to probiotic functions also overlapped with virulence factors found in pathogenic microorganisms. These findings demonstrate the genomic diversity of L. rhamnosus blood isolates and highlight the dual role of certain genetic determinants, underlining the importance of careful strain-level evaluation when selecting L. rhamnosus strains for probiotic use.