Development and validation of a species-independent functional gene microarray that targets lactic acid bacteria. - Related Documents




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846801.0000Development and validation of a species-independent functional gene microarray that targets lactic acid bacteria. During the last few years, genome-related information has become available for many microorganisms, including important food-related bacteria. Lactic acid bacteria (LAB) are important industrially in the production of fermented foods such as dairy products, sausages, sourdoughs, and vegetables. Despite their limited metabolic capacity, LAB contribute considerably to important characteristics of fermented foods, such as flavor and texture. In the present study, a species-independent functional gene microarray was developed that targets 406 genes that play key roles in the production of sugar catabolites, bacteriocins, exopolysaccharides, and aromas, in probiotic and biosafety characteristics, and in the stress response. Also, genes linked to negative traits, such as antibiotic resistance and virulence, are represented. As LAB ecosystems contain a variety of species, there was a more global focus on these specific functional properties. Thus, an algorithm was used to design gene-specific oligonucleotides that preferably hybridize with multiple LAB species, thereby allowing controlled cross-hybridization. For proof of concept, the microarray composed of 2,269 30-mer oligonucleotides focused on LAB species that are prevalent in sourdough ecosystems. Validation hybridizations using DNA and RNA from 18 LAB strains, covering 86% of all the oligonucleotides, showed that there were wide ranges in intensity and high reproducibility between microarrays.200919684161
846710.9998The Impacts of Lactiplantibacillus plantarum on the Functional Properties of Fermented Foods: A Review of Current Knowledge. One of the most varied species of lactic acid bacteria is Lactiplantibacillus plantarum (Lb. plantarum), formerly known as Lactobacillus plantarum. It is one of the most common species of bacteria found in foods, probiotics, dairy products, and beverages. Studies related to genomic mapping and gene locations of Lb. plantarum have shown the novel findings of its new strains along with their non-pathogenic or non-antibiotic resistance genes. Safe strains obtained with new technologies are a pioneer in the development of new probiotics and starter cultures for the food industry. However, the safety of Lb. plantarum strains and their bacteriocins should also be confirmed with in vivo studies before being employed as food additives. Many of the Lb. plantarum strains and their bacteriocins are generally safe in terms of antibiotic resistance genes. Thus, they provide a great opportunity for improving the nutritional composition, shelf life, antioxidant activity, flavour properties and antimicrobial activities in the food industry. Moreover, since some Lb. plantarum strains have the ability to reduce undesirable compounds such as aflatoxins, they have potential use in maintaining food safety and preventing food spoilage. This review emphasizes the impacts of Lb. plantarum strains on fermented foods, along with novel approaches to their genomic mapping and safety aspects.202235456875
463720.9998What Differentiates Probiotic from Pathogenic Bacteria? The Genetic Mobility of Enterococcus faecium Offers New Molecular Insights. Enterococcus faecium is a lactic acid bacterium with applications in food engineering and nutrigenomics, including as starter cultures in fermented foods. To differentiate the E. faecium probiotic from pathogenic bacteria, physiological analyses are often used but they do not guarantee that a bacterial strain is not pathogenic. We report here new findings and an approach based on comparison of the genetic mobility of (1) probiotic, (2) pathogenic, and (3) nonpathogenic and non-probiotic strains, so as to differentiate probiotics, and inform their safe use. The region of the 16S ribosomal DNA (rDNA) genes of different E. faecium strains native to Pernambuco-Brazil was used with the GenBank query sequence. Complete genomes were selected and divided into three groups as noted above to identify the mobile genetic elements (MGEs) (transposase, integrase, conjugative transposon protein and phage) and antibiotic resistance genes (ARGs), and to undertake pan-genome analysis and multiple genome alignment. Differences in the number of MGEs were found in ARGs, in the presence and absence of the genes that differentiate E. faecium probiotics and pathogenic bacteria genetically. Our data suggest that genetic mobility appears to be informative in differentiating between probiotic and pathogenic strains. While the present findings are not necessarily applicable to all probiotics, they offer novel molecular insights to guide future research in nutrigenomics, clinical medicine, and food engineering on new ways to differentiate pathogenic from probiotic bacteria.202032762606
