In vitro model of human mammary gland microbial colonization (MAGIC) demonstrates distinctive cytokine response to imbalanced human milk microbiota.

Despite the established concept of the human mammary gland (MG) as a habitat with its own microbiota, the exact mechanism of MG colonization is still elusive and a well-characterized in vitro model would reinforce studies of the MG microbiota development. We aimed to establish and characterize an in vitro cell model for studying MAmmary Gland mIcrobial Colonization (MAGIC) model. We used the immortalized cell line MCF10A, which expresses the strong polarized phenotype similar to MG ductal epithelium when cultured on a permeable support (Transwell). We analyzed the surface properties of the MAGIC model by gene expression analysis of E-cadherin, tight junction proteins, and mucins and by scanning electron microscopy. To demonstrate the applicability of the model, we tested the adhesion capability of the whole human milk (HM) microbial community and the cellular response of the model when challenged directly with raw HM samples. MCF10A on permeable supports differentiated and formed a tight barrier, by upregulation of CLDN8, MUC1, MUC4, and MUC20 genes. The surface of the model was covered with mucins and morphologically diverse with at least two cell types and two types of microvilli. Cells in the MAGIC model withstood the challenge with heat-treated HM samples and responded differently to the imbalanced HM microbiota by distinctive cytokine response. The microbial profile of the bacteria adhered on the MAGIC model reflected the microbiological profile of the input HM samples. The well-studied MAGIC model could be useful for studies of bacterial attachment to the MG and for in vitro studies of biofilm formation and microbiota development.IMPORTANCEThe MAGIC model may be particularly useful for studies of bacterial attachment to the surface of the mammary ducts and for in vitro studies of biofilm formation and the development of the human mammary gland (MG) microbiota. The model is also useful for immunological studies of the interaction between bacteria and MG cells. We obtained pioneering information on which of the bacteria present in the raw human milk (HM) were able to attach to the epithelium treated directly with raw HM, as well as on the effects of bacteria on the MG epithelial cells. The MAGIC cell model also offers new opportunities for research in other areas of MG physiology, such as the effects of bioactive milk components on microbial colonization of the MG, mastitis prevention, and studies of probiotic development. Since resident MG bacteria may be an important factor in breast cancer development, the MAGIC in vitro tool also offers new opportunities for cancer research.

Genomic insights into antibiotic resistance and mobilome of lactic acid bacteria and bifidobacteria.

Lactic acid bacteria (LAB) and Bifidobacterium sp. (bifidobacteria) can carry antimicrobial resistance genes (ARGs), yet data on resistance mechanisms in these bacteria are limited. The aim of our study was to identify the underlying genetic mechanisms of phenotypic resistance in 103 LAB and bifidobacteria using whole-genome sequencing. Sequencing data not only confirmed the presence of 36 acquired ARGs in genomes of 18 strains, but also revealed wide dissemination of intrinsic ARGs. The presence of acquired ARGs on known and novel mobile genetic elements raises the possibility of their horizontal spread. In addition, our data suggest that mutations may be a common mechanism of resistance. Several novel candidate resistance mechanisms were uncovered, providing a basis for further in vitro studies. Overall, 1,314 minimum inhibitory concentrations matched with genotypes in 92.4% of the cases; however, prediction of phenotype based on genotypic data was only partially efficient, especially with respect to aminoglycosides and chloramphenicol. Our study sheds light on resistance mechanisms and their transferability potential in LAB and bifidobacteria, which will be useful for risk assessment analysis.

Safety evaluation of enterococci isolated from raw milk and artisanal cheeses made in Slovenia and Serbia.

Enterococci represent a significant part of the non-starter LAB microbiota of artisanal cheeses produced mainly from raw milk. Common approaches to safety evaluation of enterococci isolates include assessment of antimicrobial resistance and virulence potential. Hence, a collection of 47 (n = 22, Serbia; n = 25, Slovenia) dairy enterococcal isolates, of which E. faecalis (n = 28), E. faecium (n = 11), E. durans (n = 5), E. casseliflavus (n = 2), and E. gallinarum (n = 1), was analyzed. The susceptibility to 12 antimicrobials was tested using a broth microdilution method, and the presence of the selected antimicrobial resistance and virulence genes was investigated using PCR. Isolates were resistant to tetracycline (TET) (25.5%), erythromycin (ERY) (17.0%), gentamycin and chloramphenicol (CHL) (∼6%). No resistance to ampicillin (AMP), ciprofloxacin (CIP), daptomycin (DAP), linezolid (LZD), teicoplanin (TEI), tigecycline (TGC) and vancomycin (VAN) was detected. Among all the resistance determinants analyzed, ermB gene was detected most frequently. All 10 virulence genes analyzed were detected with a distribution of cpd (72.3%), cob and ccf (70.2%), gelE (68.1%), hyl (59.6%), agg (53.2%) and esp (46.8%). The genes encoding cytolysin (cylA, cylM and cylB) were amplified to a lesser extent (21.3%, 21.3% and 12.8%, respectively). However, due to the limited number of enterococci isolates analyzed in the present study, further studies are still required in order to better document the safety status of dairy enterococci.

Lactic acid bacteria and bifidobacteria deliberately introduced into the agro-food chain do not significantly increase the antimicrobial resistance gene pool.

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.

Evaluation of Dietary Supplements Containing Viable Bacteria by Cultivation/MALDI-TOF Mass Spectrometry and PCR Identification.

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.

Characterization of antimicrobial resistance in lactobacilli and bifidobacteria used as probiotics or starter cultures based on integration of phenotypic and in silico data.

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.

Tetracycline resistance in lactobacilli isolated from Serbian traditional raw milk cheeses.

The aim of this study was to investigate the presence of tetracycline resistance in lactobacilli isolated from traditional Serbian white brined raw milk cheeses (Homolje, Sjenica, Zlatar). Isolation of presumptive lactobacilli was initially performed using MRS-S agar without tetracycline, or supplemented with 16 and 64 µg/mL of tetracycline. Rep-PCR (GTG)5 genotyping showed a high diversity of the isolates obtained, as examination of 233 isolates resulted in 156 different Rep-PCR fingerprints. Ninety out of 156 (57.69%) of the strains, representatives with different (GTG)5 fingerprints, were identified by MALDI-TOF MS as lactobacilli, while 66 out of 156 (42.31%) strains were identified as members of other LAB genera. All except one out of 90 Lactobacillus isolates further tested by microdilution method, demonstrated unimodal distribution of tetracycline MIC values which were equal to or lower from the breakpoint MIC values (EFSA in EFSA J 10: 1-10, 2012. 10.2903/j.efsa.2012.2740). Only one Lb. paracasei isolate showed the presence of tet(M) gene, while the other analyzed tet genes [tet(A), tet(B), tet(C) tet(K), tet(L), tet(O) and tet(W)] were not detected in any of the isolates. The results of this study indicates that lactobacilli from traditional Serbian raw milk cheeses do not present considerable tetracycline resistance reservoirs. For final conclusions about the safety of these autochthonous cheeses regarding the possible tetracycline resistance transferability, the assessment of the entire cheese microbiota is needed.