Lactic acid bacteria and bifidobacteria deliberately introduced into the food chain may act as a reservoir of antimicrobial resistance genes (ARGs), which is considered a safety concern. In the ...present study, resistance to antimicrobials of commercial probiotic strains, probiotic candidate strains, and starter cultures (n = 20) was characterised based on integration of phenotypic and in silico data. Minimum inhibitory concentrations (MICs) of 16 antimicrobials were determined for lactobacilli and bifidobacteria that were isolated from pharmaceutical products or obtained from the manufacturers or culture collections. Using different databases and bioinformatic tools, we predicted ARGs, mutations, genomic islands, and mobile genetic elements (MGEs) in their whole genome sequences. In addition, a comprehensive in silico analysis of the prevalence of the tetW gene and its genetic environment across lactobacilli and bifidobacteria (n = 1423) was conducted. Several strains exhibited phenotypic resistance to kanamycin, tetracycline, chloramphenicol, quinupristin-dalfopristin, ciprofloxacin, or neomycin. These resistances, however, did not always correspond to the presence of ARGs and vice versa. We detected an acquired tetW gene in four commercial strains of Bifidobacterium animalis subsp. lactis, whereas homologs of antimicrobial resistance (AR) proteins were predicted in all 20 proteomes. The prevalence of the tetW gene, which was often flanked by MGEs, was higher in analysed bifidobacteria (31.9%) than lactobacilli (6.3%). In addition, sequences flanking tetW were associated with putative genomic islands and were conserved in several strains, including potential pathogens. Our findings provide an insight into AR of probiotics, probiotic candidates, and starter cultures with an emphasis on tetracycline and into the safety of these strains in the context of AR.
•ARGs were detected in lactobacilli and bifidobacteria used as probiotics or starter cultures.•Genetic basis of phenotypic resistances or high-end MICs was determined.•The prevalence of tetW was higher in analysed bifidobacteria than in lactobacilli.•tetW is often placed within genomic islands and flanked by mobile genetic elements.•Sequences flanking tetW are conserved in certain strains of Firmicutes and Actinobacteria.
Adverse environmental conditions are severely limiting the use of microorganisms in food systems, such as probiotic delivery, where low pH causes a rapid decrease in the survival of ingested ...bacteria, and mixed-culture fermentation, where stepwise changes and/or metabolites of individual microbial groups can hinder overall growth and production. In our study, model probiotic lactic acid bacteria (
ATCC 8014,
GG) and yeasts native to dairy mixed cultures (
ZIM 1868) were entrapped in an optimized (cell, alginate and hardening solution concentration, electrostatic working parameters) Ca-alginate system. Encapsulated cultures were examined for short-term survival in the absence of nutrients (lactic acid bacteria) and long-term performance in acidified conditions (yeasts). In particular, the use of encapsulated yeasts in these conditions has not been previously examined. Electrostatic manufacturing allowed for the preparation of well-defined alginate microbeads (180-260 µm diameter), high cell-entrapment (95%) and viability (90%), and uniform distribution of the encapsulated cells throughout the hydrogel matrix. The entrapped
maintained improved viabilities during 180 min at pH 2.0 (19% higher when compared to the free culture), whereas,
appeared to be less robust. The encapsulated
exhibited double product yields in lactose- and lactic acid-modified MRS growth media (compared to an unfavorable growth environment for freely suspended cells). Even within a conventional encapsulation system, the pH responsive features of alginate provided superior protection and production of encapsulated yeasts, allowing several applications in lacto-fermented or acidified growth environments, further options for process optimization, and novel carrier design strategies based on inhibitor charge expulsion.
•Monolithic cation exchange HPLC proved to be a fast and efficient method for the quantification of lactoferrin in human milk.•Lactoferrin concentrations did not correlate with the milk collection ...method, i.e., by manual expression or a breast pump.•The concentration of lactoferrin in human milk was negatively correlated with the stage of lactation.