940530.9998Functional Metagenomic Screening for Antimicrobial Resistance in the Oral Microbiome. A large proportion of bacteria, from a multitude of environments, are not yet able to be grown in the laboratory, and therefore microbiological and molecular biological investigations of these bacteria are challenging. A way to circumvent this challenge is to analyze the metagenome, the entire collection of DNA molecules that can be isolated from a particular environment or sample. This collection of DNA molecules can be sequenced and assembled to determine what is present and infer functional potential, or used as a PCR template to detect known target DNA and potentially unknown regions of DNA nearby those targets; however assigning functions to new or conserved hypothetical, functionally cryptic, genes is difficult. Functional metagenomics allows researchers to determine which genes are responsible for selectable phenotypes, such as resistance to antimicrobials and metabolic capabilities, without the prerequisite needs to grow the bacteria containing those genes or to already know which genes are of interest. It is estimated that a third of the resident species of the human oral cavity is not yet cultivable and, together with the ease of sample acquisition, makes this metagenome particularly suited to functional metagenomic studies. Here we describe the methodology related to the collection of saliva samples, extraction of metagenomic DNA, construction of metagenomic libraries, as well as the description of functional assays that have previously led to the identification of new genes conferring antimicrobial resistance.202134410638
940440.9998The Application of Transposon Insertion Sequencing in Identifying Essential Genes in B. fragilis. Essential genes are those that are indispensable for the survival of organism under specific growth conditions. Investigating essential genes in pathogenic bacteria not only helps to understand vital biological networks but also provides novel targets for drug development. Availability of genetic engineering tools and high-throughput sequencing methods has enabled essential genes identification in many pathogenic gram-positive and gram-negative bacteria. Bacteroides fragilis is one of the major bacteria specific of human gastrointestinal microbiota. When B. fragilis moves out of its niche, it turns into deadly pathogen. Here, we describe detailed method for the essential gene identification in B. fragilis. Generated transposon mutant pool can be used for other applications such as identification of genes responsible for drug resistance in B. fragilis.202234709623
422050.9998Whole genome sequencing for the risk assessment of probiotic lactic acid bacteria. Probiotic bacteria exhibit beneficial effects on human and/or animal health, and have been widely used in foods and fermented products for decades. Most probiotics consist of lactic acid bacteria (LAB), which are used in the production of various food products but have also been shown to have the ability to prevent certain diseases. With the expansion of applications for probiotic LAB, there is an increasing concern with regard to safety, as cases with adverse effects, i.e., severe infections, transfer of antimicrobial resistance genes, etc., can occur. Currently, in vitro assays remain the primary way to assess the properties of LAB. However, such methodologies are not meeting the needs of strain risk assessment on a high-throughput scale, in the context of the evolving concept of food safety. Analyzing the complete genetic information, including potential virulence genes and other determinants with a negative impact on health, allows for assessing the safe use of the product, for which whole-genome sequencing (WGS) of individual LAB strains can be employed. Genomic data can also be used to understand subtle differences in the strain level important for beneficial effects, or protect patents. Here, we propose that WGS-based bioinformatics analyses are an ideal and cost-effective approach for the initial in silico microbial risk evaluation, while the technique may also increase our understanding of LAB strains for food safety and probiotic property evaluation.202335694810
463660.9998Functional screening of antibiotic resistance genes from a representative metagenomic library of food fermenting microbiota. Lactic acid bacteria (LAB) represent the predominant microbiota in fermented foods. Foodborne LAB have received increasing attention as potential reservoir of antibiotic resistance (AR) determinants, which may be horizontally transferred to opportunistic pathogens. We have previously reported isolation of AR LAB from the raw ingredients of a fermented cheese, while AR genes could be detected in the final, marketed product only by PCR amplification, thus pointing at the need for more sensitive microbial isolation techniques. We turned therefore to construction of a metagenomic library containing microbial DNA extracted directly from the food matrix. To maximize yield and purity and to ensure that genomic complexity of the library was representative of the original bacterial population, we defined a suitable protocol for total DNA extraction from cheese which can also be applied to other lipid-rich foods. Functional library screening on different antibiotics allowed recovery of ampicillin and kanamycin resistant clones originating from Streptococcus salivarius subsp. thermophilus and Lactobacillus helveticus genomes. We report molecular characterization of the cloned inserts, which were fully sequenced and shown to confer AR phenotype to recipient bacteria. We also show that metagenomics can be applied to food microbiota to identify underrepresented species carrying specific genes of interest.201425243126
