Development and application of the active surveillance of pathogens microarray to monitor bacterial gene flux. - Related Documents




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463201.0000Development 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
434010.9998Predicting antimicrobial susceptibility from the bacterial genome: A new paradigm for one health resistance monitoring. The laboratory identification of antibacterial resistance is a cornerstone of infectious disease medicine. In vitro antimicrobial susceptibility testing has long been based on the growth response of organisms in pure culture to a defined concentration of antimicrobial agents. By comparing individual isolates to wild-type susceptibility patterns, strains with acquired resistance can be identified. Acquired resistance can also be detected genetically. After many decades of research, the inventory of genes underlying antimicrobial resistance is well known for several pathogenic genera including zoonotic enteric organisms such as Salmonella and Campylobacter and continues to grow substantially for others. With the decline in costs for large scale DNA sequencing, it is now practicable to characterize bacteria using whole genome sequencing, including the carriage of resistance genes in individual microorganisms and those present in complex biological samples. With genomics, we can generate comprehensive, detailed information on the bacterium, the mechanisms of antibiotic resistance, clues to its source, and the nature of mobile DNA elements by which resistance spreads. These developments point to a new paradigm for antimicrobial resistance detection and tracking for both clinical and public health purposes.202133010049
507620.9998Diagnostic microarray for human and animal bacterial diseases and their virulence and antimicrobial resistance genes. Rapid diagnosis and treatment of disease is often based on the identification and characterization of causative agents derived from phenotypic characteristics. Current methods can be laborious and time-consuming, often requiring many skilled personnel and a large amount of lab space. The objective of our study was to develop a spotted microarray for rapid identification and characterization of bacterial pathogens and their antimicrobial resistance genes. Our spotted microarray consists of 489 70mer probes that detect 40 bacterial pathogens of medical, veterinary and zoonotic importance (including 15 NIAID Category A, B and C pathogens); associated genes that encode resistance for antimicrobial and metal resistance; and DNA elements that are important for horizontal gene transfer among bacteria. High specificity and reliability of the microarray was achieved for bacterial pathogens of animal and human importance by validating MDR pathogenic bacteria as pure cultures or by following their inoculation in complex and highly organic sample matrices, such as soil and manure.201020035807
463730.9998What 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
463140.9998Genome Analysis of an Enterococcal Prophage, Entfac.MY. BACKGROUND: Bacteriophages are bacterial parasites. Unlike lytic bacteriophages, lysogenic bacteriophages do not multiply immediately after entering the host cells and may integrate their genomes into the bacterial genomes as prophages. Prophages can include various phenotypic and genotypic effects on the host bacteria. Enterococcus spp. are Gram-positive bacteria that cause infections in humans and animals. In recent decades, these bacteria have become resistant to various antimicrobials, including vancomycin. The aim of this study was to analyze genome of an enterococcal prophage. METHODS: In this study, Enterococcus faecium EntfacYE was isolated from biological samples and its genome was analyzed using next-generation sequencing method. RESULTS: Overall, 254 prophage genes were identified in the bacterial genome. The prophage included 39 housekeeping, 41 replication and regulation, 80 structural and packaging, and 48 lysis genes. Moreover, 46 genes with unknown functions were identified. All genes were annotated in DNA Data Bank of Japan. CONCLUSION: In general, most prophage genes were linked to packaging and structure (31.5%) gene group. However, genes with unknown functions included a high proportion (18.11%), which indicated necessity of further analyses. Genomic analysis of the prophages can be effective in better understanding of their roles in development of bacterial resistance to antibiotics. Moreover, identification and study of prophages can help researchers develop genetic engineering tools and novel infection therapies.202236061127
418150.9998The 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
940560.9998Functional 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
