A survey on antimicrobial resistance genes of frequently used probiotic bacteria, 1901 to 2022.

BackgroundAntimicrobial resistance (AMR) is caused by AMR determinants, mainly genes (ARGs) in the bacterial genome. Bacteriophages, integrative mobile genetic elements (iMGEs) or plasmids can allow ARGs to be exchanged among bacteria by horizontal gene transfer (HGT). Bacteria, including bacteria with ARGs, can be found in food. Thus, it is conceivable that in the gastrointestinal tract, bacteria from the gut flora could take up ARGs from food.AimThe study objective was to gain insight into the ARG set carried by commonly used probiotic bacteria that may enter the human body with non-fermented foods, fermented foods, or probiotic dietary supplements (FFPs) and to assess ARG mobility.MethodsNext generation sequencing whole genome data from 579 isolates of 12 commonly employed probiotic bacterial species were collected from a public repository. Using bioinformatical tools, ARGs were analysed and linkage with mobile genetic elements assessed.ResultsResistance genes were found in eight bacterial species. The ratios of ARG positive/negative samples per species were: Bifidobacterium animalis (65/0), Lactiplantibacillus plantarum (18/194), Lactobacillus delbrueckii (1/40), Lactobacillus helveticus (2/64), Lactococcus lactis (74/5), Leucoconstoc mesenteroides (4/8), Levilactobacillus brevis (1/46), Streptococcus thermophilus (4/19). In 66% (112/169) of the ARG-positive samples, at least one ARG could be linked to plasmids or iMGEs. No bacteriophage-linked ARGs were found.ConclusionThe finding of potentially mobile ARGs in probiotic strains for human consumption raises awareness of a possibility of ARG HGT in the gastrointestinal tract. In addition to existing recommendations, screening FFP bacterial strains for ARG content and mobility characteristics might be considered.

Different effects of soybean protein and its derived peptides on the growth and metabolism of Bifidobacterium animalis subsp. animalis JCM 1190.

Bifidobacterium is a common probiotic that plays a vital role in the intestinal tract. This study aimed to explore the different effects of soybean protein and soybean peptides on the growth and metabolism of Bifidobacterium animalis subsp. animalis JCM 1190. Soybean protein and soybean peptides were digested in vitro, after which different nitrogen source containing media were prepared and used for the monoculture of Bifidobacterium animalis subsp. animalis JCM 1190 and the co-culture of Bifidobacterium animalis subsp. animalis JCM 1190 and Escherichia coli JCM 1649. During the culture process, the viable cell number and lactic acid and acetic acid contents were measured, while non-targeted metabonomics was used to detect the differential metabolites and metabolic pathways. The results showed that soybean protein and soybean peptides promoted the growth and metabolism of Bifidobacterium animalis subsp. animalis JCM 1190, while digested soybean peptides had a better effect. Digested soybean peptides increased the viable cell number and lactic acid and acetic acid contents in the monoculture by regulating glycine, serine, and threonine metabolism, as well as pyruvate metabolism, the TCA cycle, glycolipid metabolism, and other metabolic pathways, balanced the ability of Bifidobacterium animalis subsp. animalis JCM 1190 and Escherichia coli JCM 1649 to utilize nitrogen sources during the early period and enhanced the competitiveness of Bifidobacterium animalis subsp. animalis JCM 1190 during the later period in co-culture.

Unravelling the antimicrobial action of antidepressants on gut commensal microbes.

