Deciphering the Antibacterial Mechanisms of 5-Fluorouracil in Escherichia coli through Biochemical and Transcriptomic Analyses.

The emergence of carbapenem-resistant Gram-negative pathogens presents a clinical challenge in infection treatment, prompting the repurposing of existing drugs as an essential strategy to address this crisis. Although the anticancer drug 5-fluorouracil (5-FU) has been recognized for its antibacterial properties, its mechanisms are not fully understood. Here, we found that the minimal inhibitory concentration (MIC) of 5-FU against Escherichia coli was 32-64 µg/mL, including strains carrying blaNDM-5, which confers resistance to carbapenems. We further elucidated the antibacterial mechanism of 5-FU against E. coli by using genetic and biochemical analyses. We revealed that the mutation of uracil phosphoribosyltransferase-encoding gene upp increased the MIC of 5-FU against E. coli by 32-fold, indicating the role of the upp gene in 5-FU resistance. Additionally, transcriptomic analysis of E. coli treated with 5-FU at 8 µg/mL and 32 µg/mL identified 602 and 1082 differentially expressed genes involved in carbon and nucleic acid metabolism, DNA replication, and repair pathways. The biochemical assays showed that 5-FU induced bacterial DNA damage, significantly increased intracellular ATP levels and the NAD+/NADH ratio, and promoted reactive oxygen species (ROS) production. These findings suggested that 5-FU may exert antibacterial effects on E. coli through multiple pathways, laying the groundwork for its further development as a therapeutic candidate against carbapenem-resistant bacterial infections.

Dihydroartemisinin inhibits plasmid transfer in drug-resistant Escherichia coli via limiting energy supply.

Conjugative transfer of antibiotic resistance genes (ARGs) by plasmids is an important route for ARG dissemination. An increasing number of antibiotic and nonantibiotic compounds have been reported to aid the spread of ARGs, highlighting potential challenges for controlling this type of horizontal transfer. Development of conjugation inhibitors that block or delay the transfer of ARG-bearing plasmids is a promising strategy to control the propagation of antibiotic resistance. Although such inhibitors are rare, they typically exhibit relatively high toxicity and low efficacy in vivo and their mechanisms of action are inadequately understood. Here, we studied the effects of dihydroartemisinin (DHA), an artemisinin derivative used to treat malaria, on conjugation. DHA inhibited the conjugation of the IncI2 and IncX4 plasmids carrying the mobile colistin resistance gene ( mcr-1) by more than 160-fold in vitro in Escherichia coli, and more than two-fold (IncI2 plasmid) in vivo in a mouse model. It also suppressed the transfer of the IncX3 plasmid carrying the carbapenem resistance gene bla NDM-5 by more than two-fold in vitro. Detection of intracellular adenosine triphosphate (ATP) and proton motive force (PMF), in combination with transcriptomic and metabolomic analyses, revealed that DHA impaired the function of the electron transport chain (ETC) by inhibiting the tricarboxylic acid (TCA) cycle pathway, thereby disrupting PMF and limiting the availability of intracellular ATP for plasmid conjugative transfer. Furthermore, expression levels of genes related to conjugation and pilus generation were significantly down-regulated during DHA exposure, indicating that the transfer apparatus for conjugation may be inhibited. Our findings provide new insights into the control of antibiotic resistance and the potential use of DHA.

[Synergistic effect of ß-thujaplicin and tigecycline against tet(X4)-positive Escherichia coli in vitro].

The widespread of tigecycline resistance gene tet(X4) has a serious impact on the clinical efficacy of tigecycline. The development of effective antibiotic adjuvants to combat the looming tigecycline resistance is needed. The synergistic activity between the natural compound ß-thujaplicin and tigecycline in vitro was determined by the checkerboard broth microdilution assay and time-dependent killing curve. The mechanism underlining the synergistic effect between ß-thujaplicin and tigecycline against tet(X4)-positive Escherichia coli was investigated by determining cell membrane permeability, bacterial intracellular reactive oxygen species (ROS) content, iron content, and tigecycline content. ß-thujaplicin exhibited potentiation effect on tigecycline against tet(X4)-positive E. coli in vitro, and presented no significant hemolysis and cytotoxicity within the range of antibacterial concentrations. Mechanistic studies demonstrated that ß-thujaplicin significantly increased the permeability of bacterial cell membranes, chelated bacterial intracellular iron, disrupted the iron homeostasis and significantly increased intracellular ROS level. The synergistic effect of ß-thujaplicin and tigecycline was identified to be related to interfere with bacterial iron metabolism and facilitate bacterial cell membrane permeability. Our studies provided theoretical and practical data for the application of combined ß-thujaplicin with tigecycline in the treatment of tet(X4)-positive E. coli infection.