417870.9998Efficacy and food safety considerations of poultry competitive exclusion products. Competitive exclusion (CE) products are anaerobic cultures of bacteria that are applied to poultry hatchlings to establish a protective enteric microbiota that excludes intestinal colonization by human food-borne pathogens. For safety of the poultry flock and human consumers, the identities of bacteria in CE products need to be known. A CE product is a culture of intestinal contents from adult chickens. It may be microbiologically defined by analysis of bacteria isolated from the culture, but many bacteria are hard to reliably isolate, identify, and characterize with conventional techniques. Sequence analysis of 16S ribosomal RNA (rRNA) genes may be more reliable than conventional techniques to identify CE bacteria. Bacteria in CE products may contain antimicrobial drug resistance and virulence mechanisms that could be transferred to the enteric bacteria of the food animal and to the human consumer. Detection methods for specific antimicrobial drug resistance and virulence genes and the integrase genes of conjugative transposons, mostly utilizing PCR technology, are being developed that can be applied to assess these risks in CE bacteria. With improvements in efficacy, bacterial identification, and detection and control of the possible risks of gene transfer, CE product technology can be made a more effective food safety tool.200617039457
462980.9998Screening and in silico characterization of prophages in Helicobacter pylori clinical strains. The increase of antibiotic resistance calls for alternatives to control Helicobacter pylori, a Gram-negative bacterium associated with various gastric diseases. Bacteriophages (phages) can be highly effective in the treatment of pathogenic bacteria. Here, we developed a method to identify prophages in H. pylori genomes aiming at their future use in therapy. A polymerase chain reaction (PCR)-based technique tested five primer pairs on 74 clinical H. pylori strains. After the PCR screening, 14 strains most likely to carry prophages were fully sequenced. After that, a more holistic approach was taken by studying the complete genome of the strains. This study allowed us to identify 12 intact prophage sequences, which were then characterized concerning their morphology, virulence, and antibiotic-resistance genes. To understand the variability of prophages, a phylogenetic analysis using the sequences of all H. pylori phages reported to date was performed. Overall, we increased the efficiency of identifying complete prophages to 54.1 %. Genes with homology to potential virulence factors were identified in some new prophages. Phylogenetic analysis revealed a close relationship among H. pylori-phages, although there are phages with different geographical origins. This study provides a deeper understanding of H. pylori-phages, providing valuable insights into their potential use in therapy.202539368610
414290.9998Antimicrobial Resistance in Pasteurellaceae of Veterinary Origin. Members of the highly heterogeneous family Pasteurellaceae cause a wide variety of diseases in humans and animals. Antimicrobial agents are the most powerful tools to control such infections. However, the acquisition of resistance genes, as well as the development of resistance-mediating mutations, significantly reduces the efficacy of the antimicrobial agents. This article gives a brief description of the role of selected members of the family Pasteurellaceae in animal infections and of the most recent data on the susceptibility status of such members. Moreover, a review of the current knowledge of the genetic basis of resistance to antimicrobial agents is included, with particular reference to resistance to tetracyclines, β-lactam antibiotics, aminoglycosides/aminocyclitols, folate pathway inhibitors, macrolides, lincosamides, phenicols, and quinolones. This article focusses on the genera of veterinary importance for which sufficient data on antimicrobial susceptibility and the detection of resistance genes are currently available (Pasteurella, Mannheimia, Actinobacillus, Haemophilus, and Histophilus). Additionally, the role of plasmids, transposons, and integrative and conjugative elements in the spread of the resistance genes within and beyond the aforementioned genera is highlighted to provide insight into horizontal dissemination, coselection, and persistence of antimicrobial resistance genes. The article discusses the acquisition of diverse resistance genes by the selected Pasteurellaceae members from other Gram-negative or maybe even Gram-positive bacteria. Although the susceptibility status of these members still looks rather favorable, monitoring of their antimicrobial susceptibility is required for early detection of changes in the susceptibility status and the newly acquired/developed resistance mechanisms.201829916344
4179100.9998Epidemiology of Antimicrobial Resistance Genes in Streptococcus agalactiae Sequences from a Public Database in a One Health Perspective. Streptococcus agalactiae is a well-known pathogen in humans and food-producing animals. Therefore, this bacterium is a paradigmatic example of a pathogen to be controlled by a One Health approach. Indeed, the zoonotic and reverse-zoonotic potential of the bacteria, the prevalence of Group B Streptococci (GBS) diseases in both human and animal domains, and the threatening global situation on GBS antibiotic resistance make these bacteria an important target for control programs. An epidemiological analysis using a public database containing sequences of S. agalactiae from all over the world was conducted to evaluate the frequency and evolution of antibiotic resistance genes in those isolates. The database we considered (NCBI pathogen detection isolate browser-NPDIB) is maintained on a voluntary basis. Therefore, it does not follow strict epidemiological criteria. However, it may be considered representative of the bacterial population related to human diseases. The results showed that the number of reported sequences increased largely in the last four years, and about 50% are of European origin. The frequency data and the cluster analysis showed that the AMR genes increased in frequency in recent years and suggest the importance of verifying the application of prudent protocols for antimicrobials in areas with an increasing frequency of GBS infections both in human and veterinary medicine.202236140016