Human lactoferrin (hLF) is one of the most important whey proteins in human milk, known for its ability to modulate innate host immunity and multifunctional activities for neonatal growth. The objective of this study was to validate an efficient method for the detection and quantification of hLF using a unique technology of cation-exchange high-performance liquid chromatography (HPLC) on CIM® monolithic columns. Human milk samples were collected using manual expression or a breast pump, at different weeks of lactation. After sample preparation, hLF was detected and measured by HPLC method and further confirmed by SDS-PAGE. Selected fractions were analysed also by LC-MS/MS. Presumably, due to the high density of positive charge on the surface of the N-terminal domain, hLF binds strongly to the column and elutes last, enabling the high specificity of this method. The LC-MS/MS analysis indicated that hLF eluted in two clearly separated peaks, presumably representing two different molecular species of hLF. hLF concentration in the human milk samples ranged from 2.03 mg/mL to 5.79 mg/mL and was not significantly affected by the sample collection method whereas it was negatively correlated with the stage of lactation. These results suggest that cation exchange chromatography is an accurate, efficient, and robust method for the detection and quantification of hLF.
The aim of the present study was to characterize human milk microbiota (HMM) with 16S rRNA gene amplicon next-generation sequencing and cultivation/matrix-assisted laser desorption/ionization ...(MALDI)-time of flight (TOF) mass spectrometry (MS) identification approaches. We analyzed 31 human milk samples from healthy Slovenian mothers. To check the accuracy of MALDI-TOF MS identification, several colonies representing most abundant genera and those, which could not be reliably identified by MALDI-TOF, were subjected to Sanger sequencing of their 16S rRNA gene. We showed that cultivation/MALDI-TOF MS was a suitable tool for culture-dependent determination of HMM. With both approaches,
and
were found as predominant genera in HMM and the abundance of
was associated with decreased microbial diversity. In addition, we characterized factors that might influence HMM. The use of a breast pump was significantly associated with composition of HMM, lower microbial load, and higher abundance of cultivable staphylococci. Moreover, our study suggests that administration of probiotics to the suckling infant might influence HMM by increased abundance of lactobacilli and the presence of viable probiotic bacteria in human milk. However, since our study was observational with relatively small sample size, more targeted studies are needed to study possible transfer of probiotics to the mammary gland via an external route and the physiological relevance of these events.
Sound and timely microbial gut colonization completes newborn's healthy metabolic programming and manifests in infant appropriate growth and weight development. Feces, collected at 3, 30, and 90 days ...after birth from 60 breastfed Slovenian newborns, was submitted to microbial DNA extraction and qPCR quantification of selected gut associated taxa. Multivariate regression analysis was applied to evaluate microbial dynamics with respect to infant demographic, environmental, clinical characteristics and first year growth data. Early microbial variability was marked by the proportion of Bacilli, but diminished and converged in later samples, as bifidobacteria started to prevail. The first month proportions of enterococci were associated with maternity hospital locality and supplementation of breastfeeding with formulae, while Enterococcus faecalis proportion reflected the mode of delivery. Group Bacteroides-Prevotella proportion was associated with infant weight and ponderal index at first month. Infant mixed feeding pattern and health issues within the first month revealed the most profound and extended microbial perturbations. Our findings raise concerns over the ability of the early feeding supplementation to emulate and support the gut microbiota in a way similar to the exclusively breastfed infants. Additionally, practicing supplementation beyond the first month also manifested in higher first year weight and weight gain Z-score.
