Distribution and spread of tigecycline resistance gene tet(X4) in Escherichia coli from different sources.

Tigecycline serves as a last-resort antimicrobial agent against severe infections caused by multidrug-resistant bacteria. Tet(X) and its numerous variants encoding flavin-dependent monooxygenase can confer resistance to tigecycline, with tet(X4) being the most prevalent variant. This study aims to investigate the prevalence and characterize tigecycline resistance gene tet(X) in E. coli isolates from various origins in Yangzhou, China, to provide insights into tet(X) dissemination in this region. In 2022, we tested the presence of tet(X) in 618 E. coli isolates collected from diverse sources, including patients, pig-related samples, chicken-related samples, and vegetables in Yangzhou, China. The antimicrobial susceptibility of tet(X)-positive E. coli isolates was conducted using the agar dilution method or the broth microdilution method. Whole genome sequencing was performed on tet(X)-positive strains using Illumina and Oxford Nanopore platforms. Four isolates from pig or pork samples carried tet(X4) and exhibited resistance to multiple antimicrobial agents, including tigecycline. They were classified as ST542, ST10, ST761, and ST48, respectively. The tet(X4) gene was located on IncFIA8-IncHI1/ST17 (n=2), IncFIA18-IncFIB(K)-IncX1 (n=1), and IncX1 (n=1) plasmids, respectively. These tet(X4)-carrying plasmids exhibited high similarity to other tet(X4)-bearing plasmids with the same incompatible types found in diverse sources in China. They shared related genetic environments of tet(X4) associated with ISCR2, as observed in the first identified tet(X4)-bearing plasmid p47EC. In conclusion, although a low prevalence (0.65%) of tet(X) in E. coli strains was observed in this study, the horizontal transfer of tet(X4) among E. coli isolates mediated by pandemic plasmids and the mobile element ISCR2 raises great concerns. Thus, heightened surveillance and immediate action are imperative to curb this clinically significant resistance gene and preserve the efficacy of tigecycline.

Third-Generation Tetracyclines: Current Knowledge and Therapeutic Potential.

Tetracyclines constitute a unique class of antibiotic agents, widely prescribed for both community and hospital infections due to their broad spectrum of activity. Acting by disrupting protein synthesis through tight binding to the 30S ribosomal subunit, their interference is typically reversible, rendering them bacteriostatic in action. Resistance to tetracyclines has primarily been associated with changes in pump efflux or ribosomal protection mechanisms. To address this challenge, tetracycline molecules have been chemically modified, resulting in the development of third-generation tetracyclines. These novel tetracyclines offer significant advantages in treating infections, whether used alone or in combination therapies, especially in hospital settings. Beyond their conventional antimicrobial properties, research has highlighted their potential non-antibiotic properties, including their impact on immunomodulation and malignancy. This review will focus on third-generation tetracyclines, namely tigecycline, eravacycline, and omadacycline. We will delve into their mechanisms of action and resistance, while also evaluating their pros and cons over time. Additionally, we will explore their therapeutic potential, analyzing their primary indications of prescription, potential future uses, and non-antibiotic features. This review aims to provide valuable insights into the clinical applications of third-generation tetracyclines, thereby enhancing understanding and guiding optimal clinical use.

Structural basis for differential inhibition of eukaryotic ribosomes by tigecycline.

Tigecycline is widely used for treating complicated bacterial infections for which there are no effective drugs. It inhibits bacterial protein translation by blocking the ribosomal A-site. However, even though it is also cytotoxic for human cells, the molecular mechanism of its inhibition remains unclear. Here, we present cryo-EM structures of tigecycline-bound human mitochondrial 55S, 39S, cytoplasmic 80S and yeast cytoplasmic 80S ribosomes. We find that at clinically relevant concentrations, tigecycline effectively targets human 55S mitoribosomes, potentially, by hindering A-site tRNA accommodation and by blocking the peptidyl transfer center. In contrast, tigecycline does not bind to human 80S ribosomes under physiological concentrations. However, at high tigecycline concentrations, in addition to blocking the A-site, both human and yeast 80S ribosomes bind tigecycline at another conserved binding site restricting the movement of the L1 stalk. In conclusion, the observed distinct binding properties of tigecycline may guide new pathways for drug design and therapy.

Evaluation of role of Tigecycline among clinically significant multidrug resistant pathogens from a tertiary care hospital.

