Unlocking the potential of microbiome editing: A review of conjugation-based delivery.

In recent decades, there has been a rapid increase in the prevalence of multidrug-resistant pathogens, posing a challenge to modern antibiotic-based medicine. This has highlighted the need for novel treatments that can specifically affect the target microorganism without disturbing other co-inhabiting species, thus preventing the development of dysbiosis in treated patients. Moreover, there is a pressing demand for tools to effectively manipulate complex microbial populations. One of the approaches suggested to address both issues was to use conjugation as a tool to modify the microbiome by either editing the genome of specific bacterial species and/or the removal of certain taxonomic groups. Conjugation involves the transfer of DNA from one bacterium to another, which opens up the possibility of introducing, modifying or deleting specific genes in the recipient. In response to this proposal, there has been a significant increase in the number of studies using this method for gene delivery in bacterial populations. This MicroReview aims to provide a detailed overview on the use of conjugation for microbiome engineering, and at the same time, to initiate a discussion on the potential, limitations and possible future directions of this approach.

Integrative and Conjugative Elements and Prophage DNA as Carriers of Resistance Genes in Erysipelothrix rhusiopathiae Strains from Domestic Geese in Poland.

Goose erysipelas is a serious problem in waterfowl breeding in Poland. However, knowledge of the characteristics of Erysipelothrix rhusiopathiae strains causing this disease is limited. In this study, the antimicrobial susceptibility and serotypes of four E. rhusiopathiae strains from domestic geese were determined, and their whole-genome sequences (WGSs) were analyzed to detect resistance genes, integrative and conjugative elements (ICEs), and prophage DNA. Sequence type and the presence of resistance genes and transposons were compared with 363 publicly available E. rhusiopathiae strains, as well as 13 strains of other Erysipelothrix species. Four strains tested represented serotypes 2 and 5 and the MLST groups ST 4, 32, 242, and 243. Their assembled circular genomes ranged from 1.8 to 1.9 kb with a GC content of 36-37%; a small plasmid was detected in strain 1023. Strains 1023 and 267 were multidrug-resistant. The resistance genes detected in the genome of strain 1023 were erm47, tetM, and lsaE-lnuB-ant(6)-Ia-spw cluster, while strain 267 contained the tetM and ermB genes. Mutations in the gyrA gene were detected in both strains. The tetM gene was embedded in a Tn916-like transposon, which in strain 1023, together with the other resistance genes, was located on a large integrative and conjugative-like element of 130 kb designated as ICEEr1023. A minor integrative element of 74 kb was identified in strain 1012 (ICEEr1012). This work contributes to knowledge about the characteristics of E. rhusiopathiae bacteria and, for the first time, reveals the occurrence of erm47 and ermB resistance genes in strains of this species. Phage infection appears to be responsible for the introduction of the ermB gene into the genome of strain 267, while ICEs most likely play a key role in the spread of the other resistance genes identified in E. rhusiopathiae.

A CRISPR interference screen reveals a role for cell wall teichoic acids in conjugation in Bacillus subtilis.

Conjugative elements are widespread in bacteria and include plasmids and integrative and conjugative elements (ICEs). They transfer from donor to recipient cells via an element-encoded type IV secretion system. These elements interact with and utilize host functions for their lifecycles. We sought to identify essential host genes involved in the lifecycle of the integrative and conjugative element ICEBs1 of Bacillus subtilis. We constructed a library of strains for inducible knockdown of essential B. subtilis genes using CRISPR interference. Each strain expressed one guide RNA in ICEBs1. We induced partial interference of essential genes and identified those that caused an acute defect in acquisition of ICEBs1 by recipient cells. This screen revealed that reducing expression of genes needed for synthesis of cell wall teichoic acids caused a decrease in conjugation. Using three different ways to reduce their synthesis, we found that wall teichoic acids were necessary in both donors and recipients for efficient conjugative transfer of ICEBs1. Further, we found that depletion of wall teichoic acids caused cells involved in ICEBs1 conjugation to die, most likely from damage to the cell envelope. Our results indicate that wall teichoic acids help protect against envelope stress caused by active conjugation machines.

Examination of the horizontal gene transfer dynamics of an integrative and conjugative element encoding multidrug resistance in Histophilus somni.

