Spread Of Antimicrobial Resistance Genes Research Articles

How Gut Microbiome Perturbation Caused by Antibiotic Pre-Treatments Affected the Conjugative Transfer of Antimicrobial Resistance Genes

The global spread of antimicrobial resistance genes (ARGs) poses a significant threat to public health. While antibiotics effectively treat bacterial infections, they can also induce gut dysbiosis, the severity of which varies depending on the specific antibiotic treatment used. However, it remains unclear how gut dysbiosis affects the mobility and dynamics of ARGs. To address this, mice were pre-treated with streptomycin, ampicillin, or sulfamethazine, and then orally inoculated with Salmonella enterica serovar Typhimurium and S. Heidelberg carrying a multi-drug resistance IncA/C plasmid. The streptomycin pre-treatment caused severe microbiome perturbation, promoting the high-density colonization of S. Heidelberg and S. Typhimurium, and enabling an IncA/C transfer from S. Heidelberg to S. Typhimurium and a commensal Escherichia coli. The ampicillin pre-treatment induced moderate microbiome perturbation, supporting only S. Heidelberg colonization and the IncA/C transfer to commensal E. coli. The sulfamethazine pre-treatment led to mild microbiome perturbation, favoring neither Salmonella spp. colonization nor a conjugative plasmid transfer. The degree of gut dysbiosis also influenced the enrichment or depletion of the ARGs associated with mobile plasmids or core commensal bacteria, respectively. These findings underscore the significance of pre-existing gut dysbiosis induced by various antibiotic treatments on ARG dissemination and may inform prudent antibiotic use practices.

Evaluation of a DNA vaccine with self-designed CpG sequences against J genotype IHNV infection in rainbow trout (Oncorhynchus mykiss)

Rainbow trout suffer from infectious hematopoietic necrosis virus (IHNV) outbreaks, which lead to massive mortality and huge economic loss worldwide. The approved commercial vaccine is used for the prevention of IHN in Canada. Given that Chinese domestic J-genotype isolates are different from North American IHNV isolates, the development of an effective DNA vaccine against Chinese J-genotype isolates is urgent. In this study, we developed a DNA vaccine encoding glycoprotein based on our previously isolated IHNV GS21 strain and self-designed CpG sequences were supplemented as molecular adjuvants. The vaccinated rainbow trout were significantly protected against IHNV with approximately a relative percent survival (RPS) of 94.74 % compared to the unvaccinated group. Moreover, the specific antibody of IgM and neutralizing antibody (NAb) was significantly provoked after the vaccination. Particularly, the antiviral immune response was rapidly evoked in the early stage of vaccination including the up-regulation of Mx-1, IFN-Ⅰ, and IFN-γ. The IHNV load in vaccinated fish was apparently lower than that in the unvaccinated group. Furthermore, the integration of exogenous genes into the host chromosome and the spread of antimicrobial-resistant genes were not found. These results suggested that our vaccine enhances robust immune responses and evokes considerable protection against IHNV with limited genetically modified risk, which is an effective and promising vaccine candidate for further prevention of IHNV outbreaks.

Exploring the Role of the Environment as a Reservoir of Antimicrobial-Resistant Campylobacter: Insights from Wild Birds and Surface Waters.

Antimicrobial resistance (AMR) is a growing global health challenge, compromising bacterial infection treatments and necessitating robust surveillance and mitigation strategies. The overuse of antimicrobials in humans and farm animals has made them hotspots for AMR. However, the spread of AMR genes in wildlife and the environment represents an additional challenge, turning these areas into new AMR hotspots. Among the AMR bacteria considered to be of high concern for public health, Campylobacter has been the leading cause of foodborne infections in the European Union since 2005. This study examines the prevalence of AMR genes and virulence factors in Campylobacter isolates from wild birds and surface waters in Luxembourg. The findings reveal a significant prevalence of resistant Campylobacter strains, with 12% of C. jejuni from wild birds and 37% of C. coli from surface waters carrying resistance genes, mainly against key antibiotics like quinolones and tetracycline. This study underscores the crucial role of the environment in the spread of AMR bacteria and genes, highlighting the urgent need for enhanced surveillance and control measures to curb AMR in wildlife and environmental reservoirs and reduce transmission risks to humans. This research supports One Health approaches to tackling antimicrobial resistance and protecting human, animal, and environmental health.