4797110.9998Antibiotic resistance among clinically important gram-positive bacteria in the UK. The resistance of bacteria to antibiotics, particularly those used for first-line therapy, is an increasing cause for concern. In the UK, the prevalence of resistance to methicillin and mupirocin in Staphylococcus aureus, and to penicillin and macrolides in Streptococcus pneumoniae, appear to be increasing. There has also been an increase in the number of hospitals where glycopeptide-resistant enterococci are known to have been isolated. The increases in methicillin-resistant S. aureus and glycopeptide-resistant enterococci are due, in part, to the inter-hospital spread of epidemic strains. Although new quinolones and streptogramins with activity against Gram-positive bacteria (including strains resistant to currently available agents) are under development, there is no reason to believe that resistance to these agents will not emerge. The control of resistance in Gram-positive bacteria will require a multi-faceted approach, including continued and improved surveillance, a reduction in the unnecessary use of antibiotics, and the application of other strategies such as vaccination.19989777517
9404120.9998The 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
4341130.9998Antimicrobial Resistance in Nontyphoidal Salmonella. Non-typhoidal Salmonella is the most common foodborne bacterial pathogen in most countries. It is widely present in food animal species, and therefore blocking its transmission through the food supply is a prominent focus of food safety activities worldwide. Antibiotic resistance in non-typhoidal Salmonella arises in large part because of antibiotic use in animal husbandry. Tracking resistance in Salmonella is required to design targeted interventions to contain or diminish resistance and refine use practices in production. Many countries have established systems to monitor antibiotic resistance in Salmonella and other bacteria, the earliest ones appearing the Europe and the US. In this chapter, we compare recent Salmonella antibiotic susceptibility data from Europe and the US. In addition, we summarize the state of known resistance genes that have been identified in the genus. The advent of routine whole genome sequencing has made it possible to conduct genomic surveillance of resistance based on DNA sequences alone. This points to a new model of surveillance in the future that will provide more definitive information on the sources of resistant Salmonella, the specific types of resistance genes involved, and information on how resistance spreads.201830027887
4640140.9998Genome analysis of probiotic bacteria for antibiotic resistance genes. To date, probiotic bacteria are used in the diet and have various clinical applications. There are reports of antibiotic resistance genes in these bacteria that can transfer to other commensal and pathogenic bacteria. The aim of this study was to use whole-genome sequence analysis to identify antibiotic resistance genes in a group of bacterial with probiotic properties. Also, this study followed existing issues about the importance and presence of antibiotic resistance genes in these bacteria and the dangers that may affect human health in the future. In the current study, a collection of 126 complete probiotic bacterial genomes was analyzed for antibiotic resistance genes. The results of the current study showed that there are various resistance genes in these bacteria that some of them are transferable to other bacteria. The tet(W) tetracycline resistance gene was more than other antibiotic resistance genes in these bacteria and this gene was found in Bifidobacterium and Lactobacillus. In our study, the most numbers of antibiotic resistance genes were transferred with mobile genetic elements. We propose that probiotic companies before the use of a micro-organism as a probiotic, perform an antibiotic susceptibility testing for a large number of antibiotics. Also, they perform analysis of complete genome sequence for prediction of antibiotic resistance genes.202234989942
4633150.9998Detection of Helicobacter pylori virulence-associated genes. Helicobacter pylori is an important human pathogen and persistent colonization of the human gastric mucosa can cause severe gastrointestinal diseases. The bacterium should not be considered as a uniform organism, but as a population of closely related and yet genetically diverse bacteria. Several genes of H. pylori (such as vacA and cagA) have been identified as being virulence-associated and may have important clinical and epidemiological implications. Assessment of virulence-associated genes of H. pylori should be included in clinical and epidemiological studies as well as therapeutic trials, in order to stratify between patient groups, harboring H. pylori strains with particular virulence genotypes. Molecular determination of antibiotic resistance will be especially useful for treatment studies. Together with our increasing knowledge about the human genome, typing of H. pylori will facilitate the management of gastroenterological pathologies.200111901834