Integrative and conjugative elements (ICEs) are self-transferable mobile genetic elements that play a significant role in disseminating antimicrobial resistance between bacteria via horizontal gene transfer. A recently identified ICE in a clinical isolate of Histophilus somni (ICEHs02) is 72 914 base pairs in length and harbours seven predicted antimicrobial resistance genes conferring resistance to tetracycline (tetR-tet(H)), florfenicol (floR), sulfonamide (Sul2), aminoglycosides (APH(3″)-Ib, APH(6)-Id, APH(3')-Ia), and copper (mco). This study investigated ICEHs02 host range, assessed effects of antimicrobial stressors on transfer frequency, and examined effects of ICEHs02 acquisition on hosts. Conjugation assays examined transfer frequency of ICEHs02 to H. somni and Pasteurella multocida strains. Polymerase chain reaction assays confirmed the presence of a circular intermediate, ICE-associated core genes, and cargo genes in recipient strains. Susceptibility testing examined ICEHs02-associated resistance phenotypes in recipient strains. Tetracycline and ciprofloxacin induction significantly increased the transfer rates of ICEHs02 in vitro. The copy numbers of the circular intermediate of ICEHs02 per chromosome exhibited significant increases of ∼37-fold after tetracycline exposure and ∼4-fold after ciprofloxacin treatment. The acquisition of ICEHs02 reduced the relative fitness of H. somni transconjugants (TG) by 28% (w = 0.72 ± 0.04) and the relative fitness of P. multocida TG was decreased by 15% (w = 0.85 ± 0.01).

Upregulation of the cfiA carbapenemase gene in a Bacteroides fragilis strain by the novel integrative and conjugative element Tn7563.

We describe Tn7563, a 31,844-bp integrative and conjugative element (ICE) carrying promoters upregulating the cfiA carbapenemase gene in Bacteroides fragilis strain Tbg-22. Excision and circularization of Tn7563 was demonstrated by PCR. Previously, only insertion sequences (IS) have been shown to carry mobile promoters for cfiA.

Second-Generation Transfer Mediates Efficient Propagation of ICEBs1 in Biofilms.

Horizontal gene transfer (HGT) by integrative and conjugative elements (ICEs) is an important mechanism in the spread of antibiotic resistance genes. However, little is known about the spatiotemporal dynamic of ICE propagation in bacterial biofilms, which are multicellular structures ubiquitous in natural and clinical environments. We report here that a high level of biofilm matrix production favors ICEBs1 acquisition. Also, using a fluorescently marked ICEBs1, we observed that conjugation appears restricted to clusters of bacteria in a close neighborhood in which a high level of ICEBs1 transfer occurs. These conjugative clusters are heterogeneously distributed in the biofilm, forming close to the air-biofilm interface. Importantly, we established that transconjugant cells are the main contributors to ICEBs1 propagation in biofilms. Our findings provide a novel spatiotemporal understanding of ICEs propagation in biofilms, which should have an important role in our understanding of horizontal gene transfer in relevant settings. IMPORTANCE The transfer of mobile genetic elements between bacteria is the main cause of the spread of antibiotic resistance genes. While biofilms are the predominant bacterial lifestyle both in the environment and in clinical settings, their impact on the propagation of mobile genetic elements is still poorly understood. In this study, we examined the spatiotemporal propagation of the well-characterized ICEBs1. Using the Gram-positive Bacillus subtilis, we observed that the main actors of ICEBs1 propagation in biofilms are the newly formed transconjugants that allow rapid transfer of ICEBs1 to new recipients. Our study provides a better understanding of the spatiotemporal dynamic of conjugative transfer in biofilms.

Specificity and Selective Advantage of an Exclusion System in the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis.