463470.9998Genome analysis reveals a biased distribution of virulence and antibiotic resistance genes in the genus Enterococcus and an abundance of safe species. Enterococci are lactic acid bacteria (LAB) that, as their name implies, often are found in the gastrointestinal tract of animals. Like many other gut-dwelling LAB, for example, various lactobacilli, they are frequently found in other niches as well, including plants and fermented foods from all over the world. In fermented foods, they contribute to flavor and other organoleptic properties, help extend shelf life, and some even possess probiotic properties. There are many positive attributes of enterococci; however, they have been overshadowed by the occurrence of antibiotic-resistant and virulent strains, often reported for the two species, Enterococcus faecalis and Enterococcus faecium. More than 40,000 whole-genome sequences covering 64 Enterococcus type species are currently available in the National Center for Biotechnology Information repository. Closer inspection of these sequences revealed that most represent the two gut-dwelling species E. faecalis and E. faecium. The remaining 62 species, many of which have been isolated from plants, are thus quite underrepresented. Of the latter species, we found that most carried no potential virulence and antibiotic resistance genes, an observation that is aligned with these species predominately occupying other niches. Thus, the culprits found in the Enterococcus genus mainly belong to E. faecalis, and a biased characterization has resulted in the opinion that enterococci do not belong in food. Since enterococci possess many industrially desirable traits and frequently are found in other niches besides the gut of animals, we suggest that their use as food fermentation microorganisms is reconsidered.IMPORTANCEWe have retrieved a large number of Enterococcus genome sequences from the National Center for Biotechnology Information repository and have scrutinized these for the presence of virulence and antibiotic resistance genes. Our results show that such genes are prevalently found in the two species Enterococcus faecalis and Enterococcus faecium. Most other species do not harbor any virulence and antibiotic resistance genes and display great potential for use as food fermentation microorganisms or as probiotics. The study contributes to the current debate on enterococci and goes against the mainstream perception of enterococci as potentially dangerous microorganisms that should not be associated with food and health.202540202320
463880.9998Comprehensive Scanning of Prophages in Lactobacillus: Distribution, Diversity, Antibiotic Resistance Genes, and Linkages with CRISPR-Cas Systems. Prophage integration, release, and dissemination exert various effects on host bacteria. In the genus Lactobacillus, they may cause bacteriophage contamination during fermentation and even regulate bacterial populations in the gut. However, little is known about their distribution, genetic architecture, and relationships with their hosts. Here, we conducted prophage prediction analysis on 1,472 genomes from 16 different Lactobacillus species and found prophage fragments in almost all lactobacilli (99.8%), with 1,459 predicted intact prophages identified in 64.1% of the strains. We present an uneven prophage distribution among Lactobacillus species; multihabitat species retained more prophages in their genomes than restricted-habitat species. Characterization of the genome features, average nucleotide identity, and landscape visualization presented a high genome diversity of Lactobacillus prophages. We detected antibiotic resistance genes in more than 10% of Lactobacillus prophages and validated that the occurrence of resistance genes conferred by prophage integration was possibly associated with phenotypic resistance in Lactobacillus plantarum. Furthermore, our broad and comprehensive examination of the distribution of CRISPR-Cas systems across the genomes predicted type I and type III systems as potential antagonistic elements of Lactobacillus prophage. IMPORTANCE Lactobacilli are inherent microorganisms in the human gut and are widely used in the food processing industries due to their probiotic properties. Prophages were reportedly hidden in numerous Lactobacillus genomes and can potentially contaminate entire batches of fermentation or modulate the intestinal microecology once they are released. Therefore, a comprehensive scanning of prophages in Lactobacillus is essential for the safety evaluation and application development of probiotic candidates. We show that prophages are widely distributed among lactobacilli; however, intact prophages are more common in multihabitat species and display wide variations in genome feature, integration site, and genomic organization. Our data of the prophage-mediated antibiotic resistance genes (ARGs) and the resistance phenotype of lactobacilli provide evidence for deciphering the putative role of prophages as vectors of the ARGs. Furthermore, understanding the association between prophages and CRISPR-Cas systems is crucial to appreciate the coevolution of phages and Lactobacillus.202134060909