Background: Tigecycline, a glycylcycline antibiotic is a promising option for the treatment of single or multidrug resistant pathogens. The aim of the study was to evaluate the in-vitro Tigecycline susceptibility of various pathogens from clinical samples received at the tertiary care hospitals in South India. Methods: The analysis of specimens from patients admitted were carried out in this prospective cross sectional study. The identification and antimicrobial susceptibility testing was performed by semi-automated Vitek 2 systems and Kirby Bauer method. Pattern of data analysis was done by descriptive statistics. Results: Among 2574 isolates, 812 isolates were Gram positive pathogens and 1762 isolates were Gram negative pathogens. Resistance to Tigecycline was more common among Gram negative pathogens (18.62%) in comparison to the Gram positive pathogens (0.49%). Among 740 Extended Spectrum Beta Lactamases (ESBL) producers such as Klebsiella species & E coli, 629 isolates were susceptible, and 93 isolates were resistant to the tigecycline. All the methicillin resistant Staphylococcus aureus (MRSA) isolates were susceptible to tigecycline. Conclusion: Multidrug resistant (MDR) pathogens like Acinetobacter species, and Klebsiella species were found to be highly effective in vitro to tigecycline for elimination of infections caused by both Gram positive and Gram negative pathogens. The use of combination therapy becomes crucial to prevent the development of Pan Drug resistance.

AcrAB-TolC efflux pump overexpression and tet(A) gene mutation increase tigecycline resistance in Klebsiella pneumoniae.

Tigecycline-non-susceptible Klebsiella pneumoniae (TNSKP) is increasing and has emerged as a global public health issue. However, the mechanism of tigecycline resistance remains unclear. The objective of this study was to investigate the potential role of efflux pump system in tigecycline resistance. 29 tigecycline-non-susceptible Klebsiella pneumoniae (TNSKP) strains were collected and their minimum inhibitory concentrations (MIC) were determined by the broth microdilution method. The ramR, acrR, rpsJ, tet(A), and tet(X) were amplified by polymerase chain reaction (PCR). The mRNA expression of different efflux pump genes and regulator genes were analyzed by real-time PCR. Additionally, KP14 was selected for genome sequencing. KP14 genes without acrB, oqxB, and TetA were modified using suicide plasmids and MIC of tigecycline of KP14 with target genes knocked out was investigated. It was found that MIC of tigecycline of 20 out of the 29 TNSKP strains decreased by over four folds once combined with phenyl-arginine-ß-naphthylamide dihydrochloride (PaßN). Most strains exhibited upregulation of AcrAB and oqxAB efflux pumps. The strains with acrB, oqxB, and tetA genes knocked out were constructed, wherein the MIC of tigecycline of KP14∆acrB and KP14∆tetA was observed to be 2 µg/mL (decreased by 16 folds), the MIC of tigecycline of KP14ΔacrBΔTetA was 0.25 µg/mL (decreased by 128 folds), but the MIC of tigecycline of KP14∆oqxB remained unchanged at 32 µg/mL. The majority of TNSKP strains demonstrated increased expression of AcrAB-TolC and oqxAB, while certain strains showed mutations in other genes associated with tigecycline resistance. In KP14, both overexpression of AcrAB-TolC and tet(A) gene mutation contributed to the mechanism of tigecycline resistance.

Emergence of aztreonam/avibactam and tigecycline-resistant Pseudomonas putida group Co-producing blaIMP-1, blaAFM-4 and blaOXA-1041 with a novel sequence type ST268 in Southwestern China.

OBJECTIVES: The emergence of carbapenem-resistant Pseudomonas putida (CRPP) has raised public awareness. This study investigated two strains from the Pseudomonas putida group that were resistant to carbapenem, tigecycline, and aztreonam-avibactam (ATM-AVI), with a focus on their microbial and genomic characteristics. METHODS: We assessed the antibiotic resistance profile using broth dilution, disk diffusion, and E-test methods. Efflux pump phenotype testing and real-time quantitative PCR were employed to evaluate efflux pump activity in tigecycline resistance, while polymerase chain reaction was utilized to detect common carbapenem genes. Additionally, whole-genome sequencing was performed to analyze genomic characteristics. The transferability of blaIMP-1 and blaAFM-4 was assessed through a conjugation experiment. Furthermore, growth kinetics and biofilm formation were examined using growth curves and crystal violet staining. RESULTS: Both strains demonstrated resistance to carbapenem, tigecycline, and ATM-AVI. Notably, NMP can restore sensitivity to tigecycline. Subsequent analysis revealed that they co-produced blaIMP-1, blaAFM-4, tmexCD-toprJ, and blaOXA-1041, belonging to a novel sequence type ST268. Although they were closely related on the phylogenetic tree, they exhibited different levels of virulence. Genetic environment analysis indicated variations compared to prior studies, particularly regarding the blaIMP-1 and blaAFM-4 genes, which showed limited horizontal transferability. Moreover, it was observed that temperature exerted a specific influence on their biological factors. CONCLUSION: We initially identified two P. putida ST268 strains co-producing blaIMP-1, blaAFM-4, blaOXA-1041, and tmexCD-toprJ. The resistance to tigecycline and ATM-AVI can be attributed to the presence of multiple drug resistance determinants. These findings underscore the significance of P. putida as a reservoir for novel antibiotic resistance genes. Therefore, it is imperative to develop alternative antibiotic therapies and establish effective monitoring of bacterial resistance.