Compensatory evolution of chromosomes and plasmids counteracts the plasmid fitness cost.

Plasmids incur a fitness cost that has the potential to restrict the dissemination of resistance in bacterial pathogens. However, bacteria can overcome this disadvantage by compensatory evolution to maintain their resistance. Compensatory evolution can occur via both chromosomes and plasmids, but there are a few reviews regarding this topic, and most of them focus on plasmids. In this review, we provide a comprehensive overview of the currently reported mechanisms underlying compensatory evolution on chromosomes and plasmids, elucidate key targets regulating plasmid fitness cost, and discuss future challenges in this field. We found that compensatory evolution on chromosomes primarily arises from mutations in transcriptional regulatory factors, whereas compensatory evolution of plasmids predominantly involves three pathways: plasmid copy number regulation, conjugation transfer efficiency, and expression of antimicrobial resistance (AMR) genes. Furthermore, the importance of reasonable selection of research subjects and effective integration of diverse advanced research methods is also emphasized in our future study on compensatory mechanisms. Overall, this review establishes a theoretical framework that aims to provide innovative ideas for minimizing the emergence and spread of AMR genes.

Effect of chemotherapeutic agents on natural transformation frequency in Acinetobacter baylyi.

Natural transformation is the ability of a bacterial cell to take up extracellular DNA which is subsequently available for recombination into the chromosome (or maintenance as an extrachromosomal element). Like other mechanisms of horizontal gene transfer, natural transformation is a significant driver for the dissemination of antimicrobial resistance. Recent studies have shown that many pharmaceutical compounds such as antidepressants and anti-inflammatory drugs can upregulate transformation frequency in the model species Acinetobacter baylyi. Chemotherapeutic compounds have been shown to increase the abundance of antimicrobial resistance genes and increase colonization rates of potentially pathogenic bacteria in patient gastrointestinal tracts, indicating an increased risk of infection and providing a pool of pathogenicity or resistance genes for transformable commensal bacteria. We here test for the effect of six cancer chemotherapeutic compounds on A. baylyi natural transformation frequency, finding two compounds, docetaxel and daunorubicin, to significantly decrease transformation frequency, and daunorubicin to also decrease growth rate significantly. Enhancing our understanding of the effect of chemotherapeutic compounds on the frequency of natural transformation could aid in preventing the horizontal spread of antimicrobial resistance genes.

Characterization and Abundance of Plasmid-Dependent Alphatectivirus Bacteriophages

Antimicrobial resistance (AMR) is a major public health threat, exacerbated by the ability of bacteria to rapidly disseminate antimicrobial resistance genes (ARG). Since conjugative plasmids of the incompatibility group P (IncP) are ubiquitous mobile genetic elements that often carry ARG and are broad-host-range, they are important targets to prevent the dissemination of AMR. Plasmid-dependent phages infect plasmid-carrying bacteria by recognizing components of the conjugative secretion system as receptors. We sought to isolate plasmid-dependent phages from wastewater using an avirulent strain of Salmonella enterica carrying the conjugative IncP plasmid pKJK5. Irrespective of the site, we only obtained bacteriophages belonging to the genus Alphatectivirus. Eleven isolates were sequenced, their genomes analyzed, and their host range established using S. enterica, Escherichia coli, and Pseudomonas putida carrying diverse conjugative plasmids. We confirmed that Alphatectivirus are abundant in domestic and hospital wastewater using culture-dependent and culture-independent approaches. However, these results are not consistent with their low or undetectable occurrence in metagenomes. Therefore, overall, our results emphasize the importance of performing phage isolation to uncover diversity, especially considering the potential of plasmid-dependent phages to reduce the spread of ARG carried by conjugative plasmids, and to help combat the AMR crisis.

In Silico Detection of Integrons and Their Relationship with Resistance Phenotype of Salmonella Isolates from a Brazilian Pork Production Chain.