Fecal Carriage of Escherichia coli Harboring the tet(X4)-IncX1 Plasmid from a Tertiary Class-A Hospital in Beijing, China.

The emergence of the mobile tigecycline-resistance gene, tet(X4), poses a significant threat to public health. To investigate the prevalence and genetic characteristics of the tet(X4)-positive Escherichia coli in humans, 1101 human stool samples were collected from a tertiary class-A hospital in Beijing, China, in 2019. Eight E. coli isolates that were positive for tet(X4) were identified from clinical departments of oncology (n = 3), hepatology (n = 2), nephrology (n = 1), urology (n = 1), and general surgery (n = 1). They exhibited resistance to multiple antibiotics, including tigecycline, but remained susceptible to meropenem and polymyxin B. A phylogenetic analysis revealed that the clonal spread of four tet(X4)-positive E. coli from different periods of time or departments existed in this hospital, and three isolates were phylogenetically close to the tet(X4)-positive E. coli from animals and the environment. All tet(X4)-positive E. coli isolates contained the IncX1-plasmid replicon. Three isolates successfully transferred their tigecycline resistance to the recipient strain, C600, demonstrating that the plasmid-mediated horizontal gene transfer constitutes another critical mechanism for transmitting tet(X4). Notably, all tet(X4)-bearing plasmids identified in this study had a high similarity to several plasmids recovered from animal-derived strains. Our findings revealed the importance of both the clonal spread and horizontal gene transfer in the spread of tet(X4) within human clinics and between different sources.

Structural diversity of the ISCR2-mediated rolling-cycle transferable unit carrying tet(X4).

BACKGROUND: Mobile tigecycline-resistance gene tet(X) variants have emerged as diverse pathogens from animal, human as well as their associated environments, which could potentially threaten public health. The insertion sequence, ISCR2, carries tet(X4) for horizontal transfer by rolling-cycle (RC) transposition. However, the diversity of ISCR2 and tet(X4) isolated from different sources is largely unknown. METHODS: The tet(X4)-carrying isolates were collected from human and livestock in several multiple regions of China. The whole genomic sequences of these isolates were either obtained from NCBI GenBank or determined by Illumina Hiseq 2500 and the MinION platform. The intact transposon region, ISCR2-tet(X4)-ISCR2, observed in a small number of isolates as the reference sequence to construct the transposon phylogeny. The diversity of the genetic environments of all ISCR2-tet(X4) elements were analyzed. RESULTS: A 2760-bp element encompassing the tet(X4)-hydrolase-encoding gene, catD, located between two ISCR2 elements was highly conserved in all isolates and could form an RC transposable unit (RC-TU). ISCR2 could also capture more resistance genes and formed a larger RC-TU base on RC transposition. However, the ISCR2-mediated RC-TUs were constantly truncated and inserted by other IS elements, indicating frequent recombination events. Of these elements, IS26 disrupted both the upstream and downstream ISCR2-mediated RC-TUs, indicating that IS26 captured tet(X4), thus leading to a wider spread of tet(X4). CONCLUSIONS: These results confirmed the critical role of ISCR2 for dissemination and co-transmission of tet(X4) and other resistance genes. More effort is needed to monitor the variation tendencies of tet(X4)-carrying mobile elements and determine the driving factors for disseminating transferable tigecycline resistance.

Presence of Mobile Tigecycline Resistance Gene tet(X4) in Clinical Klebsiella pneumoniae.

