The role of mobile genetic elements in adaptation of the microbiota to the dynamic human gut ecosystem.

The human gut microbiota is a dense microbial ecosystem with extensive opportunities for bacterial contact-dependent processes such as conjugation and Type VI secretion system (T6SS)-dependent antagonism. In the gut Bacteroidales, two distinct genetic architectures of T6SS loci, GA1 and GA2, are contained on Integrative and Conjugative Elements (ICE). Despite intense interest in the T6SSs of the gut Bacteroidales, there is only a superficial understanding of their evolutionary patterns, and of their dissemination among Bacteroidales species in human gut communities. Here, we combine extensive genomic and metagenomic analyses to better understand their ecological and evolutionary dynamics. We identify new genetic subtypes, document extensive intrapersonal transfer of these ICE to Bacteroidales species within human gut microbiomes, and most importantly, reveal frequent population fixation of these newly armed strains in multiple species within a person. We further show the distribution of each of the distinct T6SSs in human populations and show there is geographical clustering. We reveal that the GA1 T6SS ICE integrates at a minimal recombination site leading to their integration throughout genomes and their frequent interruption of genes, whereas the GA2 T6SS ICE integrate at one of three different tRNA genes. The exclusion of concurrent GA1 and GA2 T6SSs in individual strains is associated with intact T6SS loci and with an ICE-encoded gene. By performing a comprehensive analysis of mobile genetic elements (MGE) in co-resident Bacteroidales species in numerous human gut communities, we identify 74 MGE that transferred to multiple Bacteroidales species within individual gut microbiomes. We further show that only three other MGE demonstrate multi-species spread in human gut microbiomes to the degree demonstrated by the GA1 and GA2 ICE. These data underscore the ubiquity and dissemination of mobile T6SS loci within Bacteroidales communities and across human populations.

Actinobacillus pleuropneumoniae is an important respiratory pathogen that can cause porcine contagious pleuropneumonia (PCP), resulting in significant economic losses in swine industry. Microorganisms are subjected to drastic changes in environmental osmolarity. In order to alleviate the drastic rise or fall of osmolarity, cells activate mechanosensitive channels MscL and MscS through tension changes. MscL not only regulates osmotic pressure but also has been reported to secrete protein and uptake aminoglycoside antibiotic. However, MscL and MscS, as the most common mechanosensitive channels, have not been characterized in A. pleuropneumoniae. In this study, the osmotic shock assay showed that MscL increased sodium adaptation by regulating cell length. The results of MIC showed that deletion of mscL decreased the sensitivity of A. pleuropneumoniae to multiple antibiotics, while deletion of mscS rendered A. pleuropneumoniae hypersensitive to penicillin. Biofilm assay demonstrated that MscL contributed the biofilm formation but MscS did not. The results of animal assay showed that MscL and MscS did not affect virulence in vivo. In conclusion, MscL is essential for sodium hyperosmotic tolerance, biofilm formation, and resistance to chloramphenicol, erythromycin, penicillin, and oxacillin. On the other hand, MscS is only involved in oxacillin resistance.IMPORTANCEBacterial resistance to the external environment is a critical function that ensures the normal growth of bacteria. MscL and MscS play crucial roles in responding to changes in both external and internal environments. However, the function of MscL and MscS in Actinobacillus pleuropneumoniae has not yet been reported. Our study shows that MscL plays a significant role in osmotic adaptation, antibiotic resistance, and biofilm formation of A. pleuropneumoniae, while MscS only plays a role in antibiotic resistance. Our findings provide new insights into the functional characteristics of MscL and MscS in A. pleuropneumoniae. MscL and MscS play a role in antibiotic resistance and contribute to the development of antibiotics for A. pleuropneumoniae.