838590.9997Function and Phylogeny of Bacterial Butyryl Coenzyme A:Acetate Transferases and Their Diversity in the Proximal Colon of Swine. Studying the host-associated butyrate-producing bacterial community is important, because butyrate is essential for colonic homeostasis and gut health. Previous research has identified the butyryl coenzyme A (CoA):acetate-CoA transferase (EC 2.3.8.3) as a gene of primary importance for butyrate production in intestinal ecosystems; however, this gene family (but) remains poorly defined. We developed tools for the analysis of butyrate-producing bacteria based on 12 putative but genes identified in the genomes of nine butyrate-producing bacteria obtained from the swine intestinal tract. Functional analyses revealed that eight of these genes had strong But enzyme activity. When but paralogues were found within a genome, only one gene per genome encoded strong activity, with the exception of one strain in which no gene encoded strong But activity. Degenerate primers were designed to amplify the functional but genes and were tested by amplifying environmental but sequences from DNA and RNA extracted from swine colonic contents. The results show diverse but sequences from swine-associated butyrate-producing bacteria, most of which clustered near functionally confirmed sequences. Here, we describe tools and a framework that allow the bacterial butyrate-producing community to be profiled in the context of animal health and disease. IMPORTANCE: Butyrate is a compound produced by the microbiota in the intestinal tracts of animals. This compound is of critical importance for intestinal health, and yet studying its production by diverse intestinal bacteria is technically challenging. Here, we present an additional way to study the butyrate-producing community of bacteria using one degenerate primer set that selectively targets genes experimentally demonstrated to encode butyrate production. This work will enable researchers to more easily study this very important bacterial function that has implications for host health and resistance to disease.201627613689
4381100.9997Specific Gene Loci of Clinical Pseudomonas putida Isolates. Pseudomonas putida are ubiquitous inhabitants of soils and clinical isolates of this species have been seldom described. Clinical isolates show significant variability in their ability to cause damage to hosts because some of them are able to modulate the host's immune response. In the current study, comparisons between the genomes of different clinical and environmental strains of P. putida were done to identify genetic clusters shared by clinical isolates that are not present in environmental isolates. We show that in clinical strains specific genes are mostly present on transposons, and that this set of genes exhibit high identity with genes found in pathogens and opportunistic pathogens. The set of genes prevalent in P. putida clinical isolates, and absent in environmental isolates, are related with survival under oxidative stress conditions, resistance against biocides, amino acid metabolism and toxin/antitoxin (TA) systems. This set of functions have influence in colonization and survival within human tissues, since they avoid host immune response or enhance stress resistance. An in depth bioinformatic analysis was also carried out to identify genetic clusters that are exclusive to each of the clinical isolates and that correlate with phenotypical differences between them, a secretion system type III-like was found in one of these clinical strains, a determinant of pathogenicity in Gram-negative bacteria.201626820467
4222110.9997Conjugal Transfer of Antibiotic Resistances in Lactobacillus spp. Lactic acid bacteria (LAB) are a heterogeneous group of bacteria which are Gram-positive, facultative anaerobes and non-motile, non-spore forming, with varied shapes from cocci to coccobacilli and bacilli. Lactobacillus is the largest and most widely used bacterial species amongst LAB in fermented foods and beverages. The genus is a common member of human gut microbiome. Several species are known to provide benefits to the human gut via synergistic interactions with the gut microbiome and their ability to survive the gut environment. This ability to confer positive health effects provide them a status of generally recognized as safe (GRAS) microorganisms. Due to their various beneficial characteristics, other factors such as their resistance acquisition were overlooked. Overuse of antibiotics has made certain bacteria develop resistance against these drugs. Antibiotic resistance was found to be acquired mainly through conjugation which is a type of lateral gene transfer. Several in vitro methods of conjugation have been discussed previously depending on their success to transfer resistance. In this review, we have addressed methods that are employed to study the transfer of resistance genes using the conjugation phenomenon in lactobacilli.202134076710
