RESUMO
OBJECTIVES: Extracellular vesicles (EVs) have become the focus of research as an emerging method of horizontal gene transfer. In recent years, studies on the association between EVs and the spread of bacterial resistance have emerged, but there is a lack of research on the role of EVs secreted by extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli in the spread of ß-lactam resistance. Therefore, the aim of this study was to investigate the role of EVs in the transmission of ß-lactam resistance. METHODS: In this study, the role of EVs in the transmission of ß-lactam resistance in E. coli was evaluated by the EVs-mediated bacterial resistance to ß-lactam antibiotics test and the EVs-mediated blaCTX-M-55 transfer experiments using EVs secreted by ESBL-E. coli. RESULTS: The results showed that ESBL-EVs were protective against ß-lactam antibiotic-susceptible bacteria, and this protective effect was dependent on the integrity of the EVs and showed dose- and time-dependent effects. At the same time, ESBL-EVs can also mediate the horizontal transmission of blaCTX-M-55, and EVs-mediated gene transfer is selective, preferring to transfer in more closely related species. CONCLUSIONS: In this study, we demonstrated the important role of EVs in the transmission of ß-lactam resistance in chicken ESBL-E. coli, and evaluated the risk of EVs-mediated horizontal gene transfer, which provided a theoretical basis for elucidating the mechanism of EVs-mediated resistance transmission.
Assuntos
Antibacterianos , Escherichia coli , Vesículas Extracelulares , Transferência Genética Horizontal , Resistência beta-Lactâmica , beta-Lactamases , beta-Lactamas , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Vesículas Extracelulares/metabolismo , beta-Lactamases/genética , beta-Lactamases/metabolismo , beta-Lactamas/farmacologia , Resistência beta-Lactâmica/genética , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Infecções por Escherichia coli/microbiologia , AnimaisRESUMO
Extended-spectrumß-lactam producing Escherichia coli (ESBL-EC) readily colonizes live poultry and serves as a major source of contamination in retail chicken meat, posing significant threats to public health. This study aims to investigate the impact of inappropriate antibiotic use on the dissemination and exacerbation of antibiotic resistance in ESBL-EC and explore the underlying molecular mechanisms. Through experimental analysis, we propose a hypothesis that inappropriate antibiotic use may exacerbate resistance by affecting vesicle formation and protein secretion. Experimental results demonstrate that under the influence of amoxicillin, the concentration of proteins secreted in outer membrane vehicles (OMVs) by ESBL-EC significantly increases, along with a significant upregulation in the expression of the CTX-M-55-type Extended-spectrum beta-lactamase (CTX-M-55). Proteomic analysis and differential gene knockout experiments identified the key protein YdcZ, associated with OMVs formation and protein transportation in ESBL-EC under amoxicillin treatment. Further investigations reveal direct interactions between YdcZ and other proteins (YdiH and BssR). Upon ydcz gene knockout, a significant decrease in protein concentration within OMVs is observed, accompanied by a noticeable reduction in protection against sensitive bacteria. These findings suggest a critical role of YdcZ in regulating the process of protein transportation to OMVs in ESBL-EC under the influence of amoxicillin. In summary, our research uncovers the significant role of inappropriate antibiotic use in promoting the secretion of OMVs by ESBL-EC, aiding the survival of antibiotic-sensitive bacteria in the vicinity of infection sites. These findings provide new insights into the mechanisms underlying antibiotic-induced bacterial resistance dissemination and offer novel avenues for exploring prevention and control strategies against bacterial resistance propagation.