Bacterial resistance to antibiotics is one of the three major health challenges of the 21st century. One of the most important reasons for the acquisition and spread of antibiotic resistance in the environment is the irrational and uncontrolled use of antibacterial drugs, not only for medical but also other purposes, and their improper disposal. The microbiome of aquatic and soil ecosystems is characterized by the acquisition of antibiotic resistance through mobile genetic elements, contact with antibacterial drugs and their residues, the action of heavy metals and environmental stress. Also, according to the literature, it is noted that the resistance of microorganisms to antibacterial drugs in the environment existed much earlier than in clinical strains. These facts can not help but worry, because antibiotic-resistant strains of the environment have an extremely negative impact on human health. Once in the human body with water and food, they significantly complicate and / or make it impossible to further treat life-threatening diseases. Also, antibacterial residues circulating in aquatic and soil ecosystems, entering the human body can cause cancer, allergic reactions or disruption of the natural intestinal microflora. These ecosystems are characterized by large-scale spread of antibiotic-resistant microorganisms, antibacterial drugs and their residues. The aim of our work was to analyze with the help of theoretical methods of scientific research the reasons for the acquisition and spread of antibiotic resistance among environmental microbiota, namely in aquatic and soil ecosystems. To determine the impact of antibiotic-resistant bacteria of these ecosystems on human health. We have found that antibacterial drugs, antibiotic-resistant strains and resistance genes are a particular problem for wastewater treatment. Antibiotics can provide a selective load, as the mechanisms that break them down can promote resilience and selectively enrich. Wastewater treatment plants can be a favorable factor for the horizontal transfer of genes and the development of bacterial polyresistance, and high-resistance genes can be preserved even after disinfection. Soil is also an important reservoir for antibiotic-resistant bacteria and resistance genes. Microorganisms are in a constant struggle for existence in this ecosystem and try to colonize the micro-scale with the most favorable for their ecotype habitat. Antibiotic-resistant soil bacteria are in close contact with other members of the microbiota, which in turn promotes the horizontal transfer of resistance genes, even between cells of different species or genera through genetic determinants. Conclusion: ecosystems are characterized by large-scale spread of antibiotic-resistant microorganisms, antibacterial drugs and their residues. Therefore, this problem should be properly addressed, as the presence of antibiotic-resistant microorganisms, antibacterial drugs and their residues in the environment can cause unpredictable environmental consequences and adversely affect human health with more severe incurable infectious diseases. Monitoring programs for antibiotic-resistant microorganisms and resistance genes in soil and aquatic ecosystems are necessary and very relevant today. After all, this microbiota poses a serious threat to both the environment and human health and can easily spread from one part of the world around the world.

Exploring the resistome, virulome, mobilome and microbiome along pork production chain using metagenomics

ABSTRACTPurposeUrinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) are a major public health concern due to their recurrent nature and antibiotic resistance. Biofilm formation plays a crucial role in UPEC persistence, yet the genetic mechanisms underlying this process remain poorly understood. This study employs next-generation sequencing (NGS) to investigate the genomic characteristics of biofilm-forming, multidrug-resistant (MDR) UPEC isolates, with a focus on antimicrobial resistance (AMR), virulence factors, and mobile genetic elements.MethodsFive biofilm-forming MDR UPEC isolates were selected for whole-genome sequencing (WGS) using the Illumina NovaSeq 6000 platform. Genome assembly and annotation were performed using SPAdes and Prokka. Multilocus sequence typing (MLST) and serotyping were conducted to determine genetic diversity, while AMR genes were identified using ResFinder. Virulence factors and biofilm-related genes were analyzed through PathogenFinder, and mobile genetic elements, including plasmids and insertion sequences, were characterized.ResultsGenomic analysis revealed significant diversity among isolates, with MLST identifying high-risk sequence types such as ST131, known for its strong association with MDR and virulence. AMR profiling indicated resistance to multiple antibiotics, including beta-lactams, aminoglycosides, and fluoroquinolones. All isolates harbored virulence genes associated with adhesion, immune evasion, and biofilm formation. Mobile genetic elements, particularly IncF-type plasmids and insertion sequences, were detected across isolates, suggesting a role in horizontal gene transfer of resistance traits. Biofilm-associated genes correlated with biofilm production capabilities, reinforcing their role in UPEC persistence.ConclusionThis study provides critical insights into the genetic landscape of biofilm-forming UPEC, highlighting the role of mobile elements in antibiotic resistance dissemination. The findings underscore the importance of genomic surveillance and the need for novel therapeutic strategies targeting biofilm-mediated resistance to combat recurrent UTIs.