4632120.9997Development and application of the active surveillance of pathogens microarray to monitor bacterial gene flux. BACKGROUND: Human and animal health is constantly under threat by emerging pathogens that have recently acquired genetic determinants that enhance their survival, transmissibility and virulence. We describe the construction and development of an Active Surveillance of Pathogens (ASP) oligonucleotide microarray, designed to 'actively survey' the genome of a given bacterial pathogen for virulence-associated genes. RESULTS: The microarray consists of 4958 reporters from 151 bacterial species and include genes for the identification of individual bacterial species as well as mobile genetic elements (transposons, plasmid and phage), virulence genes and antibiotic resistance genes. The ASP microarray was validated with nineteen bacterial pathogens species, including Francisella tularensis, Clostridium difficile, Staphylococcus aureus, Enterococcus faecium and Stenotrophomonas maltophilia. The ASP microarray identified these bacteria, and provided information on potential antibiotic resistance (eg sufamethoxazole resistance and sulfonamide resistance) and virulence determinants including genes likely to be acquired by horizontal gene transfer (e.g. an alpha-haemolysin). CONCLUSION: The ASP microarray has potential in the clinic as a diagnostic tool, as a research tool for both known and emerging pathogens, and as an early warning system for pathogenic bacteria that have been recently modified either naturally or deliberately.200818844996
4386130.9997Large-scale screening of a targeted Enterococcus faecalis mutant library identifies envelope fitness factors. Spread of antibiotic resistance among bacteria responsible for nosocomial and community-acquired infections urges for novel therapeutic or prophylactic targets and for innovative pathogen-specific antibacterial compounds. Major challenges are posed by opportunistic pathogens belonging to the low GC% gram-positive bacteria. Among those, Enterococcus faecalis is a leading cause of hospital-acquired infections associated with life-threatening issues and increased hospital costs. To better understand the molecular properties of enterococci that may be required for virulence, and that may explain the emergence of these bacteria in nosocomial infections, we performed the first large-scale functional analysis of E. faecalis V583, the first vancomycin-resistant isolate from a human bloodstream infection. E. faecalis V583 is within the high-risk clonal complex 2 group, which comprises mostly isolates derived from hospital infections worldwide. We conducted broad-range screenings of candidate genes likely involved in host adaptation (e.g., colonization and/or virulence). For this purpose, a library was constructed of targeted insertion mutations in 177 genes encoding putative surface or stress-response factors. Individual mutants were subsequently tested for their i) resistance to oxidative stress, ii) antibiotic resistance, iii) resistance to opsonophagocytosis, iv) adherence to the human colon carcinoma Caco-2 epithelial cells and v) virulence in a surrogate insect model. Our results identified a number of factors that are involved in the interaction between enterococci and their host environments. Their predicted functions highlight the importance of cell envelope glycopolymers in E. faecalis host adaptation. This study provides a valuable genetic database for understanding the steps leading E. faecalis to opportunistic virulence.201122194979
4729140.9997Newly isolated lactic acid bacteria from silage targeting biofilms of foodborne pathogens during milk fermentation. BACKGROUND: Raw milk, meat and plant materials are subjected to high risks of contamination by various pathogenic bacteria and thus their growth prevention is a great challenge in the food industry. Food fermentation by lactic acid bacteria (LAB) besides changing its organoleptic characteristics also helps to eliminate unfavorable microflora and represses growth of pathogens. To the date only few LABs has been reported to exhibit activity against bacteria embedded in the biofilms characterized by extreme resistance to antimicrobials, high exchange rate with resistance genes and represent high risk factor for foodborne disease development. RESULTS: Six novel LAB strains isolated from the clover silage exhibited pronounced antibacterial activity against biofilm embedded pathogens. We show explicitly that these strains demonstrate high acidification rate, completely repress the growth of E. coli, S. aureus and to a lesser extent P. aeruginosa as well as exhibit appropriate probiotic and milk-fermenting properties. Moreover, in contrast to the approved probiotic strain Lactobacillus plantarum 8PA3, the new isolates were able to efficiently eradicate preformed biofilms of these pathogens and prevent bacterial spreading originating from the biofilm. We suggest these strains as potential additives to the pre-cultures of conventional LAB strains as efficient tools targeting foodborne pathogens in order to prevent food contamination from either seeded raw material or biofilm-fouled equipment. CONCLUSIONS: The AG10 strain identified as L. plantarum demonstrate attractive probiotic and milk fermentation properties as well as high resistance to simulated gastric conditions thus appearing perspective as a starter culture for the prevention of bacterial contamination originating from fouled equipment during milk fermentation.201931703621
