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.

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.

Coexistence of blaIMP-4 and blaSFO-1 in an IncHI5B plasmid harbored by tigecycline-non-susceptible Klebsiella variicola strain.

BACKGROUND: Klebsiella variicola is considered a newly emerging human pathogen. Clinical isolates of carbapenemase and broad-spectrum ß-lactamase-producing K. variicola remain relatively uncommon. A strain of K. variicola 4253 was isolated from a clinical sample, and was identified to carry the blaIMP-4 and blaSFO-1 genes. This study aims to discern its antibiotic resistance phenotype and genomic characteristics. METHODS: Species identification was conducted using MALDI-TOF/MS. PCR identification confirmed the presence of the blaIMP-4 and blaSFO-1 genes. Antibiotic resistance phenotype and genomic characteristics were detected by antimicrobial susceptibility testing and whole-genome sequencing. Plasmid characterization was carried out through S1-PFGE, conjugation experiments, Southern blot, and comparative genomic analysis. RESULTS: K. variicola 4253 belonged to ST347, and demonstrated resistance to broad-spectrum ß-lactamase drugs and tigecycline while being insensitive to imipenem and meropenem. The blaIMP-4 and blaSFO-1 genes harbored on the plasmid p4253-imp. The replicon type of p4253-imp was identified as IncHI5B, representing a multidrug-resistant plasmid capable of horizontal transfer and mediating the dissemination of drug resistance. The blaIMP-4 gene was located on the In809-like integrative element (Intl1-blaIMP-4-aacA4-catB3), which circulates in Acinetobacter and Enterobacteriaceae. CONCLUSIONS: This study reports the presence of a strain of K. variicola, which is insensitive to tigecycline, carrying a plasmid harboring blaIMP-4 and blaSFO-1. It is highly likely that the strain acquired this plasmid through horizontal transfer. The blaIMP-4 array (Intl1-blaIMP-4-aacA4-catB3) is also mobile in Acinetobacter and Enterobacteriaceae. So it is essential to enhance clinical awareness and conduct epidemiological surveillance on multidrug-resistant K. variicola, conjugative plasmids carrying blaIMP-4, and the In809 integrative element.

[Analysis of epidemiological characteristics and risk factors of catheter-associated urinary tract infections in patients with perineal and/or hip burns].