The pork production chain is an important reservoir of antimicrobial resistant bacteria. This study identified and characterized integrons in Salmonella isolates from a Brazilian pork production chain and associate them with their antibiotic resistance pattern. A total of 41 whole-genome sequencing data of nontyphoidal Salmonella were analyzed using PlasmidSPAdes and IntegronFinder software. Nine isolates (21.9%) had some integrons identified (complete and/or incomplete). Six complete class 1 integrons were found, with streptomycin resistance genes (aadA1, aadA2) alone or downstream of a trimethoprim resistance gene (dfrA1, dfrA12), and some also containing resistance genes for sulfonamides (sul1, sul3) and chloramphenicol (cmlA1). Class 2 integron was detected in only one isolate, containing dfrA1-sat2-aadA1 gene cassettes. Five isolates harbored CALINs-clusters attC but lacking integrases-with antimicrobial resistance genes typically found in integron structures. In all, integrons were observed among four serotypes: Derby, Bredeney, Panama, and monophasic var. Typhimurium I 4,[5],12:i:-. The association of integrons with antibiotic resistance phenotype showed that these elements were predominantly identified in multidrug resistance isolates, and six of the seven gentamicin-resistant isolates had integrons. So, surveillance of integrons in Salmonella should be performed to identify the potential for the spread of antimicrobial resistance genes among bacteria.

Dispersion of antimicrobial resistant bacteria in pig farms and in the surrounding environment

BackgroundAntimicrobial resistance has been identified as a major threat to global health. The pig food chain is considered an important source of antimicrobial resistance genes (ARGs). However, there is still a lack of knowledge on the dispersion of ARGs in pig production system, including the external environment.ResultsIn the present study, we longitudinally followed one swine farm located in Italy from the weaning phase to the slaughterhouse to comprehensively assess the diversity of ARGs, their diffusion, and the bacteria associated with them. We obtained shotgun metagenomic sequences from 294 samples, including pig feces, farm environment, soil around the farm, wastewater, and slaughterhouse environment. We identified a total of 530 species-level genome bins (SGBs), which allowed us to assess the dispersion of microorganisms and their associated ARGs in the farm system. We identified 309 SGBs being shared between the animals gut microbiome, the internal and external farm environments. Specifically, these SGBs were characterized by a diverse and complex resistome, with ARGs active against 18 different classes of antibiotic compounds, well matching antibiotic use in the pig food chain in Europe.ConclusionsCollectively, our results highlight the urgency to implement more effective countermeasures to limit the dispersion of ARGs in the pig food systems and the relevance of metagenomics-based approaches to monitor the spread of ARGs for the safety of the farm working environment and the surrounding ecosystems.

Spread and persistence of antimicrobial resistance genes in wastewater from human and animal sources in São Paulo, Brazil.

The spread of antimicrobial resistance (AMR) through multiple reservoirs is a global concern. Wastewater is a critical AMR dissemination source, so this study aimed to assess the persistence of resistance genetic markers in wastewater using a culture-independent approach. Raw and treated wastewater samples (n = 121) from a wastewater treatment plant (WWTP), a human hospital, a veterinary hospital, and a pig farm were monthly collected and concentrated by filtration. DNA was extracted directly from filter membranes, and PCR was used in the qualitative search of 32 antimicrobial resistance genes (ARGs). Selected genes (blaCTX-M, blaKPC, qnrB, and mcr-1) were enumerated by quantitative real-time PCR (qPCR). Twenty-six ARGs were detected in the qualitative ARGs search, while quantitative data showed a low variation of the ARG's relative abundance (RA) throughout the months, especially at the human hospital and the WWTP. At the WWTP, despite significantly reducing the absolute number of gene copies/L after each treatment stage (p < 0.05), slight increases (p > 0.05) in the RAs of genes blaCTX-M, qnrB, and mcr-1 were observed in reused water (tertiary treatment) when compared with secondary effluent. Although the increase is not statistically significant, it is worth noting that there was some level of ARGs concentration after the disinfection process. No significant absolute or relative after-treatment quantification reductions were observed for any ARGs at the veterinary hospital or the pig farm. The spread of ARGs through sewage needs to be continuously addressed, because their release into natural environments may pose potential risks of exposure to resistant bacteria and impact local ecosystems.

Understanding the rapid spread of antimicrobial resistance genes mediated by IS26.