Over the past decade, there has been increasing evidence highlighting the implication of the gut microbiota in a variety of brain disorders such as depression, anxiety, and schizophrenia. Studies have shown that depression affects the stability of gut microbiota, but the impact of antidepressant treatments on microbiota structure and metabolism remains underexplored. In this study, we investigated the in vitro antimicrobial activity of antidepressants from different therapeutic classes against representative strains of human gut microbiota. Six different antidepressants: phenelzine, venlafaxine, desipramine, bupropion, aripiprazole and (S)-citalopram have been tested for their antimicrobial activity against 12 commensal bacterial strains using agar well diffusion, microbroth dilution method, and colony counting. The data revealed an important antimicrobial activity (bacteriostatic or bactericidal) of different antidepressants against the tested strains, with desipramine and aripiprazole being the most inhibitory. Strains affiliating to most dominant phyla of human microbiota such as Akkermansia muciniphila, Bifidobacterium animalis and Bacteroides fragilis were significantly altered, with minimum inhibitory concentrations (MICs) ranged from 75 to 800 µg/mL. A significant reduction in bacterial viability was observed, reaching 5 logs cycle reductions with tested MICs ranged from 400 to 600 µg/mL. Our findings demonstrate that gut microbiota could be altered in response to antidepressant drugs.

Prebiotic activity of monofloral honeys produced by stingless bees in the semi-arid region of Brazilian Northeastern toward Lactobacillus acidophilus LA-05 and Bifidobacterium lactis BB-12.

This study assessed the in vitro prebiotic effects of honeys from Ziziphus joazeiro Mart. (juazeiro; J) and Mimosa arenosa Willd Poir (jurema branca; JB) produced by native stingless bees, namely Melipona subnitida Ducke (jandaíra; J) and M. scutellaris Latrelle (uruçu; U), in the Brazilian Northeastern semi-arid region toward the probiotics Lactobacillus acidophilus LA-05 and Bifidobacterium animalis subsp. lactis BB-12. Cells of the probiotic strains were enumerated over 48 h of cultivation in broths containing each honey (JJ, JU, JBJ or JBU) as a sole carbon source. The metabolic activities of probiotic strains in these media were assessed by measuring changes in pH values and sugars, organic acids and phenolics contents. All honeys (20 or 30 g/L) exerted growth promoting effects and displayed positive prebiotic activity scores (0.94-1.22) on tested probiotics. JJ showed the highest (p < 0.05) stimulatory effects on probiotics growth and prebiotic scores. At the end of the cultivation period, counts of L. acidophilus LA-05 and B. lactis BB-12 increased (p < 0.05) more than 2 log in broths regardless the monofloral honey added. The pH values and sugars contents decreased (p < 0.05), while the organic acids contents increased (p < 0.05) during cultivation of probiotics in broths containing JJ, JU, JBJ or JBU as carbon source. After 48 h of cultivation, contents of gallic, caftaric and caffeic acid, catechin and procyanidins (B1 and B2) decreased (p < 0.05) in media containing JJ, JU, JBJ or JBU despite of the inoculated probiotic. JJ honey presented overall the better stimulatory effects on the growth and metabolism of L. acidophilus LA-05 and B. lactis BB-12. These results showed for the first time the potential prebiotic properties of four monofloral honeys produced by stingless bees in the Brazilian Northeastern semi-arid region.

Effect of 2 types of resistant starches on the quality of yogurt.

This study investigated the effects of resistant starch (RS) 2 (a high-amylose corn starch) and RS3 (physically modified corn starch) on yogurt quality. Yogurt containing Bifidobacterium BB-12 was treated with RS2 or RS3 to a final concentration of 1.5%, with the control group receiving 1.5% (wt/wt) of sucrose. Multispeckle diffusing wave spectroscopy and scanning electron microscopy were used to investigate the effect of the resistant starches on the gelation process and microstructure of yogurt. The quality of the yogurt treatments was evaluated using viable counts of Bifidobacterium BB-12 and all viable cells, titratable acidity, amount of whey separation, and viscosity during storage. The resistant starches affected the progress of gelation and microstructure and decreased the decline of viable counts of Lactobacillus. Notably, RS3 effectively protected the Bifidobacterium BB-12, increased the viscosity, and decreased titratable acidity. Our results suggested that RS could improve the quality of yogurt and have a more probiotic effect. Further studies could lead to optimization in yogurt processing by mixing these 2 types of RS to determine their best usage and explore their interactions with proteins.

Effect of full-fat goat's milk and prebiotics use on Bifidobacterium BB-12 survival and on the physical properties of spray-dried powders under storage conditions.