Assuntos
Amoxicilina , Antibacterianos , Proteínas de Escherichia coli , Escherichia coli , Transporte Proteico , beta-Lactamases , Antibacterianos/farmacologia , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , beta-Lactamases/metabolismo , beta-Lactamases/genética , Amoxicilina/farmacologia , Animais , Testes de Sensibilidade Microbiana , Proteômica , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Galinhas/microbiologia , Farmacorresistência Bacteriana , Membrana Externa Bacteriana/efeitos dos fármacos , Membrana Externa Bacteriana/metabolismo , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/tratamento farmacológicoRESUMO
Antibiotic resistance (AMR) poses a significant global health challenge, with swine farms recognized as major reservoirs of antibiotic resistance genes (ARGs). Recently, bacterial membrane vesicles (BMVs) have emerged as novel carriers mediating horizontal gene transfer. However, little is known about the ARGs carried by BMVs in swine farm environments and their transfer potential. This study investigated the distribution, sources, and microbiological origins of BMVs in three key microbial habitats of swine farms (feces, soil, and fecal wastewater), along with the ARGs and mobile genetic elements (MGEs) they harbor. Characterization of BMVs revealed particle sizes ranging from 20 to 500 nm and concentrations from 108 to 1012 particles/g, containing DNA and proteins. Metagenomic sequencing identified BMVs predominantly composed of members of the Proteobacteria phyla, including Pseudomonadaceae, Moraxellaceae, and Enterobacteriaceae, carrying diverse functional genes encompassing resistance to 14 common antibiotics and 74,340 virulence genes. Notably, multidrug resistance, tetracycline, and chloramphenicol resistance genes were particularly abundant. Furthermore, BMVs harbored various MGEs, primarily plasmids, and demonstrated the ability to protect their DNA cargo from degradation and facilitate horizontal gene transfer, including the transmission of resistance genes. In conclusion, this study reveals widespread presence of BMVs carrying ARGs and potential virulence genes in swine farm feces, soil, and fecal wastewater. These findings not only provide new insights into the role of extracellular DNA in the environment but also highlight concerns regarding the gene transfer potential mediated by BMVs and associated health risks.
RESUMO
Proteus mirabilis can transfer transposons, insertion sequences, and gene cassettes to the chromosomes of other hosts through SXT/R391 integrative and conjugative elements (ICEs), significantly increasing the possibility of antibiotic resistance gene (ARG) evolution and expanding the risk of ARGs transmission among bacteria. A total of 103 strains of P. mirabilis were isolated from 25 farms in China from 2018 to 2020. The positive detection rate of SXT/R391 ICEs was 25.2% (26/103). All SXT/R391 ICEs positive P. mirabilis exhibited a high level of overall drug resistance. Conjugation experiments showed that all 26 SXT/R391 ICEs could efficiently transfer to Escherichia coli EC600 with a frequency of 2.0 × 10-7 to 6.0 × 10-5. The acquired ARGs, genetic structures, homology relationships, and conservation sequences of 26 (19 different subtypes) SXT/R391 ICEs were investigated by high-throughput sequencing, whole-genome typing, and phylogenetic tree construction. ICEPmiChnHBRJC2 carries erm (42), which have never been found within an SXT/R391 ICE in P. mirabilis, and ICEPmiChnSC1111 carries 19 ARGs, including clinically important cfr, blaCTX-M-65, and aac(6')-Ib-cr, making it the ICE with the most ARGs reported to date. Through genetic stability, growth curve, and competition experiments, it was found that the transconjugant of ICEPmiChnSCNNC12 did not have a significant fitness cost on the recipient bacterium EC600 and may have a higher risk of transmission and dissemination. Although the transconjugant of ICEPmiChnSCSZC20 had a relatively obvious fitness cost on EC600, long-term resistance selection pressure may improve bacterial fitness through compensatory adaptation, providing scientific evidence for risk assessment of horizontal transfer and dissemination of SXT/R391 ICEs in P. mirabilis.IMPORTANCEThe spread of antibiotic resistance genes (ARGs) is a major public health concern. The study investigated the prevalence and genetic diversity of integrative and conjugative elements (ICEs) in Proteus mirabilis, which can transfer ARGs to other hosts. The study found that all of the P. mirabilis strains carrying ICEs exhibited a high level of drug resistance and a higher risk of transmission and dissemination of ARGs. The analysis of novel multidrug-resistant ICEs highlighted the potential for the evolution and spread of novel resistance mechanisms. These findings emphasize the importance of monitoring the spread of ICEs carrying ARGs and the urgent need for effective strategies to combat antibiotic resistance. Understanding the genetic diversity and potential for transmission of ARGs among bacteria is crucial for developing targeted interventions to mitigate the threat of antibiotic resistance.