Microbial communities inhabiting the breast milk microenvironment are essential in supporting mammary gland health in lactating women and in providing gut-colonizing bacterial 'inoculum' for their ...infants' gastro-intestinal development. Bacterial DNA was extracted from colostrum samples of 45 healthy Slovenian mothers. Characteristics of the communities in the samples were assessed by polymerase chain reaction (PCR) coupled with denaturing gradient gel electrophoresis (DGGE) and by quantitative real-time PCR (qPCR). PCR screening for the prevalence of bacteriocin genes was performed on DNA of culturable and total colostrum bacteria. DGGE profiling revealed the presence of Staphylococcus and Gemella in most of the samples and exposed 4 clusters based on the abundance of 3 bands: Staphylococcus epidermidis/Gemella, Streptococcus oralis/pneumonia and Streptococcus salivarius. Bacilli represented the largest proportion of the communities. High prevalence in samples at relatively low quantities was confirmed by qPCR for enterobacteria (100%), Clostridia (95.6%), Bacteroides-Prevotella group (62.2%) and bifidobacteria (53.3%). Bacterial quantities (genome equivalents ml-1) varied greatly among the samples; Staphylococcus epidermidis and staphylococci varied in the range of 4 logs, streptococci and all bacteria varied in the range of 2 logs, and other researched groups varied in the range of 1 log. The quantity of most bacterial groups was correlated with the amount of all bacteria. The majority of the genus Staphylococcus was represented by the species Staphylococcus epidermidis (on average 61%), and their abundances were linearly correlated. Determinants of salivaricin A, salivaricin B, streptin and cytolysin were found in single samples. This work provides knowledge on the colostrum microbial community composition of healthy lactating Slovenian mothers and reports bacteriocin gene prevalence.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The insufficient quality of products containing beneficial live bacteria in terms of content and viability of labelled microorganisms is an often-reported problem. The aim of this work was to ...evaluate the quality of dietary supplements containing viable bacteria available in Slovenian pharmacies using plate counting, matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and species- or subspecies-specific PCR with DNA isolated from consortia of viable bacteria, from individual isolates, or directly from the products. Twelve percent of the products (3 of 26) contained insufficient numbers of viable bacteria. Eighty-three of the labelled species (111 in total) were confirmed by PCR with DNA from the product; 74% of these were confirmed by PCR with DNA from viable consortium, and 65% of these were confirmed by MALDI-TOF MS analysis of colonies. Certain species in multi-strain products were confirmed by PCR with DNA from viable consortia but not by MALDI-TOF MS, suggesting that the number of isolates examined (three per labelled strain) was too low. With the exception of
Lacticaseibacillus casei
and closely related species (
Lacticaseibacillus rhamnosus
and
Lacticaseibacillus zeae
), PCR and MALDI-TOF identification results agreed for 99% of the isolates examined, although several MALDI-TOF results had lower score values (1.700–1.999), indicating that the species identification was not reliable. The species
L. zeae
, which appeared in 20 matches of the Biotyper analysis, was identified as
L. rhamnosus
by PCR. The MALDI-TOF MS analysis was also unsuccessful in detecting
Lactobacillus acidophilus
La-5 and
Bacillus coagulans
due to missing peaks and unreliable identification, respectively. Mislabelling was detected by both methods for two putative
L. casei
strains that turned out to belong to the species
Lacticaseibacillus paracasei
. PCR remains more successful in subspecies-level identification as long as the database of MALDI-TOF MS spectra is not expanded by building in-house databases. The lack of positive PCR results with viable consortia or colonies, but positive PCR results with DNA isolated directly from the products observed in 10% (11/112) of the labelled strains, suggests the presence of non-culturable bacteria in the products. MALDI-TOF MS is a faster and simpler alternative to PCR identification, provided that a sufficient number of colonies are examined. Generation of in-house library may further improve the identification accuracy at the species and sub-species level.
Matsoni is a fermented milk common in the Caucasian region. In this study, whole-genome sequencing was performed with the aim of determining the taxonomic position of 25 previously selected candidate ...Matsoni starter cultures, identifying bacteriocin genes and prophages in their genomes, and evaluating their safety. In silico prediction of antibiotic resistance genes, virulence factors, bacteriocin genes and prophages was performed using dedicated databases and bioinformatics tools. With the exception of Enterococcus faecalis, which contained the mobile genetic element Tn916 with tetM and virulence factors, and Streptococcus thermophilus with mefE, the isolates were found to be safe for use as starter cultures. Thermophilin A, thermophilin 13, streptide and Blp genes were predicted in isolates of Streptococcus thermophilus, and genes for enterocins A, B, and X were found in Enterococcus faecium, but inhibition between strains was not observed. Several complete prophages were also detected in the genomes of Lactobacillus delbrueckii ssp. lactis. Lacticaseibacillus rhamnosus, Limosilactobacillus fermentum, Streptococcus thermophilus, Enterococcus faecalis, and Enterococcus faecium. Some of these isolates are good candidates for customised autochthonous Matsoni starter cultures which are an alternative to the commercial starter cultures currently used in the industry.
