Lactic acid bacteria can act as reservoirs of antibiotic resistance genes that can be ultimately transferred to pathogens. The present work reports on the minimum inhibitory concentration (MIC) of 16 antibiotics to 25 LAB isolates of five Lactobacillus and one Bifidobacterium species from the human vagina. Acquired resistances were detected to kanamycin, streptomycin, chloramphenicol, gentamicin, and ampicillin. A PCR analysis of lactobacilli failed to identify genetic determinants involved in any of these resistances. Surprisingly, a tet(W) gene was detected by PCR in two Bifidobacterium bifidum strains, although they proved to be tetracycline-susceptible. In agreement with the PCR results, no acquired genes were identified in the genome of any of the Lactobacillus spp. strains sequenced. A genome analysis of B. bifidum VA07-1AN showed an insertion of two guanines in the middle of tet(W) interrupting the open reading frame. By growing the strain in the presence of tetracycline, stable tetracycline-resistant variants were obtained. An amino acid substitution in the ribosomal protein S12 (K43R) was further identified as the most likely cause of VA07-1AN being streptomycin resistance. The results of this work expand our knowledge of the resistance profiles of vaginal LAB and provide evidence for the genetic basis of some acquired resistances.

Edible mushrooms, including wild mushrooms, are currently being investigated as natural sources to evaluate their prebiotic potential. This study aimed to evaluate the prebiotic potential of crude polysaccharides (CPSs) extracted from wild Lentinus squarrosulus UBU_LS1 and Lentinus polychrous UBU_LP2 and their application as cryoprotectants in the freeze-drying process to formulate a novel synbiotic product. Based on fruiting body morphology and molecular identification, two wild edible mushrooms named UBU_LS1 and UBU_LP2 were identified as Lentinus squarrosulus and Lentinus polychrous, respectively. L. squarrosulus UBU_LS1 and L. polychrous UBU_LP2 contained high amounts of CPS after hot water extraction. Monosaccharide component analysis showed that CPS_UBU_LS1 and CPS_UBU_LP2 were typical heteropolysaccharides. CPS_UBU_LS1 and CPS_UBU_LP2 showed hydrolysis tolerance to the simulated human gastric acidic pH solution, indicating that these CPSs are capable of reaching the lower gastrointestinal tract. Antioxidant activity determined using the 1,1-diphenyl-2-picrylhydrazyl assay revealed that the CPS_UBU_LS1 and CPS_UBU_LP2 displayed greater antioxidant activity comparable with that of ascorbic acid. It was found that CPS_UBU_LS1 and CPS_UBU_LP2 have a high potential for stimulating growth in all probiotic strains. Moreover, both CPS compounds could possibly be used as cryoprotectants in freeze drying, since the viability of the selected probiotic L. fermentum 47-7 exhibited cell survival of greater than 70% after 90 days of storage at 4 °C. These results highlight that wild edible mushrooms L. squarrosulus UBU_LS1 and L. polychrous UBU_LP2 are potential natural sources of prebiotics and can be applied as cryoprotectants in the freeze-drying process. The crude polysaccharide derived from this study could also be considered as a potent antioxidative compound. Therefore, our study provides evidence to support the application of CPSs from wild edible mushrooms in synbiotic product development and in various functional foods. Finally, further evaluation of these prebiotics, including the determination of the potential rehabilitation of beneficial gut microbes in diseased individuals, is currently being conducted by our research group.

There is an increasing interest to develop various lactic acid bacteria (LAB) species as mucosal delivery vehicles, for which the development of a variety of cloning and expression systems for these bacteria is of primary importance. This study reports the complete nucleotide sequence of the cryptic plasmid pRCEID7.6 derived from the chicken probiotic LAB strain Lactobacillus casei TISTR1341. Sequence analysis and comparison showed that pRCEID7.6 is composed of nine putative open reading frames. The replicon origin of pRCEID7.6 consisted of untranslated origin of replication and translated replication protein B sequences. This region was used to construct Escherichia coli/L. casei shuttle vectors carrying erythromycin and chloramphenicol resistance genes as selective markers. Segregation and structural stability of the vectors in L. casei was sufficient for most genetic applications. The feasibility of this vector for heterologous protein expression in L. casei was determined by cloning in pRCEID-LC7.6, the gene encoding the nucleocapsid protein (NP), from the influenza A virus under the control of the homologous promoter from the lactate dehydrogenase gene. L. casei carrying this recombinant plasmid was shown to successfully express the NP protein. Therefore, this shuttle vector can be used for further study in the development of mucosal delivery vehicles.

Lyophilization is one of the most used methods for bacterial preservation. In this process, the cryoprotectant not only largely decreases cellular damage but also plays an important part in the conservation of viability during freeze-drying. This study investigated using cryoprotectant and a mixture of the cryoprotectant to maintain probiotic activity. Seven probiotic strains were considered: (Limosilactobacillus reuteri KUKPS6103; Lacticaseibacillus rhamnosus KUKPS6007; Lacticaseibacillus paracasei KUKPS6201; Lactobacillus acidophilus KUKPS6107; Ligilactobacillus salivarius KUKPS6202; Bacillus coagulans KPSTF02; Saccharomyces cerevisiae subsp. boulardii KUKPS6005) for the production of a multi-strain probiotic and the complex medium for the lyophilized synbiotic production. Cholesterol removal, antioxidant activity, biofilm formation and gamma aminobutyric acid (GABA) production of the probiotic strains were analyzed. The most biofilm formation occurred in L. reuteri KUKPS6103 and the least in B. coagulans KPSTF02. The multi-strain probiotic had the highest cholesterol removal. All the probiotic strains had GABA production that matched the standard of γ-aminobutyric acid. The lyophilized synbiotic product containing complex medium as a cryoprotectant and wall material retained a high viability of 7.53 × 10⁸ CFU/g (8.89 log CFU/g) after 8 weeks of storage. We found that the survival rate of the multi-strain probiotic after freeze-drying was 15.37% in the presence of complex medium that was used as high performing wall material. Our findings provided a new type of wall material that is safer and more effective and, can be extensively applied in relevant food applications.