Integrative and conjugative elements (ICEs) are mobile genetic elements capable of transferring their own and other DNA. They contribute to the spread of antibiotic resistance and other important traits for bacterial evolution. Exclusion is a mechanism used by many conjugative plasmids and a few ICEs to prevent their host cell from acquiring a second copy of the cognate element. ICEBs1 of Bacillus subtilis has an exclusion mechanism whereby the exclusion protein YddJ in a potential recipient inhibits the activity of the ICEBs1-encoded conjugation machinery in a potential donor. The target of YddJ-mediated exclusion is the conjugation protein ConG (a VirB6 homolog). Here, we defined the regions of YddJ and ConG that confer exclusion specificity and determined the importance of exclusion to host cells. Using chimeras that had parts of ConG from ICEBs1 and the closely related ICEBat1, we identified a putative extracellular loop of ConG that conferred specificity for exclusion by the cognate YddJ. Using chimeras of YddJ from ICEBs1 and ICEBat1, we identified two regions in YddJ needed for exclusion specificity. We also found that YddJ-mediated exclusion reduced the death of donor cells following conjugation into recipients. Donor death was dependent on the ability of transconjugants to themselves become donors and was reduced under osmoprotective conditions, indicating that death was likely due to alterations in the donor cell envelope caused by excessive conjugation. We postulate that elements that can have high frequencies of transfer likely evolved exclusion mechanisms to protect the host cells from excessive death.IMPORTANCE Horizontal gene transfer is a driving force in bacterial evolution, responsible for the spread of many traits, including antibiotic and heavy metal resistance. Conjugation, one type of horizontal gene transfer, involves DNA transfer from donor to recipient cells through conjugation machinery and direct cell-cell contact. Exclusion mechanisms allow conjugative elements to prevent their host from acquiring additional copies of the element and are highly specific, enabling hosts to acquire heterologous elements. We defined regions of the exclusion protein and its target in the conjugation machinery that convey high specificity of exclusion. We found that exclusion protects donors from cell death during periods of high transfer. This is likely important for the element to enter new populations of cells.

Characterization of TMexCD3-TOprJ3, an RND-Type Efflux System Conferring Resistance to Tigecycline in Proteus mirabilis, and Its Associated Integrative Conjugative Element.

The emergence and transmission of novel antimicrobial resistance genes pose a great threat to public health globally. Recently, the plasmid-encoding RND efflux pump TMexCD1-TOprJ1 in Klebsiella pneumoniae was reported to reduce the sensitivity of multiple antimicrobials. Here, we identified a pandrug-resistant Proteus mirabilis isolate that harbored the novel tmexCD3-toprJ3 gene cluster located on SXT/R391 ICE. This study expands current knowledge of the transfer mechanisms of tmexCD1-toprJ1-like gene clusters among P. mirabilis isolates and warrants further genomic epidemiology investigations.

Antimicrobial resistance phenotypes and genotypes of Streptococcus suis isolated from clinically healthy pigs from 2017 to 2019 in Jiangxi Province, China.

AIMS: This study aimed to investigate the antimicrobial resistance (AMR) profiles and genotypes of Streptococcus suis from Jiangxi Province, China. METHODS AND RESULTS: A total of 314 nasal swab samples were collected from clinically healthy pigs, with a positive isolation rate of S. suis of 34·08%. Antimicrobial susceptibility testing showed that more than 80% of the isolates were susceptible to vancomycin, penicillin, minocycline and chloramphenicol. A high frequency of resistance to clindamycin, tetracycline, clarithromycin and erythromycin was observed. All of the isolates were resistant to three or more categories of antimicrobials. The erm(B) and tet(O) served as the most frequent genotypes that contributed to lincosamide, macrolide and tetracycline resistances. A part of macrolide-resistant genotypes could not exhibit specific phenotypes. Finally, integrative and conjugative elements (ICEs) were identified in 28·97% of the isolates. CONCLUSIONS: The multidrug resistance of S. suis has widely emerged in Jiangxi Province. The most prevalent resistance genes and genotypes were similar to those in other regions or countries. The presence of ICEs is increasing the risk of horizontal transfer of AMR genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings could provide guidance for the rational use of antimicrobial drugs and be helpful for monitoring the AMR information of S. suis in China.

Antibiotic resistance in Pseudomonas aeruginosa - Mechanisms, epidemiology and evolution.