Recent research has revealed that horizontal gene transfer and biofilm formation are connected processes. Although published research investigating this interconnectedness is still limited, we will review this subject in order to highlight the potential of these observations because of their believed importance in the understanding of the adaptation and subsequent evolution of social traits in bacteria. Here, we discuss current evidence for such interconnectedness centred on plasmids. Horizontal transfer rates are typically higher in biofilm communities compared with those in planktonic states. Biofilms, furthermore, promote plasmid stability and may enhance the host range of mobile genetic elements that are transferred horizontally. Plasmids, on the other hand, are very well suited to promote the evolution of social traits such as biofilm formation. This, essentially, transpires because plasmids are independent replicons that enhance their own success by promoting inter-bacterial interactions. They typically also carry genes that heighten their hosts' direct fitness. Furthermore, current research shows that the so-called mafia traits encoded on mobile genetic elements can enforce bacteria to maintain stable social interactions. It also indicates that horizontal gene transfer ultimately enhances the relatedness of bacteria carrying the mobile genetic elements of the same origin. The perspective of this review extends to an overall interconnectedness between horizontal gene transfer, mobile genetic elements and social evolution of bacteria.

After more than 150 years of research in microbiology, new technologies and new insights into the microbial world have sparked a revolution in the field. This is a much needed development, not only to renew interest in prokaryote research, but also to meet many emerging challenges in medicine, agriculture and industrial processes. Although many microbiologists—such as Emil von Behring, Robert Koch, Jacques Monod, Francois Jacob, Andre Lwoff, Alexander Fleming, Selman A. Waksman and Joshua Lederberg—grace the list of Nobel laureates, attention moved away from microbiology as biologists focused their interest on eukaryotic cells and higher organisms in the 1970s and 1980s. Furthermore, from the beginning, research on prokaryotes has suffered from an anthropocentric view, regarding as interesting only those organisms that cause disease or that can be exploited for industrial or agricultural use. But the advent of new technologies, some of which have been driven by a need to understand eukaryotes, may change this. We are increasingly realizing how little we know about microbes in general, their diversity, the mechanisms of their evolution and adaptation and their modes of existence within, and communication with, their environment and higher organisms. As bacteria have succeeded in occupying virtually all ecological niches on this planet, ranging from arctic regions to oceanic hot springs, they hold an immense wealth of genetic information that we have barely started to explore and that may provide many useful applications for humans. The new technologies that allow us to sequence and annotate whole genomes more rapidly and to analyse the expression of thousands of genes in a single experiment are likely to speed up this change, particularly as microbes are well suited for high‐throughput analysis. Any microbial genome can now be sequenced within a few hours and, in the near future, new bioinformatics tools will enable scientists not …

Bacterial virulence and antibiotic resistance have a significant influence on disease severity and treatment options during bacterial infections. Frequently, the underlying genetic determinants are encoded on mobile genetic elements (MGEs). In the leading human pathogen Staphylococcus aureus, MGEs that contain antibiotic resistance genes commonly do not contain genes for virulence determinants. The phenol-soluble modulins (PSMs) are staphylococcal cytolytic toxins with a crucial role in immune evasion. While all known PSMs are core genome-encoded, we here describe a previously unidentified psm gene, psm-mec, within the staphylococcal methicillin resistance-encoding MGE SCCmec. PSM-mec was strongly expressed in many strains and showed the physico-chemical, pro-inflammatory, and cytolytic characteristics typical of PSMs. Notably, in an S. aureus strain with low production of core genome-encoded PSMs, expression of PSM-mec had a significant impact on immune evasion and disease. In addition to providing high-level resistance to methicillin, acquisition of SCCmec elements encoding PSM-mec by horizontal gene transfer may therefore contribute to staphylococcal virulence by substituting for the lack of expression of core genome-encoded PSMs. Thus, our study reveals a previously unknown role of methicillin resistance clusters in staphylococcal pathogenesis and shows that important virulence and antibiotic resistance determinants may be combined in staphylococcal MGEs.

Dissemination of antibiotic resistance genes (ARGs) via integrons in Escherichia coli: A risk to human health

The global widespread use of antimicrobials and accompanying increase in resistant bacterial strains is of major public health concern. Wastewater systems and wastewater treatment plants are considered a niche for antibiotic resistance genes (ARGs), with diverse microbial communities facilitating ARG transfer via mobile genetic element (MGE). In contrast to hospital sewage, wastewater from other health care facilities is still poorly investigated. At the instance of a nursing home located in south-west Germany, in the present study, shotgun metagenomics was used to investigate the impact on wastewater of samples collected up- and down-stream in different seasons. Microbial composition, ARGs and MGEs were analyzed using different annotation approaches with various databases, including Antibiotic Resistance Ontologies (ARO), integrons and plasmids. Our analysis identified seasonal differences in microbial communities and abundance of ARG and MGE between samples from different seasons. However, no obvious differences were detected between up- and downstream samples. The results suggest that, in contrast to hospitals, sewage from the nursing home does not have a major impact on ARG or MGE in wastewater, presumably due to much less intense antimicrobial usage. Possible limitations of metagenomic studies using high-throughput sequencing for detection of genes that seemingly confer antibiotic resistance are discussed.