Prevalence and genomic characterization of clinical Escherichia coli strains that harbor the plasmid-borne tet(X4) gene in China.

The tigecycline resistance gene tet(X4) has been widely reported in animals and animal products in some Asian countries including China in recent years but only sporadically detected in human. In this study, we investigated the prevalence and genetic features of tet(X4)-positive clinical E. coli strains. A total of 462 fecal samples were collected from patients in four hospitals located in four provinces in China in 2023. Nine tet(X4)-positive E. coli strains were isolated and subjected to characterization of their genetic and phenotypic features by performing antimicrobial susceptibility test, whole-genome sequencing, bioinformatic and phylogenetic analysis. The majority of the test strains were found to exhibit resistance to multiple antimicrobial agents including tigecycline but remained susceptible to colistin and meropenem. A total of seven different sequence types (STs) and an unknown ST type were identified among the nine tet(X4)-positive strains. Notably, the tet(X4) gene in six out of these nine tet(X4)-positive E. coli strains was located in a IncFIA-HI1A-HI1B hybrid plasmid, which was an tet(X4)-bearing epidemic plasmid responsible for dissemination of the tet(X4) gene in China. Furthermore, the tet(X4) gene in four out of nine tet(X4)-positive E. coli isolates could be successfully transferred to E. coli EC600 through conjugation. In conclusion, this study characterized the epidemic tet(X4)-bearing plasmids and tet(X4)-associated genetic environment in clinical E. coli strains, suggested the importance of continuous surveillance of such tet(X4)-bearing plasmids to control the increasingly widespread dissemination of tigecycline-resistant pathogens in clinical settings in China.

Large-scale bacterial genomic and metagenomic analysis reveals Pseudomonas aeruginosa as potential ancestral source of tigecycline resistance gene cluster tmexCD-toprJ.

BACKGROUND: The global dissemination of the multidrug resistance efflux pump gene cluster tmexCD-toprJ has greatly weakened the effects of multiple antibiotics, including tigecycline. However, the potential origin and transmission mechanisms of the gene cluster remain unclear. METHODS: Here, we concluded a comprehensive bioinformatics analysis on integrated 73,498 bacterial genomes, including Pseudomonas spp., Klebsiella spp., Aeromonas spp., Proteus spp., and Citrobacter spp., along with 1,152 long-read metagenomic datasets to trace the origin and propagation of tmexCD-toprJ. RESULTS: Our results demonstrated that tmexCD-toprJ was predominantly found in Pseudomonas aeruginosa sourced from human hosts in Asian countries and North American countries. Phylogenetic and genomic feature analyses showed that tmexCD-toprJ was likely evolved from mexCD-oprJ of some special clones of P. aeruginosa. Furthermore, metagenomic analysis confirmed that P. aeruginosa is the only potential ancestral bacterium for tmexCD-toprJ. A putative mobile genetic structure harboring tmexCD-toprJ, int-int-hp-hp-tnfxB-tmexCD-toprJ, was the predominant genetic context of tmexCD-toprJ across various bacterial genera, suggesting that the two integrase genes play a pivotal role in the horizontal transmission of tmexCD-toprJ. CONCLUSIONS: Based on these findings, it is almost certain that the tmexCD-toprJ gene cluster was derived from P. aeruginosa and further spread to other bacteria.

Repurposing harmaline as a novel approach to reverse tmexCD1-toprJ1-mediated tigecycline resistance against klebsiella pneumoniae infections.