Objective: To explore the epidemiological characteristics and risk factors of catheter-associated urinary tract infections in patients with perineal and/or hip burns. Methods: This study was a retrospective case series study. From January 2018 to December 2022, 260 patients with perineal and/or hip burns and urinary catheters indwelling who met the inclusion criteria were admitted to the Department of Burns and Wound Repair of the Second Affiliated Hospital of Zhejiang University School of Medicine, including 192 males and 68 females, aged 20-93 years. The total incidence of catheter-associated urinary tract infections in patients with perineal and/or hip burns, the detection of pathogenic bacteria, and the resistance of major Gram-negative and Gram-positive bacteria to commonly used antimicrobial drugs in clinic were recorded. According to whether catheter-associated urinary tract infection occurred or not, the patients were divided into infection group (43 cases) and non-infection group (217 cases). The basic conditions including gender, age, total burn area, depth of perineal burn, depth of hip burn, and burn site on admission, complications of diabetes mellitus, inhalation injury, and hypoproteinaemia, invasive operations including tracheotomy and non-perineal/hip debridement/skin transplantation surgery, duration of catheter retention, number of urethral catheterization, and bladder irrigation of patients between the two groups were compared, and the independent risk factors influencing the occurrence of catheter-associated urinary tract infections in patients with perineal and/or hip burns were screened. Results: The total incidence of catheter-associated urinary tract infections in patients with perineal and/or hip burns in this study was 16.5% (43/260). The pathogens detected were predominantly Gram-negative, followed by fungi; the main Gram-negative bacterium was Klebsiella pneumoniae, and the main Gram-positive bacterium was Enterococcus faecalis. The resistance rates of Klebsiella pneumoniae to amoxicillin/clavulanic acid, amitraz, amikacin, ciprofloxacin, ceftriaxone, and levofloxacin were higher than 70.0%, the resistance rates of Klebsiella pneumoniae to cefoxitin, cefoperazone/sulbactam, cefepime, meropenem, imipenem, and piperacillin/tazobactam ranged from 56.3% to 68.8%, and the resistance rates of Klebsiella pneumoniae to ceftazidime and tigecycline were lower than 50.0%. The resistance rates of Enterococcus faecalis to ciprofloxacin and penicillin were both 85.7%, the resistance rates of Enterococcus faecalis to erythromycin, clindamycin, moxifloxacin, and tetracycline ranged from 14.3% to 57.1%, and the resistance rates of Enterococcus faecalis to linezolid, tigecycline, and vancomycin were all 0. The differences were statistically significant between the two groups in terms of gender, status of complication of hypoproteinaemia, depth of perineal burn, status of non-perineal/hip debridement/skin transplantation surgery, status of bladder irrigation, number of urethral catheterization, and duration of catheter retention of patients (with χ2 values of 7.80, 4.85, 10.68, 9.11, and 16.48, respectively, and Z values of -4.88 and -5.42, respectively, P<0.05). There were no statistically significant differences in the age, total burn area, complications of diabetes mellitus and inhalation injury, burn site, depth of hip burns, and status of tracheotomy of patients between the two groups (P>0.05). Multifactorial logistic regression analysis showed that gender, deep partial-thickness perineal burns, non-perineal/hip debridement/skin transplantation surgery, bladder irrigation, and duration of catheter retention were the independent risk factors for catheter-associated urinary tract infections in patients with perineal and/or hip burns (with odds ratios of 2.86, 2.63, 2.79, 2.34, and 1.04, respectively, with 95% confidence intervals of 1.21-6.73, 1.03-6.71, 1.03-7.59, 1.05-5.22, and 1.02-1.06, respectively, P<0.05). Conclusions: The incidence of catheter-associated urinary tract infections is high in patients with perineal and/or hip burns, with Klebsiella pneumoniae as the predominant pathogenic bacteria having a high resistance rate to commonly used antimicrobial drugs in clinic. Gender, deep partial-thickness perineal burns, non-perineal/hip debridement/skin transplantation surgery, bladder irrigation, and duration of catheter retention are the independent risk factors for catheter-associated urinary tract infections in patients with perineal and/or hip burns.

Discovery of the tigecycline resistance gene cluster tmexCD3-toprJ1 in Pasteurella multocida strains isolated from pigs in China.

Pasteurella multocida is a leading cause of respiratory disorders in pigs. However, the genotypes and antimicrobial resistance characteristics of P. multocida from pigs in China have not been reported frequently. In this study, we investigated 381 porcine strains of P. multocida collected in China between 2013 and 2022. These strains were assigned to capsular genotypes A (69.55%, n = 265), D (27.82%, n =106), and F (2.62%, n = 10); or lipopolysaccharide genotypes L1 (1.31%, n = 5), L3 (24.41%, n = 93), and L6 (74.28%, n = 283). Overall, P. multocida genotype A:L6 (46.46%) was the most-commonly identified type, followed by D:L6 (27.82%), A:L3 (21.78%), F:L3 (2.62%), and A:L1 (1.31%). Antimicrobial susceptibility testing showed that a relatively high proportion of strains were resistant to tetracycline (66.67%, n = 254), and florfenicol (35.17%, n = 134), while a small proportion of strains showed resistance phenotypes to enrofloxacin (10.76%, n = 41), ampicillin (8.40%, n = 32), tilmicosin (7.09%, n = 27), and ceftiofur (2.89%, n = 11). Notably, Illumina short-read and Nanopore long-read sequencing identified a chromosome-borne tigecycline-resistance gene cluster tmexCD3-toprJ1 in P. multocida. The structure of this cluster was highly similar to the respective structures found in several members of Proteus or Pseudomonas. It is assumed that the current study identified the tmexCD3-toprJ1 cluster for the first time in P. multocida.

Tigecycline-associated hypofibrinogenemia: A single center, retrospective, controlled study.