The effects of full-fat goat's milk and/or inulin and/or oligofructose, as carrier agents, were investigated to improve the survival rates of Bifidobacterium BB-12, and the physical properties of the microcapsules under storage conditions. On the day of their manufacture, the microcapsules were evaluated for morphology, particle size, and distribution of fat and bifidobacteria. The viability of the bifidobacteria, moisture and fat content, water activity, solubility, bulk and tapped density, flowability, cohesiveness and color properties were evaluated for 120 days at 4 °C and 25 °C. The full-fat goat's milk powder with or without inulin as encapsulating agents showed the highest survival rates of Bifidobacterium BB-12 after spray drying and storage. Considering the bifidobacteria survival, both of these spray-dried powders showed the most desirable physical properties, i.e., lowest water activity and solubility, respectively. Both properties are highlighted for better stability of spray-dried powders, with microcapsules, during storage time. These results are credited to full-fat goat's milk (200 g L-1) and the presence of inulin (100 g L-1), besides the fat content showing a high correlation with the solubility values. The lowest volume occupied by the spray-dried powders was noted when oligofructose was used as the carrier agent. The samples that showed the presence of cracks influenced negatively on the bifidobacteria viability. These cracks were responsible by the greater water escape, resulting in powders with more desirable lower water activity. In relation to the color parameters, lower stability was noted when oligofructose was used, while the best stability was also noted for the powders with full-fat goat's milk and/or inulin. During storage time, the best performance was achieved by the microencapsulation process that used only full-fat goat's milk and/or inulin and storage at 4 °C.

Staying alive: growth and survival of Bifidobacterium animalis subsp. animalis under in vitro and in vivo conditions.

Members of the Bifidobacterium genus are widely used as probiotics in fermented milk products. Bifidobacterium animalis subsp. animalis CNCM I-4602 grows and survives poorly in reconstituted skimmed milk (RSM). Availing of genome and transcriptome information, this poor growth and survival phenotype in milk was substantially improved by the addition of certain compounds, such as yeast extract, uric acid, glutathione, cysteine, ferrous sulfate, and a combination of magnesium sulfate and manganese sulfate. Carbohydrate utilization of CNCM I-4602 was also investigated, allowing the identification of several carbohydrate utilization gene clusters, and highlighting this strain's inability to utilize lactose, unlike the type strain of this subspecies, B. animalis subsp. animalis ATCC25527 and the B. animalis subsp. lactis subspecies. In addition, the ability of B. animalis subsp. animalis CNCM I-4602 to colonize a murine model was investigated, which showed that this strain persists in the murine gut for a period of at least 4 weeks. Associated in vivo transcriptome analysis revealed that, among other genes, a gene cluster encoding a predicted type IVb tight adherence (Tad) pilus was upregulated, indicating that this extracellular structure plays a role in the colonization/adaptation of the murine gastrointestinal tract by this strain.

Transcriptional and Functional Analysis of Bifidobacterium animalis subsp. lactis Exposure to Tetracycline.

Commercial probiotic bacteria must be tested for acquired antibiotic resistance elements to avoid potential transfer to pathogens. The European Food Safety Authority recommends testing resistance using microdilution culture techniques previously used to establish inhibitory thresholds for the Bifidobacterium genus. Many Bifidobacterium animalis subsp. lactis strains exhibit increased resistance to tetracycline, historically attributed to the ribosomal protection gene tet(W). However, some strains that harbor genetically identical tet(W) genes show various inhibition levels, suggesting that other genetic elements also contribute to observed differences. Here, we adapted several molecular assays to confirm the inhibition of B. animalis subsp. lactis strains Bl-04 and HN019 and employed RNA sequencing to assess the transcriptional differences related to genomic polymorphisms. We detected specific stress responses to the antibiotic by correlating ATP concentration to number of viable genome copies from droplet digital PCR and found that the bacteria were still metabolically active in high drug concentrations. Transcriptional analyses revealed that several polymorphic regions, particularly a novel multidrug efflux transporter, were differentially expressed between the strains in each experimental condition, likely having phenotypic effects. We also found that the tet(W) gene was upregulated only during subinhibitory tetracycline concentrations, while two novel tetracycline resistance genes were upregulated at high concentrations. Furthermore, many genes involved in amino acid metabolism and transporter function were upregulated, while genes for complex carbohydrate utilization, protein metabolism, and clustered regularly interspaced short palindromic repeat(s) (CRISPR)-Cas systems were downregulated. These results provide high-throughput means for assessing antibiotic resistances of two highly related probiotic strains and determine the genetic network that contributes to the global tetracycline response.IMPORTANCEBifidobacterium animalis subsp. lactis is widely used in human food and dietary supplements. Although well documented to be safe, B. animalis subsp. lactis strains must not contain transferable antibiotic resistance elements. Many B. animalis subsp. lactis strains have different resistance measurements despite being genetically similar, and the reasons for this are not well understood. In the current study, we sought to examine how genomic differences between two closely related industrial B. animalis subsp. lactis strains contribute to different resistance levels. This will lead to a better understanding of resistance, identify future targets for analysis of transferability, and expand our understanding of tetracycline resistance in bacteria.