BACKGROUND: A novel plasmid-mediated resistance-nodulation-division (RND) efflux pump gene cluster tmexCD1-toprJ1 in Klebsiella pneumoniae tremendously threatens the use of convenient therapeutic options in the post-antibiotic era, including the "last-resort" antibiotic tigecycline. RESULTS: In this work, the natural alkaloid harmaline was found to potentiate tigecycline efficacy (4- to 32-fold) against tmexCD1-toprJ1-positive K. pneumoniae, which also thwarted the evolution of tigecycline resistance. Galleria mellonella and mouse infection models in vivo further revealed that harmaline is a promising candidate to reverse tigecycline resistance. Inspiringly, harmaline works synergistically with tigecycline by undermining tmexCD1-toprJ1-mediated multidrug resistance efflux pump function via interactions with TMexCD1-TOprJ1 active residues and dissipation of the proton motive force (PMF), and triggers a vicious cycle of disrupting cell membrane integrity and metabolic homeostasis imbalance. CONCLUSION: These results reveal the potential of harmaline as a novel tigecycline adjuvant to combat hypervirulent K. pneumoniae infections.

Tigecycline Opposes Bortezomib Effect on Myeloma Cells Decreasing Mitochondrial Reactive Oxygen Species Production.

Multiple myeloma is an incurable plasma cell malignancy. Most patients end up relapsing and developing resistance to antineoplastic drugs, like bortezomib. Antibiotic tigecycline has activity against myeloma. This study analyzed tigecycline and bortezomib combination on cell lines and plasma cells from myeloma patients. Apoptosis, autophagic vesicles, mitochondrial mass, mitochondrial superoxide, cell cycle, and hydrogen peroxide were studied by flow cytometry. In addition, mitochondrial antioxidants and electron transport chain complexes were quantified by reverse transcription real-time PCR (RT-qPCR) or western blot. Cell metabolism and mitochondrial activity were characterized by Seahorse and RT-qPCR. We found that the addition of tigecycline to bortezomib reduces apoptosis in proportion to tigecycline concentration. Supporting this, the combination of both drugs counteracts bortezomib in vitro individual effects on the cell cycle, reduces autophagy and mitophagy markers, and reverts bortezomib-induced increase in mitochondrial superoxide. Changes in mitochondrial homeostasis and MYC upregulation may account for some of these findings. These data not only advise to avoid considering tigecycline and bortezomib combination for treating myeloma, but caution on the potential adverse impact of treating infections with this antibiotic in myeloma patients under bortezomib treatment.

Molecular mechanisms of tigecycline-resistance among Enterobacterales.

The global emergence of antimicrobial resistance to multiple antibiotics has recently become a significant concern. Gram-negative bacteria, known for their ability to acquire mobile genetic elements such as plasmids, represent one of the most hazardous microorganisms. This phenomenon poses a serious threat to public health. Notably, the significance of tigecycline, a member of the antibiotic group glycylcyclines and derivative of tetracyclines has increased. Tigecycline is one of the last-resort antimicrobial drugs used to treat complicated infections caused by multidrug-resistant (MDR) bacteria, extensively drug-resistant (XDR) bacteria or even pan-drug-resistant (PDR) bacteria. The primary mechanisms of tigecycline resistance include efflux pumps' overexpression, tet genes and outer membrane porins. Efflux pumps are crucial in conferring multi-drug resistance by expelling antibiotics (such as tigecycline by direct expelling) and decreasing their concentration to sub-toxic levels. This review discusses the problem of tigecycline resistance, and provides important information for understanding the existing molecular mechanisms of tigecycline resistance in Enterobacterales. The emergence and spread of pathogens resistant to last-resort therapeutic options stands as a major global healthcare concern, especially when microorganisms are already resistant to carbapenems and/or colistin.

Detection and genomic characterization of Klebsiella pneumoniae and Escherichia coli harboring tet(X4) in black kites (Milvus migrans) in Pakistan.