BACKGROUND: Tigecycline-associated hypofibrinogenemia has been reported as an important adverse effect in recent years, but controlled studies minimizing confounding factors are needed. The objective of our study was to assess changes in fibrinogen levels in patients for hospitalization, comparing two antibiotic episodes (tigecycline and other) within the same patients. METHODS: The retrospective, self-controlled case series study was conducted at our University Hospitals. The study compared the change in fibrinogen levels during the patient's hospitalization for tigecycline (TigePer) and another antibiotic period (OtherPer). In addition, bleeding events, bleeding risk (determined by the IMPROVE bleeding risk score), as well as 15- and 30-day mortality rates between TigePer and OtherPer were compared. RESULTS: The study enrolled 50 patients with 100 episodes of antibiotic treatment. The median age (interquartile range) of the patients was 68.5 (21.5) years, and 38 % were female. As compared to OtherPer, TigePer had a statistically significant reduction in fibrinogen levels (p < 0.001), with a hypofibrinogenemia rate of 40 % in TigePer as compared to 2 % in OtherPer (p < 0.001). TigePer demonstrated a significantly higher 15-day mortality rate (p = 0.006). No significant differences were observed between the two periods in terms of bleeding risk, rate of bleeding events, and 30-day mortality rate (p > 0.05). CONCLUSION: Hypofibrinogenemia and other coagulopathies, without associated bleeding events, are more frequently observed in patients receiving tigecycline. Therefore, it is crucial for clinicians to monitor fibrinogen levels during tigecycline use.

Comparison of tet(X4)-containing contigs assembled from metagenomic sequencing data with plasmid sequences of isolates from a cohort of healthy subjects.

Recently discovered tet(X) gene variants have provided new insights into microbial antibiotic resistance mechanisms and their potential consequences for public health. This study focused on detection, analysis, and characterization of Tet(X4)-positive Enterobacterales from the gut microbiota of a healthy cohort of individuals in Singapore using cultivation-dependent and cultivation-independent approaches. Twelve Tet(X4)-positive Enterobacterales strains that were previously obtained from the cohort were fully genome-sequenced and comparatively analyzed. A metagenomic sequencing (MS) data set of the same samples was mined for contigs that harbored the tet(X4) resistance gene. The sequences of tet(X4)-containing contigs and plasmids sequences were compared. The presence of the resistance genes floR and estT (previously annotated as catD) was detected in the same cassette in 10 and 12 out of the 12 tet(X4)-carrying plasmids, respectively. MS detected tet(X4)-containing contigs in 2 out of the 109 subjects, while cultivation-dependent analysis previously reported a prevalence of 10.1%. The tet(X4)-containing sequences assembled from MS data are relatively short (~14 to 33 kb) but show high similarity to the respective plasmid sequences of the isolates. Our findings show that MS can complement efforts in the surveillance of antibiotic resistance genes for clinical samples, while it has a lower sensitivity than a cultivation-based method when the target organism has a low abundance. Further optimization is required if MS is to be utilized in antibiotic resistance surveillance.IMPORTANCEThe global rise in antibiotic resistance makes it necessary to develop and apply new approaches to detect and monitor the prevalence of antibiotic resistance genes in human populations. In this regard, of particular interest are resistances against last-resort antibiotics, such as tigecycline. In this study, we show that metagenomic sequencing can help to detect high abundance of the tigecycline resistance gene tet(X4) in fecal samples from a cohort of healthy human subjects. However, cultivation-based approaches currently remain the most reliable and cost-effective method for detection of antibiotic-resistant bacteria.

Occurrence and mechanisms of tigecycline resistance in carbapenem- and colistin-resistant Klebsiella pneumoniae in Thailand.

Tigecycline has been regarded as one of the most important last-resort antibiotics for the treatment of infections caused by extensively drug-resistant (XDR) bacteria, particularly carbapenem- and colistin-resistant Klebsiella pneumoniae (C-C-RKP). However, reports on tigecycline resistance have been growing. Overall, ~ 4000 K. pneumoniae clinical isolates were collected over a five-year period (2017-2021), in which 240 isolates of C-C-RKP were investigated. Most of these isolates (91.7%) were resistant to tigecycline. Notably, a high-risk clone of ST16 was predominantly identified, which was associated with the co-harboring of blaNDM-1 and blaOXA-232 genes. Their major mechanism of tigecycline resistance was the overexpression of efflux pump acrB gene and its regulator RamA, which was caused by mutations in RamR (M184V, Y59C, I141T, A28T, C99/C100 insertion), in RamR binding site (PI) of ramA gene (C139T), in MarR (S82G), and/or in AcrR (L154R, R13Q). Interestingly, four isolates of ST147 carried the mutated tet(A) efflux pump gene. To our knowledge, this is the first report on the prevalence and mechanisms of tigecycline resistance in C-C-RKP isolated from Thailand. The high incidence of tigecycline resistance observed among C-C-RKP in this study reflects an ongoing evolution of XDR bacteria against the last-resort antibiotics, which demands urgent action.