Antibiotics are powerful drugs used in the treatment of bacterial infections. The inappropriate use of these medicines has driven the dissemination of antibiotic resistance (AR) in most bacteria. Pseudomonas aeruginosa is an opportunistic pathogen commonly involved in environmental- and difficult-to-treat hospital-acquired infections. This species is frequently resistant to several antibiotics, being in the "critical" category of the WHO's priority pathogens list for research and development of new antibiotics. In addition to a remarkable intrinsic resistance to several antibiotics, P. aeruginosa can acquire resistance through chromosomal mutations and acquisition of AR genes. P. aeruginosa has one of the largest bacterial genomes and possesses a significant assortment of genes acquired by horizontal gene transfer (HGT), which are frequently localized within integrons and mobile genetic elements (MGEs), such as transposons, insertion sequences, genomic islands, phages, plasmids and integrative and conjugative elements (ICEs). This genomic diversity results in a non-clonal population structure, punctuated by specific clones that are associated with significant morbidity and mortality worldwide, the so-called high-risk clones. Acquisition of MGEs produces a fitness cost in the host, that can be eased over time by compensatory mutations during MGE-host coevolution. Even though plasmids and ICEs are important drivers of AR, the underlying evolutionary traits that promote this dissemination are poorly understood. In this review, we provide a comprehensive description of the main strategies involved in AR in P. aeruginosa and the leading drivers of HGT in this species. The most recently developed genomic tools that allowed a better understanding of the features contributing for the success of P. aeruginosa are discussed.

Lower Respiratory Tract Microbiome and Resistome of Bovine Respiratory Disease Mortalities.

Bovine respiratory disease (BRD) continues to be a serious health problem in beef cattle production. A multifactorial condition, BRD encompasses several types of pneumonia that are associated with multiple viral and bacterial agents. Comprehensive identification of microbes associated with BRD fatalities could enhance our understanding of the range of pathogens that contribute to the disease and identify new therapeutic targets. This study used metagenomic analysis to describe the lower respiratory tract microbiome and resistome of 15 feedlot cattle BRD and 3 non-BRD mortalities along with any affiliated integrative and conjugative elements (ICEs). Known bacterial pathogens associated with BRD, including Histophilus somni, Mannheimia haemolytica, and Mycoplasma bovis, were relatively abundant (> 5%) in most, but not all samples. Other relatively abundant genera (> 1%) included Acinetobacter, Bacillus, Bacteroides, Clostridium, Enterococcus, and Pseudomonas. Antimicrobial resistance genes (ARGs) comprised up to 0.5% of sequences and many of these genes were associated with ICEs previously described within the Pasteurellaceae family. A total of 20 putative ICEs were detected among 16 samples. These results document the wide diversity of microorganisms in the lower respiratory tract of cattle that have succumbed to BRD. The data also strongly suggest that antimicrobial-resistant Pasteurellaceae strains are prevalent in BRD cases in Alberta and that the resistance observed is associated with ICEs. The presence of ICEs harboring a wide array of ARGs holds significant consequence for the effectiveness of drug therapies for the control of BRD in beef cattle.

Assembly and transfer of tripartite integrative and conjugative genetic elements.

Integrative and conjugative elements (ICEs) are ubiquitous mobile genetic elements present as "genomic islands" within bacterial chromosomes. Symbiosis islands are ICEs that convert nonsymbiotic mesorhizobia into symbionts of legumes. Here we report the discovery of symbiosis ICEs that exist as three separate chromosomal regions when integrated in their hosts, but through recombination assemble as a single circular ICE for conjugative transfer. Whole-genome comparisons revealed exconjugants derived from nonsymbiotic mesorhizobia received three separate chromosomal regions from the donor Mesorhizobium ciceri WSM1271. The three regions were each bordered by two nonhomologous integrase attachment (att) sites, which together comprised three homologous pairs of attL and attR sites. Sequential recombination between each attL and attR pair produced corresponding attP and attB sites and joined the three fragments to produce a single circular ICE, ICEMcSym1271 A plasmid carrying the three attP sites was used to recreate the process of tripartite ICE integration and to confirm the role of integrase genes intS, intM, and intG in this process. Nine additional tripartite ICEs were identified in diverse mesorhizobia and transfer was demonstrated for three of them. The transfer of tripartite ICEs to nonsymbiotic mesorhizobia explains the evolution of competitive but suboptimal N2-fixing strains found in Western Australian soils. The unheralded existence of tripartite ICEs raises the possibility that multipartite elements reside in other organisms, but have been overlooked because of their unusual biology. These discoveries reveal mechanisms by which integrases dramatically manipulate bacterial genomes to allow cotransfer of disparate chromosomal regions.

Networking in microbes: conjugative elements and plasmids in the genus Alteromonas.