4180150.9997Toward integrative genomics study of genetic resistance to Salmonella and Campylobacter intestinal colonization in fowl. Salmonella enterica serotypes Enteritidis and Typhimurium and Campylobacter jejuni are responsible for most cases of food poisoning in Europe. These bacteria do not cause severe disease symptoms in chicken, but they are easily propagated by symptomless chicken carriers which cannot be easily isolated. This animal tolerance is detrimental to food safety. In this particular case, increasing animal's resistance is not sufficient, since some animals considered as resistant are able to carry bacteria during several weeks without displaying disease symptoms. We review studies aimed at evaluating the resistance of chicken to Salmonella and Campylobacter intestinal colonization, either a few days or several weeks after infection. While studies of the genetic control of Campylobacter colonization are only beginning, mostly due to technical difficulties in infection protocols, genetic studies of Salmonella colonization have been conducted for now more than 20 years. They have initially reported an estimation of the genetic parameters associated with resistance to Salmonella colonization and are now aimed at identifying the genomic regions controlling variation of this trait in experimental lines and commercial populations. With the advent of high-throughput genomics, we are closer than ever to identify the true genes controlling resistance to Enterobacteria colonization in chicken. The comparison of genes involved in early resistance to intestinal colonization with genes controlling resistance to bacteria persistence several weeks after infection (i.e., carrier-state) should soon highlight the differences between the molecular mechanisms underlying those two distinct phenotypes. It will also be highly interesting to compare the genes or genomic regions controlling Campylobacter and Salmonella, in order to evaluate the feasibility of a selection conducted on both bacteria simultaneously.201223412643
4221160.9997Antibiotic resistance in probiotic bacteria. Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. The main probiotic bacteria are strains belonging to the genera Lactobacillus and Bifidobacterium, although other representatives, such as Bacillus or Escherichia coli strains, have also been used. Lactobacillus and Bifidobacterium are two common inhabitants of the human intestinal microbiota. Also, some species are used in food fermentation processes as starters, or as adjunct cultures in the food industry. With some exceptions, antibiotic resistance in these beneficial microbes does not constitute a safety concern in itself, when mutations or intrinsic resistance mechanisms are responsible for the resistance phenotype. In fact, some probiotic strains with intrinsic antibiotic resistance could be useful for restoring the gut microbiota after antibiotic treatment. However, specific antibiotic resistance determinants carried on mobile genetic elements, such as tetracycline resistance genes, are often detected in the typical probiotic genera, and constitute a reservoir of resistance for potential food or gut pathogens, thus representing a serious safety issue.201323882264
4635170.9997A Gene Homologous to rRNA Methylase Genes Confers Erythromycin and Clindamycin Resistance in Bifidobacterium breve. Bifidobacteria are mutualistic intestinal bacteria, and their presence in the human gut has been associated with health-promoting activities. The presence of antibiotic resistance genes in this genus is controversial, since, although bifidobacteria are nonpathogenic microorganisms, they could serve as reservoirs of resistance determinants for intestinal pathogens. However, until now, few antibiotic resistance determinants have been functionally characterized in this genus. In this work, we show that Bifidobacterium breve CECT7263 displays atypical resistance to erythromycin and clindamycin. In order to delimit the genomic region responsible for the observed resistance phenotype, a library of genomic DNA was constructed and a fragment of 5.8 kb containing a gene homologous to rRNA methylase genes was able to confer erythromycin resistance in Escherichia coli This genomic region seems to be very uncommon, and homologs of the gene have been detected in only one strain of Bifidobacterium longum and two other strains of B. breve In this context, analysis of shotgun metagenomics data sets revealed that the gene is also uncommon in the microbiomes of adults and infants. The structural gene and its upstream region were cloned into a B. breve-sensitive strain, which became resistant after acquiring the genetic material. In vitro conjugation experiments did not allow us to detect gene transfer to other recipients. Nevertheless, prediction of genes potentially acquired through horizontal gene transfer events revealed that the gene is located in a putative genomic island.IMPORTANCEBifidobacterium breve is a very common human intestinal bacterium. Often described as a pioneer microorganism in the establishment of early-life intestinal microbiota, its presence has been associated with several beneficial effects for the host, including immune stimulation and protection against infections. Therefore, some strains of this species are considered probiotics. In relation to this, because probiotic