Insertion sequences (ISs) promote the transmission of antimicrobial resistance genes (ARGs) across bacterial populations. However, their contributions and dynamics during the transmission of resistance remain unclear. In this study, we selected IS26 as a representative transposable element to decipher the relationship between ISs and ARGsand to investigate their transfer features and transmission trends. We retrieved 2656 translocatable IS 26 -bounded units with ARGs (tIS26-bUs-ARGs) in complete bacterial genomes from theNCBI RefSeq database. In total, 124 ARGs spanning 12 classes of antibiotics were detected, and the average contribution rate of IS26 to these genes was 41.2%. We found that IS 26 -bounded units (IS26-bUs) mediated extensive ARG dissemination within the bacteria of theGammaproteobacteria class, showing strong transfer potential between strains, species, and even phyla. The IS26-bUs expanded in bacterial populations over time, and their temporal expansion trend was significantly correlated with antibiotic usage. This wide dissemination could be due to the nonspecific target site preference of IS26. Finally, we experimentally confirmed that the introduction of a single copy of IS26 could lead to the formation of a composite transposon mediating the transmission of "passenger" genes. These observations extend our knowledge of the IS26and provide new insights into the mediating role of ISs in the dissemination of antibiotic resistance.

Contrary effects of increasing temperatures on the spread of antimicrobial resistance in river biofilms.

River microbial communities regularly act as the first barrier of defense against the spread of antimicrobial resistance genes (ARGs) that enter environmental microbiomes through wastewater. However, how the invasion dynamics of wastewater-borne ARGs into river biofilm communities will shift due to climate change with increasing average and peak temperatures remains unknown. Here, we aimed to elucidate the effects of increasing temperatures on the naturally occurring river biofilm resistome, as well as the invasion success of foreign ARGs entering through wastewater. Natural biofilms were grown in a low-anthropogenic impact river and transferred to artificial laboratory recirculation flume systems operated at three different temperatures (20°C, 25°C, and 30°C). After 1 week of temperature acclimatization, significant increases in the abundance of the naturally occurring ARGs in biofilms were detected at higher temperatures. After this acclimatization period, biofilms were exposed to a single pulse of wastewater, and the invasion dynamics of wastewater-borne ARGs were analyzed over 2 weeks. After 1 day, wastewater-borne ARGs were able to invade the biofilms successfully with no observable effect of temperature on their relative abundance. However, thereafter, ARGs were lost at a far increased rate at 30°C, with ARG levels dropping to the initial natural levels after 14 days. Contrary to the lower temperatures, ARGs were either lost at slower rates or even able to establish themselves in biofilms with stable relative abundances above natural levels. Hence, higher temperatures come with contrary effects on river biofilm resistomes: naturally occurring ARGs increase in abundance, while foreign, invading ARGs are lost at elevated speeds.IMPORTANCEInfections with bacteria that gained resistance to antibiotics are taking millions of lives annually, with the death toll predicted to increase. River microbial communities act as a first defense barrier against the spread of antimicrobial resistance genes (ARGs) that enter the environment through wastewater after enrichment in human and animal microbiomes. The global increase in temperature due to climate change might disrupt this barrier effect by altering microbial community structure and functions. We consequently explored how increasing temperatures alter ARG spread in river microbial communities. At higher temperatures, naturally occurring ARGs increased in relative abundance. However, this coincided with a decreased success rate of invading foreign ARGs from wastewater to establish themselves in the communities. Therefore, to predict the effects of climate change on ARG spread in river microbiomes, it is imperative to consider if the river ecosystem and its resistome are dominated by naturally occurring or invading foreign ARGs.

Host- plasmid network structure in wastewater is linked to antimicrobial resistance genes

As mobile genetic elements, plasmids are central for our understanding of antimicrobial resistance spread in microbial communities. Plasmids can have varying fitness effects on their host bacteria, which will markedly impact their role as antimicrobial resistance vectors. Using a plasmid population model, we first show that beneficial plasmids interact with a higher number of hosts than costly plasmids when embedded in a community with multiple hosts and plasmids. We then analyse the network of a natural host-plasmid wastewater community from a Hi-C metagenomics dataset. As predicted by the model, we find that antimicrobial resistance encoding plasmids, which are likely to have positive fitness effects on their hosts in wastewater, interact with more bacterial taxa than non-antimicrobial resistance plasmids and are disproportionally important for connecting the entire network compared to non- antimicrobial resistance plasmids. This highlights the role of antimicrobials in restructuring host-plasmid networks by increasing the benefits of antimicrobial resistance carrying plasmids, which can have consequences for the spread of antimicrobial resistance genes through microbial networks. Furthermore, that antimicrobial resistance encoding plasmids are associated with a broader range of hosts implies that they will be more robust to turnover of bacterial strains.

Prevalence of diverse antimicrobial resistance genes and bacteria in sewage treatment plant-derived sludge environment.