The emergence of plasmid-mediated tigecycline resistance gene tet(X4) among clinically relevant bacteria has promoted significant concerns, as tigecycline is considered a last-resort drug against serious infections caused by multidrug-resistant bacteria. We herein focused on the isolation and molecular characterization of tet(X4)-positive Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli) in wild bird populations with anthropogenic interaction in Faisalabad, Pakistan. A total of 150 birds including black kites (Milvus migrans) and house crows (Corvus splendens) were screened for the presence of tigecycline resistance K. pneumoniae and E. coli. We found two K. pneumoniae and one E. coli isolate carrying tet(X4) originating from black kites. A combination of short- and long-read sequencing strategies showed that tet(X4) was located on a broad host range IncFII plasmid family in K. pneumoniae isolates whereas on an IncFII-IncFIB hybrid plasmid in E. coli. We also found an integrative and conjugative element ICEKp2 in K. pneumoniae isolate KP8336. We demonstrate the first description of tet(X4) gene in the WHO critical-priority pathogen K. pneumoniae among wild birds. The convergence of tet(X4) and virulence associated ICEKp2 in a wild bird with known anthropogenic contact should be further investigated to evaluate the potential epidemiological implications. The potential risk of global transmission of tet(X4)-positive K. pneumoniae and E. coli warrant comprehensive evaluation and emphasizes the need for effective mitigation strategies to reduce anthropogenic-driven dissemination of AMR in the environment.

Tandem amplification of a plasmid-borne tet(A) variant gene confers tigecycline resistance in Escherichia coli.

OBJECTIVES: To elucidate the mechanism of tigecycline resistance in Escherichia coli that is mediated by the tet(A) variant gene. METHODS: E. coli strain 573 carried a plasmid-borne tet(A) variant gene, tentatively designated tet(A)TIG, that conferred decreased tigecycline susceptibility (MIC 0.5 mg/L). When exposed to increasing concentrations of tigecycline (0.25-8 mg/L), mutants growing at 2, 4 and 8 mg/L were obtained and sequenced. Copies of plasmid and tet(A)TIG relative to the chromosomal DNA in the mutants were determined by WGS and quantitative PCR (qPCR). Expression of tet(A)TIG in the mutants was evaluated by RT-qPCR. The tet(A)TIG-carrying plasmids were visualized by S1-PFGE and Southern blot hybridization. PCR served for the detection of a tet(A)TIG-carrying unconventional circularizable structure (UCS). RESULTS: Tigecycline resistance with maximum MICs of 16 mg/L was seen in E. coli mutants selected in the presence of tigecycline. Compared with the parental strain, the relative copy number and transcription level of tet(A)TIG in the mutants increased significantly in the presence of 2, 4 and 8 mg/L tigecycline, respectively. With increasing tigecycline selection pressure, the tet(A)TIG-carrying plasmids in the mutants increased in size, correlating with the number of tandem amplificates of a ΔTnAs1-flanked UCS harbouring tet(A)TIG. These tandem amplificates were not stable in the absence of tigecycline. CONCLUSIONS: Tigecycline resistance is due to the tandem amplification of a ΔTnAs1-flanked tet(A)TIG-carrying plasmid-borne segment in E. coli. The gain/loss of the tandem amplificates in the presence/absence of tigecycline represents an economic way for the bacteria to survive in the presence of tigecycline.

Promoter regulatory mode evolution enhances the high multidrug resistance of tmexCD1-toprJ1.

Antibiotic resistance could rapidly emerge from acquiring the mobile antibiotic resistance genes, which are commonly evolved from an intrinsic gene. The emergence of the plasmid-borne mobilized efflux pump gene cluster tmexCD1-toprJ1 renders the last-resort antibiotic tigecycline ineffective, although its evolutionary mechanism remains unclear. In this study, we investigate the regulatory mechanisms of the progenitor NfxB-MexCD-OprJ, a chromosomally encoded operon that does not mediate antibiotic resistance in the wild-type version, and its homologs, TNfxB1-TMexCD1-TOprJ1 mediating high-level tigecycline resistance, and TNfxB3-TMexCD3-TOprJ1. Mechanistic studies demonstrated that in nfxB-mexCD-oprJ, MexCD expression was under a weaker promoter, PmexC and inhibited by a strong repressor NfxB. For tmexCD1-toprJ1, TMexCD1 was highly expressed owing to the presence of a strong promoter, PtmexC1, and an inactive suppressor, TNfxB1, with a T39R mutation that rendered it unable to bind to promoter DNA. In tnfxB3-tmexCD3-toprJ1b, TMexCD3 expression was intermediate because of the local regulator TNfxB3, which binds to two inverted repeat sequences of PtmexC. Additionally, TNfxB3 exhibited lower protein expression and weaker DNA binding affinity than its ancestor NfxB, together with their promoter activities difference explaining the different expression levels of tmexCD-toprJ homologs. Distinct fitness burdens on these homologs-carrying bacteria were observed due to the corresponding expression level, which might be associated with their global prevalence. In summary, our data depict the mechanisms underlying the evolution and dissemination of an important mobile antibiotic resistance gene from an intrinsic chromosomal gene.IMPORTANCEAs antibiotic resistance seriously challenges global health, tigecycline is one of the few effective drugs in the pipeline against infections caused by multidrug-resistant pathogens. Our previous work identified a novel tigecycline resistance efflux pump gene cluster tmexCD1-toprJ1 in animals and humans, together with its various variants, a rising clinical concern. Herein, this study focused on how the local regulation modes of tmexCD1-toprJ1 evolved to a highly expressed efflux pump. Through comparative analysis between three tnfxB-tmexCD-toprJ homologs and their progenitor nfxB-mexCD-oprJ, modes, we demonstrated the evolutionary dynamics from a chromosomal silent gene to an active state. We found the de-repression of the local regulator and an increase of the promoter activity work together to promote a high production of drug efflux machines and enhance multidrug resistance. Our findings revealed that TMexCD1-TOprJ1 adopts a distinct evolutionary path to achieve higher multidrug resistance, urgently needing tight surveillance.