The effect of triclosan on intergeneric horizontal transmission of plasmid-mediated tigecycline resistance gene tet(X4) from Citrobacter freundii isolated from grass carp gut.

The transmission of antibiotic resistance genes (ARGs) in pathogenic bacteria affects culture animal health, endangers food safety, and thus gravely threatens public health. However, information about the effect of disinfectants - triclosan (TCS) on ARGs dissemination of bacterial pathogens in aquatic animals is still limited. One Citrobacter freundii (C. freundii) strain harboring tet(X4)-resistant plasmid was isolated from farmed grass carp guts, and subsequently conjugative transfer frequency from C. freundii to Escherichia coli C600 (E. coli C600) was analyzed under different mating time, temperature, and ratio. The effect of different concentrations of TCS (0.02, 0.2, 2, 20, 200 and 2000 µg/L) on the conjugative transfer was detected. The optimum conditions for conjugative transfer were at 37 °C for 8h with mating ratio of 2:1 or 1:1 (C. freundii: E. coli C600). The conjugative transfer frequency was significantly promoted under TCS treatment and reached the maximum value under 2.00 µg/L TCS with 18.39 times that of the control group. Reactive oxygen species (ROS), superoxide dismutase (SOD) and catalase (CAT) activities, cell membrane permeability of C. freundii and E. coli C600 were obviously increased under TCS stress. Scanning electron microscope showed that the cell membrane surface of the conjugative strains was wrinkled and pitted, even broken at 2.00 µg/L TCS, while lysed or even ruptured at 200.00 µg/L TCS. In addition, TCS up-regulated expression levels of oxidative stress genes (katE, hemF, bcp, hemA, katG, ahpF, and ahpC) and cell membrane-related genes (fimC, bamE and ompA) of donor and recipient bacteria. Gene Ontology (GO) enrichment demonstrated significant changes in categories relevant to pilus, porin activity, transmembrane transporter activity, transferase activity, hydrolase activity, material transport and metabolism. Taken together, a tet(X4)-resistant plasmid could horizontal transmission among different pathogens, while TCS can promote the propagation of the resistant plasmid.

Dual-targeting tigecycline nanoparticles for treating intracranial infections caused by multidrug-resistant Acinetobacter baumannii.

Multidrug-resistant (MDR) Acinetobacter baumannii (A. baumannii) is a formidable pathogen responsible for severe intracranial infections post-craniotomy, exhibiting a mortality rate as high as 71%. Tigecycline (TGC), a broad-spectrum antibiotic, emerged as a potential therapeutic agent for MDR A. baumannii infections. Nonetheless, its clinical application was hindered by a short in vivo half-life and limited permeability through the blood-brain barrier (BBB). In this study, we prepared a novel core-shell nanoparticle encapsulating water-soluble tigecycline using a blend of mPEG-PLGA and PLGA materials. This nanoparticle, modified with a dual-targeting peptide Aß11 and Tween 80 (Aß11/T80@CSs), was specifically designed to enhance the delivery of tigecycline to the brain for treating A. baumannii-induced intracranial infections. Our findings demonstrated that Aß11/T80@CSs nanocarriers successfully traversed the BBB and effectively delivered TGC into the cerebrospinal fluid (CSF), leading to a significant therapeutic response in a model of MDR A. baumannii intracranial infection. This study offers initial evidence and a platform for the application of brain-targeted nanocarrier delivery systems, showcasing their potential in administering water-soluble anti-infection drugs for intracranial infection treatments, and suggesting promising avenues for clinical translation.

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.

Regulation of metastatic potential by drug repurposing and mitochondrial targeting in colorectal cancer cells.