The recently emerged plasmid-mediated tigecycline resistance gene tet(X4) has mainly been detected in Escherichia coli but never in Klebsiella pneumoniae. Herein, we identified a clinical K. pneumoniae isolate that harbored the tet(X4) gene located on a non-self-transferable IncFII-type plasmid, which could be cotransferred with a conjugative plasmid to E. coli C600. The extending of bacterial species carrying tet(X4) suggested the increasing risk of spreading mobile tigecycline resistance genes among important pathogens in clinical settings. IMPORTANCE Tigecycline, the first member of glycylcycline class antibiotic, is often considered one of the effective antibiotics against multidrug-resistant (MDR) infections. However, the emergence and wide distribution of two novel plasmid-mediated tigecycline resistance genes, tet(X3) and tet(X4), pose a great threat to the clinical use of tigecycline. The newly tet(X) variants have been identified from multiple different bacterial species, but the tet(X) variant in the Klebsiella pneumoniae strain has been reported only once before. In this study, we identified a clinical K. pneumoniae isolate that harbored a non-self-transferable tet(X4)-carrying plasmid. This plasmid has never been found in other tet(X4)-harboring strains and could be cotransferred with a conjugative plasmid to the recipient strain. Our findings indicate that the tet(X4) gene breaks through its original bacterial species and spreads to some important nosocomial pathogens, which posed a serious threat to public health.

Novel IS26-mediated hybrid plasmid harbouring tet(X4) in Escherichia coli.

OBJECTIVES: As the spread of antimicrobial resistance genes becomes an increasing global threat, improved understanding of genetic structure and transferability of the resistant plasmids becomes more critical. The newly description of several plasmid-mediated tet(X) variant genes, tet(X3), tet(X4) and tet(X5), poses a considerable risk for public health. This study aimed to investigate the recombination event that occurred during the conjugation process of a tet(X4)-bearing plasmid. METHODS: A Tet(X4)-producing Escherichia coli isolate, 2019XSD11, was subjected to susceptibility testing, S1-PFGE and whole genome sequencing. The genetic features of plasmids and the recombination event were analysed by sequence comparison and annotation. We performed electrotransformation assay to further test the transferability of the tet(X4)-bearing plasmid. RESULTS: A novel type of fusion tet(X4)-bearing plasmid was discovered from the transconjugant, plasmid p2019XSD11-TC2-284 (∼280kbp). The sequence of this plasmid consisted of a hybrid episome of two plasmids p2019XSD11-190 (∼190kbp) harbouring tet(X4) and p2019XSD11-92 (∼92kbp) harbouring blaCTX-M-55 originated from 2019XSD11. The two plasmids were concatenated by IS26 elements. Analyses of the genetic constitution of the plasmids essential for transmission showed the plasmid p2019XSD11-190 lacked an intact type IV secretion system. Beyond this, the origin of transfer region and relaxase genes in plasmid p2019XSD11-190 had no sequence similarity with those in plasmid p2019XSD11-92. CONCLUSIONS: The fusion of the two plasmids probably formed through IS26 homologous recombination. Such recombination events presumably play an important role in the dissemination of the tet(X4). Molecular surveillance of tet(X) variant genes and genetic structures warrants further investigation to evaluate the underlying public health risk.

Identification of the novel tigecycline resistance gene tet(X6) and its variants in Myroides, Acinetobacter and Proteus of food animal origin.

OBJECTIVES: To report a novel tigecycline resistance gene, tet(X6), and its variants in four bacterial species isolated from chickens and pigs in China. METHODS: WGS was conducted to identify the suspected resistance genes in the tigecycline-resistant Myroides phaeus 18QD1AZ29W. Functional cloning, homology modelling and molecular docking were performed to compare the function with other Tet(X) variants. Retrospective screening for tet(X6) was conducted for 80 isolates in our WGS data collection, and all genomic environments of tet(X6)-positive isolates were analysed. RESULTS: The tigecycline-resistant M. phaeus 18QD1AZ29W isolated from a pig farm in Shandong in 2018 was positive for tet(X2) and a novel tet(X) gene, designated tet(X6). Tet(X6) could increase the MICs of all tested tetracyclines/glycylcyclines for Escherichia coli only 2- to 4-fold, which was possibly due to a lower tetracycline binding capacity of Tet(X6) compared with that of other Tet(X) variants. Retrospective screening showed that seven other isolates (7/80, 8.8%), comprising four Proteus spp. and three Acinetobacter spp. from chickens and pigs in Shandong and Guangdong, were positive for three different variants of tet(X6). The analysis of the genomic environment revealed that two tet(X6)-positive isolates from M. phaeus and Proteus cibarius, respectively, contained ISCR2, which may play a role in tet(X6) transmission. CONCLUSIONS: This study identified a novel type of tigecycline resistance gene, tet(X6), in Myroides, Acinetobacter and Proteus from chickens and swine. Tet(X6) conferred lower tetracycline/glycylcycline MICs than other Tet(X) variants, and ISCR2 may play a role in the transmission of tet(X6).