The pool of mobile genetic elements (MGE) in microbial communities consists of plasmids, bacteriophages and other elements that are either self-transmissible or use mobile plasmids and phages as vehicles for their dissemination. By facilitating horizontal gene exchange, the horizontal gene pool (HGP) promotes the evolution and adaptation of microbial communities. Efforts to characterise MGE from bacterial populations resident in a variety of ecological habitats have revealed a surprisingly vast and seemingly untapped diversity. MGE, conferring such selectable traits as mercury or antibiotic resistance and degradative functions, have been readily acquired from diverse microbial communities. To circumvent the need to isolate microbial hosts, polymerase chain reaction (PCR)-based detection methods have frequently been used to assess the prevalence of MGE-specific sequences resident in the 'microbial community' HGP. As studies continue to reveal novel and distinct MGE, sequencing of newly isolated MGE from diverse habitats is essential for the continued development of DNA probes, PCR primers as well as for gene array and proteomics-based approaches. This minireview highlights insight gained from different methodological approaches, biased albeit largely toward plasmids in Gram-negative bacteria, used to study the HGP of naturally occurring microbial communities from various aquatic and terrestrial habitats.

The prevalence and diversity of mobile genetic elements in bacterial communities of different environmental habitats: insights gained from different methodological approaches

Elizabethkingia anophelis is an emerging multidrug resistant pathogen that has caused several global outbreaks. E. anophelis belongs to the large family of Flavobacteriaceae, which contains many bacteria that are plant, bird, fish, and human pathogens. Several antibiotic resistance genes are found within the E. anophelis genome, including a chloramphenicol acetyltransferase (CAT). CATs play important roles in antibiotic resistance and can be transferred in genetic mobile elements. They catalyse the acetylation of the antibiotic chloramphenicol, thereby reducing its effectiveness as a viable drug for therapy. Here, we determined the high-resolution crystal structure of a CAT protein from the E. anophelis NUHP1 strain that caused a Singaporean outbreak. Its structure does not resemble that of the classical Type A CATs but rather exhibits significant similarity to other previously characterized Type B (CatB) proteins from Pseudomonas aeruginosa, Vibrio cholerae and Vibrio vulnificus, which adopt a hexapeptide repeat fold. Moreover, the CAT protein from E. anophelis displayed high sequence similarity to other clinically validated chloramphenicol resistance genes, indicating it may also play a role in resistance to this antibiotic. Our work expands the very limited structural and functional coverage of proteins from Flavobacteriaceae pathogens which are becoming increasingly more problematic.

Impact of bacteriophage Saint3 carriage on the immune evasion capacity and hemolytic potential of Staphylococcus aureus CC398

Antibiotic resistance is a rapidly escalating global healthcare crisis. Bacteria employ diverse strategies to evade both antibiotics and the host immune system. One of these tools is the use of bacterial extracellular vesi­ cles that mediate bacterial survival under antibiotic and immune stress. This review analyzes the impact of bacterial extracellular vesicles on antibiotic resistance and immune evasion. Bacterial extracellular vesicles of gram-positive and gram-negative bacteria have a number of structural differences, as well as different mechanisms for implementing their functions. Bacterial extracellular vesicles contribute to antibiotic resistance by acting as antibiotic targets, carrying resistance genes (horizontal gene transfer), and removing/degrading antibacterial agents. By implementing the strategy of evading immune surveillance, bacterial extracellular vesicles can participate in the formation of biofilms, provoke pro- and anti-inflammatory reactions, influence the secretion of cytokines by immune cells, creating more favorable conditions for colonization and development. Therefore, bacterial extracellular vesicles significantly contribute to bacterial survival under antibiotic and immune stress. However, the molecular mechanisms underlying bacterial extracellular vesi­ cles biogenesis and host responses to bacterial extracellular vesicles remain poorly understood, highlighting the need for further research to combat antibiotic resistance and bacterial infections.