Antimicrobial resistance (AMR) contamination in the environment is one of the most significant worldwide threats of the 21st century. Since sludge is heavily exposed to diverse contaminants, including pharmaceuticals, the inhabitant bacterial population is expected to exhibit resistance to antimicrobial agents. In this study, sewage treatment plant (STP) sludge samples were analyzed to assess the antibiotic-resistant bacterial population, abundance of AMR genes (ermF, qnrS, Sul1, blaGES, blaCTX-M, and blaNDM), and mobile genetic elements (intl1 and IS26). Out of 16, six bacterial isolates exhibited resistance to 13 antibiotics with a high multiple antibiotic resistance index (MARI) (0.93) and high metal tolerance. Quantitative polymerase chain reaction showed the abundance of target genes ranging from 6.6×103 to 6.5×108 copies g-1 sludge. The overall outcome reveals that STP sludge comprised varied multidrug-resistant bacterial populations. It will give insights into the functions of heavy metals and biofilm development in the selection and spread of AMR genes and the associated bacteria. Therefore, the application of sludge needs proper screening for AMR and metal contamination prior to its countless applications. This study will contribute immensely to the risk analysis of STP effluents on environmental health, including control of AMR transmission.

Microbial evolution through horizontal gene transfer by mobile genetic elements.

Mobile genetic elements (MGEs) are crucial for horizontal gene transfer (HGT) in bacteria and facilitate their rapid evolution and adaptation. MGEs include plasmids, integrative and conjugative elements, transposons, insertion sequences and bacteriophages. Notably, the spread of antimicrobial resistance genes (ARGs), which poses a serious threat to public health, is primarily attributable to HGT through MGEs. This mini-review aims to provide an overview of the mechanisms by which MGEs mediate HGT in microbes. Specifically, the behaviour of conjugative plasmids in different environments and conditions was discussed, and recent methodologies for tracing the dynamics of MGEs were summarised. A comprehensive understanding of the mechanisms underlying HGT and the role of MGEs in bacterial evolution and adaptation is important to develop strategies to combat the spread of ARGs.

IncX3 plasmid carrying blaSHV-12 and qnrS1 in a Japanese racehorse-origin Escherichia coli isolate

Plasmids play an important role in the spread of antimicrobial resistance genes (ARGs) in bacteria. This study reports the complete sequence of the IncX3 plasmid identified in Escherichia coli isolated from faeces of a Japanese racehorse. Sequence analysis revealed that this plasmid harbours the blaSHV-12 gene, which encodes an extended spectrum ?-lactamase, and the quinolone resistance gene qnrS1. The IncX3 plasmids carrying ARGs have been previously identified in bacteria isolated from humans, animals and a variety of environments. This is the first report of the complete sequence of the IncX3 plasmid carrying blaSHV-12 and qnrS1 genes from a Japanese racehorse, which provides insights into understanding the spread and mechanism of antimicrobial resistance particularly in Japanese racehorses.

Metagenomic comparison of effects of mesophilic and thermophilic manure anaerobic digestion on antimicrobial resistance genes and mobile genetic elements

This study evaluated the long-term effects of mesophilic (MAD) and thermophilic (TAD) anaerobic digestion on the fate and evolution of antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) in cattle manure following a metagenomic approach. The results indicated that MAD and TAD lowered ARG levels in fresh cattle manure by over 50 % and MGEs by over 65 %. However, no statistically significant difference was observed between the MAD and TAD processes in terms of their ability to reduce ARG and MGE. Co-occurrence analysis suggested facultative anaerobic species belonging to the Bacillota and Actinomycetota phyla as the primary potential hosts of ARGs and MGEs. Analyses of co-assembled contigs revealed the presence of transposon and toxin-antitoxin systems in close proximity to ARGs typically found in anaerobic digesters. The identification of these systems near ARGs is particularly significant, as it highlights a potential mechanism for the persistence and spread of ARGs in such environments. Overall, the results indicated that digested cattle manure (whether under mesophilic or thermophilic conditions) would pose a much lower risk of dissemination of antimicrobial resistance than untreated manures. Since TAD did not outperform MAD at reducing ARGs and MGEs, thermophilic temperatures may not be necessary to improve ARG reduction rates in cattle manure AD. These valuable insights could help develop strategies to reduce the dissemination of antibiotic resistance due to manure treatment and disposal.