BACKGROUND: To develop evolutionary models for the free living bacterium Alteromonas the genome sequences of isolates of the genus have been extensively analyzed. However, the main genetic exchange drivers in these microbes, conjugative elements (CEs), have not been considered in detail thus far. In this work, CEs have been searched in several complete Alteromonas genomes and their sequence studied to understand their role in the evolution of this genus. Six genomes are reported here for the first time. RESULTS: We have found nine different plasmids of sizes ranging from 85 to 600 Kb, most of them were found in a single strain. Networks of gene similarity could be established among six of the plasmids that were also connected with another cluster of plasmids found in Shewanella strains. The cargo genes found in these plasmids included cassettes found before in chromosome flexible genomic islands of Alteromonas strains. We describe also the plasmids pAMCP48-600 and pAMCP49-600, the largest found in Alteromonas thus far (ca. 600 Kb) and containing all the hallmarks to be classified as chromids. We found in them some housekeeping genes and a cluster that code for an exocellular polysaccharide. They could represent the transport vectors for the previously described replacement flexible genomic islands. Integrative and conjugative elements (ICEs) were more common than plasmids and showed similar patterns of variation with cargo genes coding for components of additive flexible genomic islands. A nearly identical ICE was found in A. mediterranea MED64 and Vibrio cholera AHV1003 isolated from a human pathogen, indicating the potential exchange of these genes across phylogenetic distances exceeding the family threshold. CONCLUSION: We have seen evidence of how CEs can be vectors to transfer gene cassettes acquired in the chromosomal flexible genomic islands, both of the additive and replacement kind. These CEs showed evidence of how genetic material is exchanged among members of the same species but also (albeit less frequently) across genus and family barriers. These gradients of exchange frequency are probably one of the main drivers of species origin and maintenance in prokaryotes and also provide these taxa with large genetic diversity.

Diversity of Integrative and Conjugative Elements of Streptococcus salivarius and Their Intra- and Interspecies Transfer.

Integrative and conjugative elements (ICEs) are widespread chromosomal mobile genetic elements which can transfer autonomously by conjugation in bacteria. Thirteen ICEs with a conjugation module closely related to that of ICESt3 of Streptococcus thermophilus were characterized in Streptococcus salivarius by whole-genome sequencing. Sequence comparison highlighted ICE evolution by shuffling of 3 different integration/excision modules (for integration in the 3' end of the fda, rpsI, or rpmG gene) with the conjugation module of the ICESt3 subfamily. Sequence analyses also pointed out a recombination occurring at oriT (likely mediated by the relaxase) as a mechanism of ICE evolution. Despite a similar organization in two operons including three conserved genes, the regulation modules show a high diversity (about 50% amino acid sequence divergence for the encoded regulators and presence of unrelated additional genes) with a probable impact on the regulation of ICE activity. Concerning the accessory genes, ICEs of the ICESt3 subfamily appear particularly rich in restriction-modification systems and orphan methyltransferase genes. Other cargo genes that could confer a selective advantage to the cell hosting the ICE were identified, in particular, genes for bacteriocin synthesis and cadmium resistance. The functionality of 2 ICEs of S. salivarius was investigated. Autonomous conjugative transfer to other S. salivarius strains, to S. thermophilus, and to Enterococcus faecalis was observed. The analysis of the ICE-fda border sequence in these transconjugants allowed the localization of the DNA cutting site of the ICE integrase.IMPORTANCE The ICESt3 subfamily of ICEs appears to be widespread in streptococci and targets diverse chromosomal integration sites. These ICEs carry diverse cargo genes that can confer a selective advantage to the host strain. The maintenance of these mobile genetic elements likely relies in part on self-encoded restriction-modification systems. In this study, intra- and interspecies transfer was demonstrated for 2 ICEs of S. salivarius Closely related ICEs were also detected in silico in other Streptococcus species (S. pneumoniae and S. parasanguinis), thus indicating that diffusion of ICESt3-related elements probably plays a significant role in horizontal gene transfer (HGT) occurring in the oral cavity but also in the digestive tract, where S. salivarius is present.

Analysis of Streptococcus agalactiae pan-genome for prevalence, diversity and functionality of integrative and conjugative or mobilizable elements integrated in the tRNA(Lys CTT) gene.