4386160.9998Large-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
4265170.9998Bacteriophages as vehicles of the resistome in cystic fibrosis. Environmental microbial communities and human microbiota represent a huge reservoir of mobilizable genes, the 'mobilome', including a pool of genes encoding antimicrobial resistance, the 'resistome'. Whole-genome sequencing of bacterial genomes from cystic fibrosis (CF) patients has demonstrated that bacteriophages contribute significantly to bacterial genome alterations, and metagenomic analysis of respiratory tract DNA viral communities has revealed the presence of genes encoding antimicrobial resistance in bacteriophages of CF patients. CF airways should now be considered as the site of complex microbiota, where bacteriophages are vehicles for the adaptation of bacteria to this specific environment and for the emergence and selection of multidrug-resistant bacteria with chimeric repertoires. As phages are already known to be mobilized during chronic infection of the lungs of patients with CF, it seems particularly important to improve the understanding of the mechanisms of phage induction to prevent the spread of virulence and/or antimicrobial resistance determinants within the CF population as well as in the community. Such a modern point of view may be a seminal reflection for clinical practice in the future since current antimicrobial therapy guidelines in the context of CF may lead to the emergence of genes encoding antimicrobial resistance.201121816766
4628180.9998Genomic Analysis of Molecular Bacterial Mechanisms of Resistance to Phage Infection. To optimize phage therapy, we need to understand how bacteria evolve against phage attacks. One of the main problems of phage therapy is the appearance of bacterial resistance variants. The use of genomics to track antimicrobial resistance is increasingly developed and used in clinical laboratories. For that reason, it is important to consider, in an emerging future with phage therapy, to detect and avoid phage-resistant strains that can be overcome by the analysis of metadata provided by whole-genome sequencing. Here, we identified genes associated with phage resistance in 18 Acinetobacter baumannii clinical strains belonging to the ST-2 clonal complex during a decade (Ab2000 vs. 2010): 9 from 2000 to 9 from 2010. The presence of genes putatively associated with phage resistance was detected. Genes detected were associated with an abortive infection system, restriction-modification system, genes predicted to be associated with defense systems but with unknown function, and CRISPR-Cas system. Between 118 and 171 genes were found in the 18 clinical strains. On average, 26% of these genes were detected inside genomic islands in the 2000 strains and 32% in the 2010 strains. Furthermore, 38 potential CRISPR arrays in 17 of 18 of the strains were found, as well as 705 proteins associated with CRISPR-Cas systems. A moderately higher presence of these genes in the strains of 2010 in comparison with those of 2000 was found, especially those related to the restriction-modification system and CRISPR-Cas system. The presence of these genes in genomic islands at a higher rate in the strains of 2010 compared with those of 2000 was also detected. Whole-genome sequencing and bioinformatics could be powerful tools to avoid drawbacks when a personalized therapy is applied. In this study, it allows us to take care of the phage resistance in A. baumannii clinical strains to prevent a failure in possible phage therapy.202135250902
4626190.9998Prophages Present in Acinetobacter pittii Influence Bacterial Virulence, Antibiotic Resistance, and Genomic Rearrangements. Introduction: Antibiotic resistance and virulence are common among bacterial populations, posing a global clinical challenge. The bacterial species Acinetobacter pittii, an infectious agent in clinical environments, has shown increasing rates of antibiotic resistance. Viruses that integrate as prophages into A. pittii could be a potential cause of this pathogenicity, as they often contain antibiotic resistance or virulence factor gene sequences. Methods: In this study, we analyzed 25 A. pittii strains for potential prophages. Using virulence factor databases, we identified many common and virulent prophages in A. pittii. Results: The analysis also included a specific catalogue of the virulence factors and antibiotic resistance genes contributed by A. pittii prophages. Finally, our results illustrate multiple similarities between A. pittii and its bacterial relatives with regard to prophage integration sites and prevalence. Discussion: These findings provide a broader insight into prophage behavior that can be applied to future studies on similar species in the Acinetobacter calcoaceticus-baumannii complex.202236161193