Mobilizable plasmids drive the spread of antimicrobial resistance genes and virulence genes in Klebsiella pneumoniae

BackgroundKlebsiella pneumoniae is a notorious clinical pathogen and frequently carries various plasmids, which are the main carriers of antimicrobial resistance and virulence genes. In comparison to self-transmissible conjugative plasmids, mobilizable plasmids have received much less attention due to their defects in conjugative elements. However, the contribution of mobilizable plasmids to the horizontal transfer of antimicrobial resistance genes and virulence genes of K. pneumoniae remains unclear. In this study, the transfer, stability, and cargo genes of the mobilizable plasmids of K. pneumoniae were examined via genetic experiments and genomic analysis.MethodsCarbapenem-resistant (CR) plasmid pHSKP2 and multidrug-resistant (MDR) plasmid pHSKP3 of K. pneumoniae HS11286, virulence plasmid pRJF293 of K. pneumoniae RJF293 were employed in conjugation assays to assess the transfer ability of mobilizable plasmids. Mimic mobilizable plasmids and genetically modified plasmids were constructed to confirm the cotransfer models. The plasmid morphology was evaluated through XbaI and S1 nuclease pulsed-field gel electrophoresis and/or complete genome sequencing. Mobilizable plasmid stability in transconjugants was analyzed via serial passage culture. In addition, in silico genome analysis of 3923 plasmids of 1194 completely sequenced K. pneumoniae was performed to investigate the distribution of the conjugative elements, the cargo genes, and the targets of the CRISPR-Cas system. The mobilizable MDR plasmid and virulence plasmid of K. pneumoniae were investigated, which carry oriT but lack other conjugative elements.ResultsOur results showed that mobilizable MDR and virulence plasmids carrying oriT but lacking the relaxase gene were able to cotransfer with a helper conjugative CR plasmid across various Klebsiella and Escherichia coli strains. The transfer and stability of mobilizable plasmids rather than conjugative plasmids were not interfered with by the CRISPR–Cas system of recipient strains. According to the in silico analysis, the mobilizable plasmids carry about twenty percent of acquired antimicrobial resistance genes and more than seventy-five percent of virulence genes in K. pneumoniae.ConclusionsOur work observed that a mobilizable MDR or virulence plasmid that carries oriT but lacks the relaxase genes transferred with the helper CR conjugative plasmid and mobilizable plasmids escaped from CRISPR–Cas defence and remained stable in recipients. These results highlight the threats of mobilizable plasmids as vital vehicles in the dissemination of antibiotic resistance and virulence genes in K. pneumoniae.

209 Prevalence of Antimicrobial Resistance Genes and Mobile Genetic Elements in the Swine Gut Microbiome

Abstract Antimicrobial resistance (AMR) is one of the most pressing threats to public health. It contributes to over a million deaths a year currently and by 2050 it is expected to cause more annual death than cancer. The swine industry has taken important steps to address the AMR crisis by phasing out the use of antimicrobial growth promoters. Nonetheless, the use of antimicrobials for controlling infectious diseases on swine farms remains high, particularly during the nursery stage of pig production. This has raised concerns about the potential role of the swine industry in the emergence and spread of antimicrobial resistance genes (ARGs) capable of conferring resistance to medically important antimicrobials. While traditionally ARGs have been studied in the context of pathogens, there is growing evidence that the natural microbiome of humans and animals can also serve as a significant reservoir of ARGs. Of particular importance is the association of ARGs with different classes of mobile genetic elements (MGEs) which can facilitate the horizontal transfer of genes among commensal bacteria and pathogens. In this study, we evaluated the diversity and distribution of ARGs and their associated MGEs in the swine gut microbiome. To achieve this, we isolated and sequenced the whole genomes of 130 unique bacterial isolates from stool, digesta, and mucosa of healthy adult sows and their piglets. Screening of genomes for the presence of ARGs was performed using the Resistance Gene Identifier (RGI) and Comprehensive Antibiotic Resistance Database (CARD), resulting in the identification of 323 ARGs across 117 genomes. Among these, genes predicted to confer resistance against tetracycline, lincosamide, beta-lactams, and fluoroquinolone were the most prevalent across all genomes. Importantly, we observed that several strictly anaerobic species belonging to gut-specific lineages including Lachnospiraceae, Megasphaeraceae, and Desulfovibrionaceae contained large ARGs pools spanning more than five different classes of antimicrobials. Further, we screened the flanking regions of ARGs for the presence of signature genes of MGEs including recombinases, phage integrases and capsids, and type IV secretion system. A total of 60 ARGs were identified to be associated with different classes of MGEs, including integrative and conjugative elements (ICEs; n = 30), bacteriophages (n = 8), integrative mobile elements (IMEs; n = 6), and plasmids (n = 3). Overall, our study highlights that ARGs, including those conferring resistance to medically important antimicrobials, are prevalent across different lineages of the swine gut microbiota. The association of the identified ARGs with MGEs warrants further in vitro experiments to evaluate the risk of transmission of resistance to zoonotic pathogens capable of infecting humans and animals.