Streptococcus agalactiae is the first cause of invasive infections in human neonates and is also a major bovine and fish pathogen. High genomic diversity was observed in this species that hosts numerous mobile genetic elements, in particular elements transferable by conjugation. This works aims to evaluate the contribution of these elements to GBS genome diversity. Focusing on genomic islands integrated in the tRNA(Lys) (CTT) gene, a known hotspot of recombination, an extensive in silico search was performed on the sequenced genome of 303 strains of S. agalactiae isolated from different hosts. In all the isolates (except 9), whatever their origin (human, bovine, camel, dog, gray seal, dolphin, fish species or bullfrog), this locus carries highly diverse genomic islands transferable by conjugation such as integrative and conjugative elements (ICEs), integrative and mobilizable elements (IMEs), CIs-mobilizable elements (CIMEs) or composite elements. Transfer of an ICE from an ST67 bovine strain to a phylogenetically distant ST23 human isolate was obtained experimentally indicating that there was no barrier to ICE transfer between strains from different hosts. Interestingly, a novel family of putative IMEs that site-specifically integrate in the nic site of oriT of ICEs belonging to Tn916/ICESt3 superfamily was detected in silico. These elements carry an antibiotic resistance gene (lsa(C)) already described to confer cross-resistance to lincosamides, streptogramins A and pleuromutilins. Further work is needed to evaluate the impact of these IMEs on the transfer of targeted ICEs and the mobility and the dissemination of these IMEs.

Degenerate primer MOB typing of multiresistant clinical isolates of E. coli uncovers new plasmid backbones.

Degenerate Primer MOB Typing is a PCR-based protocol for the classification of γ-proteobacterial transmissible plasmids in five phylogenetic relaxase MOB families. It was applied to a multiresistant E. coli collection, previously characterized by PCR-based replicon-typing, in order to compare both methods. Plasmids from 32 clinical isolates of multiresistant E. coli (19 extended spectrum beta-lactamase producers and 13 non producers) and their transconjugants were analyzed. A total of 95 relaxases were detected, at least one per isolate, underscoring the high potential of these strains for antibiotic-resistance transmission. MOBP12 and MOBF12 plasmids were the most abundant. Most MOB subfamilies detected were present in both subsets of the collection, indicating a shared mobilome among multiresistant E. coli. The plasmid profile obtained by both methods was compared, which provided useful data upon which decisions related to the implementation of detection methods in the clinic could be based. The phylogenetic depth at which replicon and MOB-typing classify plasmids is different. While replicon-typing aims at plasmid replication regions with non-degenerate primers, MOB-typing classifies plasmids into relaxase subfamilies using degenerate primers. As a result, MOB-typing provides a deeper phylogenetic depth than replicon-typing and new plasmid groups are uncovered. Significantly, MOB typing identified 17 plasmids and an integrative and conjugative element, which were not detected by replicon-typing. Four of these backbones were different from previously reported elements.

Integrative and conjugative elements of Pasteurella multocida: Prevalence and signatures in population evolution.

Pasteurella multocida (P. multocida) is a bacterial pathogen responsible for a range of infections in humans and various animal hosts, causing significant economic losses in farming. Integrative and conjugative elements (ICEs) are important horizontal gene transfer elements, potentially enabling host bacteria to enhance adaptability by acquiring multiple functional genes. However, the understanding of ICEs in P. multocida and their impact on the transmission of this pathogen remains limited. In this study, 42 poultry-sourced P. multocida genomes obtained by high-throughput sequencing together with 393 publicly available P. multocida genomes were used to analyse the horizontal transfer of ICEs. Eighty-two ICEs were identified in P. multocida, including SXT/R391 and Tn916 subtypes, as well as three subtypes of ICEHin1056 family, with the latter being widely prevalent in P. multocida and carrying multiple resistance genes. The correlations between insertion sequences and resistant genes in ICEs were also identified, and some ICEs introduced the carbapenem gene blaOXA-2 and the bleomycin gene bleO to P. multocida. Phylogenetic and collinearity analyses of these bioinformatics found that ICEs in P. multocida were transmitted vertically and horizontally and have evolved with host specialization. These findings provide insight into the transmission and evolution mode of ICEs in P. multocida and highlight the importance of understanding these elements for controlling the spread of antibiotic resistance.

Evolution of the Tn4371 ICE family: traR-mediated coordination of cargo gene upregulation and horizontal transfer.