Development of a Multiplex Real-Time PCR Assay for Rapid Detection of Tigecycline Resistance Gene tet(X) Variants from Bacterial, Fecal, and Environmental Samples.

We developed a multiplex real-time SYBR green-based PCR assay for rapid detection of tet(X) and its variants, including tet(X1) and tet(X2) and high-level tigecycline resistance genes tet(X3), tet(X4), and tet(X5). We showed that the real-time PCR assay developed had high linearity (R2 ≥ 0.996), sensitivity (low detection limit), and specificity (only the target gene could be amplified significantly) and further evaluated it using bacterial, fecal, and environmental samples.

Co-existence of two novel phosphoethanolamine transferase gene variants in Aeromonas jandaei from retail fish.

Two novel phosphoethanolamine transferase genes, eptAv7 and eptAv3, were identified in the chromosome of an Aeromonas jandaei isolate from retail fish. The variants showed 79.9% and 80.0% amino acid identity to MCR-7.1 and MCR-3.1, respectively, and increased colistin resistance 128- to 256-fold in Aeromonas salmonicida. The two variants with no mobile genetic element in the flanking regions were also observed in other Aeromonas species. This finding supports the view that Aeromonas is a reservoir for MCR-3 and MCR-7 mobile colistin resistance.

Novel Plasmid-Mediated tet(X5) Gene Conferring Resistance to Tigecycline, Eravacycline, and Omadacycline in a Clinical Acinetobacter baumannii Isolate.

A novel, plasmid-mediated, high-level tigecycline resistance tet(X) gene variant, tet(X5), was detected in a clinical Acinetobacter baumannii isolate from China in 2017. Tet(X5) shows 84.5% and 90.5% amino acid identity to Tet(X3) and Tet(X4), respectively, with similar binding sites and a comparable affinity for tetracyclines. The tet(X5)-containing plasmid could only be transferred to A. baumannii via electrotransformation. This report follows the recent study describing the identification of tet(X3) and tet(X4).

Emergence of plasmid-mediated high-level tigecycline resistance genes in animals and humans.

Tigecycline is a last-resort antibiotic that is used to treat severe infections caused by extensively drug-resistant bacteria. tet(X) has been shown to encode a flavin-dependent monooxygenase that modifies tigecycline1,2. Here, we report two unique mobile tigecycline-resistance genes, tet(X3) and tet(X4), in numerous Enterobacteriaceae and Acinetobacter that were isolated from animals, meat for consumption and humans. Tet(X3) and Tet(X4) inactivate all tetracyclines, including tigecycline and the newly FDA-approved eravacycline and omadacycline. Both tet(X3) and tet(X4) increase (by 64-128-fold) the tigecycline minimal inhibitory concentration values for Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii. In addition, both Tet(X3) (A. baumannii) and Tet(X4) (E. coli) significantly compromise tigecycline in in vivo infection models. Both tet(X3) and tet(X4) are adjacent to insertion sequence ISVsa3 on their respective conjugative plasmids and confer a mild fitness cost (relative fitness of >0.704). Database mining and retrospective screening analyses confirm that tet(X3) and tet(X4) are globally present in clinical bacteria-even in the same bacteria as blaNDM-1, resulting in resistance to both tigecycline and carbapenems. Our findings suggest that both the surveillance of tet(X) variants in clinical and animal sectors and the use of tetracyclines in food production require urgent global attention.