Importance of mobile genetic elements for dissemination of antimicrobial resistance in metagenomic sewage samples across the world.

We are facing an ever-growing threat from increasing antimicrobial resistance (AMR) in bacteria. To mitigate this, we need a better understanding of the global spread of antimicrobial resistance genes (ARGs). ARGs are often spread among bacteria by horizontal gene transfer facilitated by mobile genetic elements (MGE). Here we use a dataset consisting of 677 metagenomic sequenced sewage samples from 97 countries or regions to study how MGEs are geographically distributed and how they disseminate ARGs worldwide. The ARGs, MGEs, and bacterial abundance were calculated by reference-based read mapping. We found systematic differences in the abundance of MGEs and ARGs, where some elements were prevalent on all continents while others had higher abundance in separate geographic areas. Different MGEs tended to be localized to temperate or tropical climate zones, while different ARGs tended to separate according to continents. This suggests that the climate is an important factor influencing the local flora of MGEs. MGEs were also found to be more geographically confined than ARGs. We identified several integrated MGEs whose abundance correlated with the abundance of ARGs and bacterial genera, indicating the ability to mobilize and disseminate these genes. Some MGEs seemed to be more able to mobilize ARGs and spread to more bacterial species. The host ranges of MGEs seemed to differ between elements, where most were associated with bacteria of the same family. We believe that our method could be used to investigate the population dynamics of MGEs in complex bacterial populations.

Characterization of two novel VIM-type metallo-β-lactamases, VIM-84 and VIM-85, associated with the spread of IncP-2 megaplasmids in Pseudomonas aeruginosa.

This study aimed to characterize two novel VIM-type metallo-β-lactamases, VIM-84 and VIM-85, and reveal the important role of the IncP-2 type megaplasmids in the spread of antimicrobial resistance (AMR) genes. VIM-84 and VIM-85 were encoded by two novel genes bla VIM-84 and bla VIM-85 which showed similarity to bla VIM-24. Both bla VIM-84 and bla VIM-85 are harbored into class 1 integrons embedded into the Tn1403 transposon. The bla VIM-85 gene was identified in a megaplasmid, which was related to 17 megaplasmid sequences with sizes larger than 430 kb, deposited previously in Genbank. A comparative analysis of complete plasmid sequences showed highly similar backbone regions and various AMR genes. A phylogenetic tree revealed that these megaplasmids, which were widely distributed globally, were vehicles for the spread of AMR genes. The bla VIM-24, bla VIM-84, and bla VIM-85 genes were cloned into pGK1900, and the recombinant vectors were further transformed into Escherichia coli DH5α and Pseudomonas aeruginosa PAO1. The antimicrobial susceptibility test of the cloning strains showed high levels of resistance to β-lactams while they remained susceptible to aztreonam. Enzymatic tests revealed that both, VIM-84 and VIM-85, exhibited higher activity in hydrolyzing β-lactams compared to VIM-24. A D117N mutation found in VIM-24 affected binding to the antibiotics. IMPORTANCE The metallo-β-lactamases-producing Pseudomonas aeruginosa strains play an important role in hospital outbreaks and the VIM-type enzyme is the most prevalent in European countries. Two novel VIM-type enzymes in our study, VIM-84 and VIM-85, have higher levels of resistance to β-lactams and greater hydrolytic activities for most β-lactams compared with VIM-24. Both bla VIM-84 and bla VIM-85 are harbored into class 1 integrons embedded into the Tn1403 transposon. Notably, the genes bla VIM-85 are carried by three different IncP-2-type megaplasmids which are distributed locally and appear responsible for the spread of antimicrobial resistance genes in hospital settings.