ICEKKS102Tn4677 carries a bph operon for the mineralization of polychlorinated biphenyls (PCBs)/biphenyl and belongs to the Tn4371 ICE (integrative and conjugative element) family. In this study, we investigated the role of the traR gene in ICE transfer. The traR gene encodes a LysR-type transcriptional regulator, which is conserved in sequence, positioning, and directional orientation among Tn4371 family ICEs. The traR belongs to the bph operon, and its overexpression on solid medium resulted in modest upregulation of traG (threefold), marked upregulation of xis (80-fold), enhanced ICE excision and, most notably, ICE transfer frequency. We propose the evolutional roles of traR, which upon insertion to its current position, might have connected the cargo gene activation and ICE transfer. This property of ICE, i.e., undergoing transfer under environmental conditions that lead to cargo gene activation, would instantly confer fitness advantages to bacteria newly acquiring this ICE, thereby resulting in efficient dissemination of the Tn4371 family ICEs.IMPORTANCEOnly ICEKKS102Tn4677 is proven to transfer among the widely disseminating Tn4371 family integrative and conjugative elements (ICEs) from ß and γ-proteobacteria. We showed that the traR gene in ICEKKS102Tn4677, which is conserved in the ICE family with fixed location and direction, is co-transcribed with the cargo gene and activates ICE transfer. We propose that capturing of traR by an ancestral ICE to the current position established the Tn4371 family of ICEs. Our findings provide insights into the evolutionary processes that led to the widespread distribution of the Tn4371 family of ICEs across bacterial species.

Macrolide Resistance in the Aerococcus urinae Complex: Implications for Integrative and Conjugative Elements.

The recognition of the Aerococcus urinae complex (AUC) as an emerging uropathogen has led to growing concerns due to a limited understanding of its disease spectrum and antibiotic resistance profiles. Here, we investigated the prevalence of macrolide resistance within urinary AUC isolates, shedding light on potential genetic mechanisms. Phenotypic testing revealed a high rate of macrolide resistance: 45%, among a total of 189 urinary AUC isolates. Genomic analysis identified integrative and conjugative elements (ICEs) as carriers of the macrolide resistance gene ermA, suggesting horizontal gene transfer as a mechanism of resistance. Furthermore, comparison with publicly available genomes of related pathogens revealed high ICE sequence homogeneity, highlighting the potential for cross-species dissemination of resistance determinants. Understanding mechanisms of resistance is crucial for developing effective surveillance strategies and improving antibiotic use. Furthermore, the findings underscore the importance of considering the broader ecological context of resistance dissemination, emphasizing the need for community-level surveillance to combat the spread of antibiotic resistance within the urinary microbiome.

Streptococcus suis serotype 4: a population with the potential pathogenicity in humans and pigs.

Streptococcus suis is a major bacterial pathogen in pigs and an emerging zoonotic pathogen. Different S. suis serotypes exhibit diverse characteristics in population structure and pathogenicity. Surveillance data highlight the significance of S. suis serotype 4 (SS4) in swine streptococcusis, a pathotype causing human infections. However, except for a few epidemiologic studies, the information on SS4 remains limited. In this study, we investigated the population structure, pathogenicity, and antimicrobial characteristics of SS4 based on 126 isolates, including one from a patient with septicemia. We discovered significant diversities within this population, clustering into six minimum core genome (MCG) groups (1, 2, 3, 4, 7-2, and 7-3) and five lineages. Two main clonal complexes (CCs), CC17 and CC94, belong to MCG groups 1 and 3, respectively. Numerous important putative virulence-associated genes are present in these two MCG groups, and 35.00% (7/20) of pig isolates from CC17, CC94, and CC839 (also belonging to MCG group 3) were highly virulent (mortality rate ≥ 80%) in zebrafish and mice, similar to the human isolate ID36054. Cytotoxicity assays showed that the human and pig isolates of SS4 strains exhibit significant cytotoxicity to human cells. Antimicrobial susceptibility testing showed that 95.83% of strains isolated from our labs were classified as multidrug-resistant. Prophages were identified as the primary vehicle for antibiotic resistance genes. Our study demonstrates the public health threat posed by SS4, expanding the understanding of SS4 population structure and pathogenicity characteristics and providing valuable information for its surveillance and prevention.