Safety evaluation of HOWARU® Restore (Lactobacillus acidophilus NCFM, Lactobacillus paracasei Lpc-37, Bifidobacterium animalis subsp. lactis Bl-04 and B. lactis Bi-07) for antibiotic resistance, genomic risk factors, and acute toxicity.

Although probiotic lactobacilli and bifidobacteria are generally considered safe by various regulatory agencies, safety properties, such as absence of transferable antibiotic resistance, must still be determined for each strain prior to market introduction as a probiotic. Safety requirements for probiotics vary regionally and evaluation methods are not standardized, therefore methodologies are often adopted from food ingredients or chemicals to assess microbial safety. Four individual probiotic strains, Lactobacillus acidophilus NCFM®, Lactobacillus paracasei Lpc-37®, Bifidobacterium animalis subsp. lactis strains Bl-04®, and Bi-07®, and their combination (HOWARU® Restore) were examined for antibiotic resistance by broth microdilution culture, toxin genes by PCR and genome mining, and acute oral toxicity in rats. Only B. lactis Bl-04 exhibited antibiotic resistance above a regulated threshold due to a tetW gene previously demonstrated to be non-transferable. Genomic mining did not reveal any bacterial toxin genes known to harm mammalian hosts in any of the strains. The rodent studies did not indicate any evidence of acute toxicity following a dose of 1.7-4.1 × 1012 CFU/kg body weight. Considering a 100-fold safety margin, this corresponds to 1.2-2.8 × 1012 CFU for a 70 kg human. Our findings demonstrate a comprehensive approach of in vitro, in silico, and in vivo safety testing for probiotics.

Effects of the Food Manufacturing Chain on the Viability and Functionality of Bifidobacterium animalis through Simulated Gastrointestinal Conditions.

The viability and functionality of probiotics may be influenced by industrial production processes resulting in a decrease in probiotic efficiency that benefit the health of humans. This study aimed to investigate the probiotic characteristics of Bifidobacterium strains isolated from fecal samples of healthy Thai infants. In the present work, three local strains (BF014, BF052, and BH053) belonging to Bifidobacterium animalis showed a great resistance against conditions simulating the gastrointestinal tract. Among these, B. animalis BF052 possessed considerable probiotic properties, including high acid and bile tolerance, strong adhesion capability to Caco-2 cells, and inhibitory activity against pathogens including Salmonella typhimurium and Vibrio cholerae. This strain also exhibited a high survival rate compared to commercial strains during storage in a wide variety of products, including pasteurized milk, soy milk, drinking yogurt, and orange juice. The impact of food processing processes as well as the freeze-drying process, storage of freeze-dried powders, and incorporation of freeze-dried cells in food matrix on probiotic properties was also determined. The stability of the probiotic properties of the BF052 strain was not affected by food processing chain, especially its resistance in the simulated gastrointestinal conditions and its adherence ability to Caco-2 cells. It indicates that it satisfies the criteria as a potential probiotic and may be used as an effective probiotic starter in food applications.