Clinical distribution of carbapenem genotypes and resistance to ceftazidime-avibactam in Enterobacteriaceae bacteria.

Introduction: Bacterial resistance is a major threat to public health worldwide. To gain an understanding of the clinical infection distribution, drug resistance information, and genotype of CRE in Dongguan, China, as well as the resistance of relevant genotypes to CAZ-AVI, this research aims to improve drug resistance monitoring information in Dongguan and provide a reliable basis for the clinical control and treatment of CRE infection. Methods: VITEK-2 Compact automatic analyzer was utilized to identify 516 strains of CRE collected from January 2017 to June 2023. To determine drug sensitivity, the K-B method, E-test, and MIC methods were used. From June 2022 to June 2023, 80 CRE strains were selected, and GeneXpert Carba-R was used to detect and identify the genotype of the carbapenemase present in the collected CRE strains. An in-depth analysis was conducted on the CAZ-AVI in vitro drug sensitivity activity of various genotypes of CRE, and the results were statistically evaluated using SPSS 23.0 and WHONET 5.6 software. Results: This study identified 516 CRE strains, with the majority (70.16%) being K.pneumoniae, followed by E.coli (18.99%). Respiratory specimens had highest detection rate with 53.77% identified, whereas urine specimens had the second highest detection rate with 17.99%. From June 2022 to June 2023, 95% of the strains tested using the CRE GeneXpert Carba-R assay possessed carbapenemase genes, of which 32.5% were blaNDM strains and 61.25% blaKPC strains. The results showed that CRE strains containing blaKPC had a significantly higher rate of resistance to amikacin, cefepime, and aztreonam than those harboring blaNDM. Conclusions: The CRE strains isolated from Dongguan region demonstrated a high resistance rate to various antibiotics used in clinical practice but a low resistance rate to tigecycline. These strains produce Class A serine carbapenemases and Class B metals ß-lactamases, with the majority of them carrying blaNDM and blaKPC. Notably, CRE strains with blaKPC and blaNDM had significantly lower resistance rates to tigecycline. CAZ-AVI showed a good sensitivity rate with no resistance to CRE strains carrying blaKPC. Therefore, CAZ-AVI and tigecycline should be used as a guide for rational use of antibiotics in clinical practice to effectively treat CRE.

Evaluation of the synergistic effect of eravacycline and tigecycline against carbapenemase-producing carbapenem-resistant Klebsiella pneumoniae.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a substantial healthcare challenge. This study assessed the in vitro efficacy of selected antibiotic combinations against CRKP infections. METHODS: Our research involved the evaluation of 40 clinical isolates of CRKP, with half expressing Klebsiella pneumoniae carbapenemase (KPC) and half producing Metallo-ß-lactamase (MBL), two key enzymes contributing to carbapenem resistance. We determined the minimum inhibitory concentrations (MICs) of four antibiotics: eravacycline, tigecycline, polymyxin-B, and ceftazidime/avibactam. Synergistic interactions between these antibiotic combinations were examined using checkerboard and time-kill analyses. RESULTS: We noted significant differences in the MICs of ceftazidime/avibactam between KPC and MBL isolates. Checkerboard analysis revealed appreciable synergy between combinations of tigecycline (35%) or eravacycline (40%) with polymyxin-B. The synergy rates for the combination of tigecycline or eravacycline with polymyxin-B were similar among the KPC and MBL isolates. These combinations maintained a synergy rate of 70.6% even against polymyxin-B resistant isolates. In contrast, combinations of tigecycline (5%) or eravacycline (10%) with ceftazidime/avibactam showed significantly lower synergy than combinations with polymyxin-B (P < 0.001 and P = 0.002, respectively). Among the MBL CRKP isolates, only one exhibited synergy with eravacycline or tigecycline and ceftazidime/avibactam combinations, and no synergistic activity was identified in the time-kill analysis for these combinations. The combination of eravacycline and polymyxin-B demonstrated the most promising synergy in the time-kill analysis. CONCLUSION: This study provides substantial evidence of a significant synergy when combining tigecycline or eravacycline with polymyxin-B against CRKP strains, including those producing MBL. These results highlight potential therapeutic strategies against CRKP infections.