•Whole-genome sequencing enables reliable phylogenetic classification of Matsoni isolates.•Isolates of lactobacilli from Matsoni proved to be free of antibiotic resistance genes.•Acquired mefE found in Streptococcus thermophilus does not confer macrolide resistance.•Enterococcus faecalis from Matsoni contains prophages, virulence factors, and acquired ARGs.•Lactobacilli and Streptococcus thermophilus from Matsoni contain prophages (LF1, phiJB, TP-J34).
Lactic acid bacteria (LAB) and bifidobacteria may serve as reservoirs of antimicrobial resistance, but the risk posed by strains intentionally introduced into the agro-food chain has not yet been ...thoroughly investigated. The aim of our study was to evaluate whether probiotics, starter and protective cultures, and feed additives represent a risk to human health. In addition to commercial strains of LAB and bifidobacteria, isolates from human milk or colostrum, intestinal mucosa or feces, and fermented products were analyzed. Phenotypic susceptibility data of 474 strains showed that antimicrobial resistance was more common in intestinal isolates than in commercial strains. Antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) were characterized in the whole genome sequences of 1114 strains using comparative genomics. Intrinsic ARGs were abundant in enterococci, bifidobacteria, and lactococci but were considered non-risky due to the absence of MGEs. The results revealed that 13.8% of commercial strains contained acquired ARGs, most frequently for tetracycline. We associated 75.5% of the acquired ARGs with known or novel MGEs, and their potential for transmission was assessed by examining metagenomic sequences. We confirmed that ARGs and MGEs were not as abundant or diverse in commercial strains as in human intestinal isolates or isolates from human milk, suggesting that strains intentionally introduced into the agro-food chain do not pose a significant threat. However, attention should be paid especially to individual probiotic strains containing elements that have been shown to have high potential for transferability in the gut microbiota.
Abbreviations: ARG, antimicrobial resistance gene; ICE, integrative and conjugative element; IME, integrative and mobilizable element; LAB, lactic acid bacteria; MDR, multidrug resistance; MIC, minimum inhibitory concentration; MGE, mobile genetic element; TRRPP, tetracycline-resistant ribosomal protection protein; WGS, whole genome sequences
Probiotics are live microorganisms that can have beneficial effects on humans. Encapsulation offers them a better chance of survival. Therefore, nozzle-free electrospinning was introduced for their ...embedding in nanofibrous material. Probiotic
Lactobacillus paragasseri
K7 in lyophilized and fresh form, with and without inulin as prebiotic, was added to a polymer solution of sodium alginate (NaAlg) and polyethylene oxide (PEO). Conductivity, viscosity, pH, and surface tension were determined to define the optimal concentration and volume ratio for smooth electrospinning. The success of the formed nanoscale materials was examined by scanning electron microscope (SEM), while the entrapment of probiotics in the nanofibrous mats was detected by attenuated total reflection—Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS). Spontaneous diffusion of bacteria from electrospun samples in PBS buffer pH 7.4 was studied by plate counting on MRS agar. By exposing polymer solutions containing
L. paragasseri
K7 and inulin to a high electric field, the nanofilm was formed on a polypropylene substrate, used as collecting material. When polymer solutions without inulin were used, the bead-like nanofibers may have become visible. The SEM results suggest that inulin, in addition to K7 strain, additionally lowers the conductivity of spinning macromolecular solution and hinders the nanofiber formation. The results of ATR-FTIR confirmed the presence of
L. paragasseri
K7 embedded in nanocomposites by the appearance of characteristic peaks. The samples containing the probiotic regardless of its form with inulin had similar surface composition, except that the sodium content was higher in the samples with fresh probiotic, probably due to greater and thus less easy embedding of the bacteria in NaAlg. Within 2 h, the largest amount of probiotic strain K7 was spontaneously released from the electrospun sample containing the inulin and probiotic in freeze-dried form (44%), while the amount released from the nanofibrous sample, which also contained the inulin and probiotic in fresh form, was significantly lower (21%). These preliminary results demonstrate the potential of nozzle-free electrospinning technology for the development of probiotic delivery systems for short-term use, such as feminine hygiene materials (tampons, pads, napkins).
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