bacteria are used for human and animal consumption, one of the safety concerns over these bacteria is the presence of antibiotic resistance genes, since the human gut is a densely populated habitat that could favor the transfer of genetic material to potential pathogens. In this study, we analyzed the genetic basis responsible for the erythromycin and clindamycin resistance phenotype of B. breve CECT7263. We were able to identify and characterize a novel gene homologous to rRNA methylase genes which confers erythromycin and clindamycin resistance. This gene seems to be very uncommon in other bifidobacteria and in the gut microbiomes of both adults and infants. Even though conjugation experiments showed the absence of transferability under in vitro conditions, it has been predicted to be located in a putative genomic island recently acquired by specific bifidobacterial strains.201829500262
4181180.9997The place of molecular genetic methods in the diagnostics of human pathogenic anaerobic bacteria. A minireview. Anaerobic infections are common and can cause diseases associated with severe morbidity, but are easily overlooked in clinical settings. Both the relatively small number of infections due to exogenous anaerobes and the much larger number of infections involving anaerobic species that are originally members of the normal flora, may lead to a life-threatening situation unless appropriate treatment is instituted. Special laboratory procedures are needed for the isolation, identification and susceptibility testing of this diverse group of bacteria. Since many anaerobes grow more slowly than the facultative or aerobic bacteria, and particularly since clinical specimens yielding anaerobic bacteria commonly contain several organisms and often very complex mixtures of aerobic and anaerobic bacteria, considerable time may elapse before the laboratory is able to provide a final report. Species definition based on phenotypic features is often time-consuming and is not always easy to carry out. Molecular genetic methods may help in the everyday clinical microbiological practice in laboratories dealing with the diagnostics of anaerobic infections. Methods have been introduced for species diagnostics, such as 16S rRNA PCR-RFLP profile determination, which can help to distinguish species of Bacteroides, Prevotella, Actinomyces, etc. that are otherwise difficult to differentiate. The use of DNA-DNA hybridization and the sequencing of special regions of the 16S rRNA have revealed fundamental taxonomic changes among anaerobic bacteria. Some anaerobic bacteria are extremely slow growing or not cultivatable at all. To detect them in special infections involving flora changes due to oral malignancy or periodontitis, for instance, a PCR-based hybridization technique is used. Molecular methods have demonstrated the spread of specific resistance genes among the most important anaerobic bacteria, the members of the Bacteroides genus. Their detection and investigation of the IS elements involved in their expression may facilitate following of the spread of antibiotic resistance among anaerobic bacteria involved in infections and in the normal flora members. Molecular methods (a search for toxin genes and ribotyping) may promote a better understanding of the pathogenic features of some anaerobic infections, such as the nosocomial diarrhoea caused by C. difficile and its spread in the hospital environment and the community. The investigation of toxin production at a molecular level helps in the detection of new toxin types. This mini-review surveys some of the results obtained by our group and others using molecular genetic methods in anaerobic diagnostics.200616956128
4224190.9997The Genus Enterococcus: Between Probiotic Potential and Safety Concerns-An Update. A considerable number of strains belonging to different species of Enterococcus are highly competitive due to their resistance to wide range of pH and temperature. Their competitiveness is also owed to their ability to produce bacteriocins recognized for their wide-range effectiveness on pathogenic and spoilage bacteria. Enterococcal bacteriocins have attracted great research interest as natural antimicrobial agents in the food industry, and as a potential drug candidate for replacing antibiotics in order to treat multiple drugs resistance pathogens. However, the prevalence of virulence factors and antibiotic-resistance genes and the ability to cause disease could compromise their application in food, human and animal health. From the current regulatory point of view, the genus Enterococcus is neither recommended for the QPS list nor have GRAS status. Although recent advances in molecular biology and the recommended methods for the safety evaluation of Enterococcus strains allowed the distinction between commensal and clinical clades, development of highly adapted methods and legislations are still required. In the present review, we evaluate some aspects of Enterococcus spp. related to their probiotic properties and safety concerns as well as the current and potential application in food systems and treatment of infections. The regulatory status of commensal Enterococcus candidates for food, feed, probiotic use, and recommended methods to assess and ensure their safety are also discussed.201830123208