Characterization of genes related to the efflux pump and porin in multidrug-resistant Escherichia coli strains isolated from patients with COVID-19 after secondary infection.

BACKGROUND: Escherichia coli (E. coli) is a multidrug resistant opportunistic pathogen that can cause secondary bacterial infections in patients with COVID-19. This study aimed to determine the antimicrobial resistance profile of E. coli as a secondary bacterial infection in patients with COVID-19 and to assess the prevalence and characterization of genes related to efflux pumps and porin. METHODS: A total of 50 nonduplicate E. coli isolates were collected as secondary bacterial infections in COVID-19 patients. The isolates were cultured from sputum samples. Confirmation and antibiotic susceptibility testing were conducted by Vitek 2. PCR was used to assess the prevalence of the efflux pump and porin-related genes in the isolates. The phenotypic and genotypic evolution of antibiotic resistance genes related to the efflux pump was evaluated. RESULTS: The E. coli isolates demonstrated high resistance to ampicillin (100%), cefixime (62%), cefepime (62%), amoxicillin-clavulanic acid (60%), cefuroxime (60%), and ceftriaxone (58%). The susceptibility of E. coli to ertapenem was greatest (92%), followed by imipenem (88%), meropenem (86%), tigecycline (80%), and levofloxacin (76%). Regarding efflux pump gene combinations, there was a significant association between the acrA gene and increased resistance to levofloxacin, between the acrB gene and decreased resistance to meropenem and increased resistance to levofloxacin, and between the ompF and ompC genes and increased resistance to gentamicin. CONCLUSIONS: The antibiotics ertapenem, imipenem, meropenem, tigecycline, and levofloxacin were effective against E. coli in patients with COVID-19. Genes encoding efflux pumps and porins, such as acrA, acrB, and outer membrane porins, were highly distributed among all the isolates. Efflux pump inhibitors could be alternative antibiotics for restoring tetracycline activity in E. coli isolates.

Antimicrobial activity of eravacycline and other comparative agents on aerobic and anaerobic bacterial pathogens in Taiwan: A clinical microbiological study.

OBJECTIVES: Eravacycline, a new tetracycline derivative, exhibits broad-spectrum antimicrobial susceptibility. This study aimed to comprehensively investigate in vitro activities of eravacycline, tigecycline, and ertapenem against various Gram-positive, Gram-negative, and anaerobic bacteria. METHODS: Minimum inhibitory concentrations (MICs) were determined using the broth microdilution method. The following bacterial species were collected: vancomycin-sensitive (VS) Enterococci species, vancomycin-resistant Enterococci species (VRE), Staphylococcus aureus, Streptococcus anginosus, Bacteroides species, Clostridioides difficile, Clostridium innocuum, Clostridium perfringens, Parabacteroides distasonis, and Stenotrophomonas maltophilia. RESULTS: We found that eravacycline exhibited superior in vitro activity compared to tigecycline and ertapenem. Notably, it exhibited the lowest MIC90 for several bacterial species, including VS E. faecalis (0.12 µg/mL), VS E. faecium (0.12 µg/mL), and others. Besides, VRE was susceptible to eravacycline (MIC90:0.12 µg/mL) and tigecycline (MIC90:0.12 µg/mL), but was all resistant to ertapenem (MIC90 > 64 µg/mL). S. aureus was also susceptible to eravacycline (MIC90:0.5 µg/mL) as well as tigecycline (MIC90:1.0 µg/mL). Furthermore, S. anginosus showed higher susceptibility to eravacycline (MIC90:2.0 µg/mL) and tigecycline (MIC90:4.0 µg/mL), but lower to ertapenem (MIC90:32.0 µg/mL). Eravacycline and tigecycline also demonstrated good susceptibility to anaerobes, including Bacteroides species (susceptibility rate: 100%), P. distasonis (100%), C. difficile (94.1‒100%), C. innocuum (94.1‒96.1%), and C. perfringens (88.9‒96.3%). For S. maltophilia, both tigecycline and eravacycline showed an MIC90 of 2 µg/mL. A moderate-to-strong correlation (rho = 0.608-0.804, P < 0.001) was noted between the MIC values of eravacycline and tigecycline against various bacterial species. CONCLUSIONS: Our study highlights the potential of eravacycline as an effective treatment option for multidrug-resistant bacterial infections.

Antimicrobial efficacy of eravacycline against emerging extensively drug-resistant (XDR) Acinetobacter baumannii isolates.

PURPOSE: Drug-resistant Acinetobacter baumannii is an emerging threat. This study has been conducted to observe the efficacy of eravacycline along with the RND-efflux pump system. METHODS: A cross-sectional study was done collecting 48 clinical isolates of Acinetobacter baumannii. MICs of 15 antibiotics were detected along with BMD of tigecycline and eravacycline. PCR products of drug-resistant regulatory genes were sequenced and analyzed. RESULTS: Of the total 48 Isolates, 35 (72.91%) were XDR and 13 (27.08%) were MDR. Out of all, 60.41% of isolates were found to be susceptible to eravacycline by BMD according to both FDA and EUCAST guidelines. A 2-fold decline of MIC50/90 was observed with the use of eravacycline compared to tigecycline. RND-efflux genes like AdeC in 30 (62.5%) isolates and Regulatory gene AdeS in 29 (60.41%) isolates were detected, explaining the existing resistance mechanism. CONCLUSIONS: XDR Acinetobacter poses an escalating threat due to its resistance to multiple antibiotics, raising serious concerns in healthcare settings. Eravacycline is an encouraging new drug for empirical use in severe infection caused due to the same. Molecular investigation and strict antimicrobial stewardship should be followed to control the emergence, and a better understanding of mechanisms of resistance to prevent the spread of drug-resistant isolates.

Gramine sensitizes Klebsiella pneumoniae to tigecycline killing.

BACKGROUND: The presence of plasmid-mediated resistance-nodulation-division (RND) efflux pump gene cluster tmexCD1-toprJ1 and its related variants has been associated with heightened resistance to tigecycline, thus diminishing its effectiveness. In this study, we explored the potential of gramine, a naturally occurring indole alkaloid, as an innovative adjuvant to enhance the treatment of infections caused by K. pneumoniae carrying tmexCD-toprJ-like gene clusters. METHODS: The synergistic potential of gramine in combination with antibiotics against both planktonic and drug-tolerant multidrug-resistant Enterobacterales was evaluated using the checkerboard microbroth dilution technique and time-killing curve analyses. Afterwards, the proton motive force (PMF) of cell membrane, the function of efflux pump and the activity of antioxidant system were determined by fluorescence assay and RT-PCR. The intracellular accumulation of tigecycline was evaluated by HPLC-MS/MS. The respiration rate, bacterial ATP level and the NAD+/NADH ratio were investigated to reveal the metabolism state. Finally, the safety of gramine was assessed through hemolytic activity and cytotoxicity assays. Two animal infection models were used to evaluate the in vivo synergistic effect. RESULTS: Gramine significantly potentiated tigecycline and ciprofloxacin activity against tmexCD1-toprJ1 and its variants-positive pathogens. Importantly, the synergistic activity was also observed against bacteria in special physiological states such as biofilms and persister cells. The mechanism study showed that gramine possesses the capability to augment tigecycline accumulation within cells by disrupting the proton motive force (PMF) and inhibiting the efflux pump functionality. In addition, the bacterial respiration rate, intracellular ATP level and tricarboxylic acid cycle (TCA) were promoted under the treatment of gramine. Notably, gramine effectively restored tigecycline activity in multiple animal infection models infected by tmexCD1-toprJ1 positive K. pneumoniae (RGF105-1). CONCLUSION: This study provides the first evidence of gramine's therapeutic potential as a novel tigecycline adjuvant for treating infections caused by K. pneumoniae carrying tmexCD-toprJ-like gene clusters.