BACKGROUND: Increased mitochondrial activities contributing to cancer cell proliferation, invasion, and metastasis have been reported in different cancers; however, studies on the therapeutic targeting of mitochondria in regulating cell proliferation and invasiveness are limited. Because mitochondria are believed to have evolved through bacterial invasion in mammalian cells, antibiotics could provide an alternative approach to target mitochondria, especially in cancers with increased mitochondrial activities. In this study, we investigated the therapeutic potential of bacteriostatic antibiotics in regulating the growth potential of colorectal cancer (CRC) cells, which differ in their metastatic potential and mitochondrial functions. METHODS: A combination of viability, cell migration, and spheroid formation assays was used to measure the effect on metastatic potential. The effect on mitochondrial mechanisms was investigated by measuring mitochondrial DNA copy number by qPCR, biogenesis (by qPCR and immunoblotting), and functions by measuring reactive oxygen species, membrane potential, and ATP using standard methods. In addition, the effect on assembly and activities of respiratory chain (RC) complexes was determined using blue native gel electrophoresis and in-gel assays, respectively). Changes in metastatic and cell death signaling were measured by immunoblotting with specific marker proteins and compared between CRC cells. RESULTS: Both tigecycline and tetracycline effectively reduced the viability, migration, and spheroid-forming capacity of highly metastatic CRC cells. This increased sensitivity was attributed to reduced mtDNA content, mitochondrial biogenesis, ATP content, membrane potential, and increased oxidative stress. Specifically, complex I assembly and activity were significantly inhibited by these antibiotics in high-metastatic cells. Significant down-regulation in the expression of mitochondrial-mediated survival pathways, such as phospho-AKT, cMYC, phospho-SRC, and phospho-FAK, and upregulation in cell death (apoptosis and autophagy) were observed, which contributed to the enhanced sensitivity of highly metastatic CRC cells toward these antibiotics. In addition, the combined treatment of the CRC chemotherapeutic agent oxaliplatin with tigecycline/tetracycline at physiological concentrations effectively sensitized these cells at early time points. CONCLUSION: Altogether, our study reports that bacterial antibiotics, such as tigecycline and tetracycline, target mitochondrial functions specifically mitochondrial complex I architecture and activity and would be useful in combination with cancer chemotherapeutics for high metastatic conditions.

Resensitizing tigecycline- and colistin-resistant Escherichia coli using an engineered conjugative CRISPR/Cas9 system.

Tigecycline and colistin were referred to as the "last resort" antibiotics in defending against carbapenem-resistant, Gram-negative bacterial infections, and are currently widely used in clinical treatment. However, the emergence and prevalence of plasmid-mediated tet(X4) and mcr-1 genes pose a serious threat to the therapeutic application of tigecycline and colistin, respectively. In this research, a tigecycline- and colistin-resistant bacteria resensitization system was developed based on efficient and specific DNA damage caused by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Associated Protein 9 (Cas9) nucleases. A conjugation method was used to deliver the resensitization system, which harbors two single-guide RNAs targeting tet(X4) and mcr-1 genes and constitutively expressed Cas9. The conjugation efficiency was nearly 100% after conjugation condition optimization in vitro, and the resensitivity efficiency for clinical isolates was over 90%. In addition, when performing resensitization in vivo, the resistance marker was replaced with a glutamate-based, chromosomal, plasmid-balanced lethal system to prevent the introduction of additional resistance genes in clinical settings, making this strategy a therapeutic approach to combat the in vivo spread of antibiotic resistance genes (ARGs) among bacterial pathogens. As a proof of concept, this resensitive system can significantly decrease the counts of tigecycline- and colistin-resistant bacteria to 1% in vivo. Our study demonstrates the efficacy and adaptability of CRISPR-Cas systems as powerful and programmable antimicrobials in resensitizing tet(X4)- and mcr-1-mediated, tigecycline- and colistin-resistant strains, and opens up new pathways for the development of CRISPR-based tools for selective bacterial pathogen elimination and precise microbiome composition change. IMPORTANCE: The emergence of plasmid-encoded tet(X4) and mcr-1 isolated from human and animal sources has affected the treatment of tigecycline and colistin, and has posed a significant threat to public health. Tigecycline and colistin are considered as the "last line of defense" for the treatment of multidrug-resistant (MDR) Gram-negative bacterial infections, so there is an urgent need to find a method that can resensitize tet(X4)-mediated tigecycline-resistant and mcr-1-mediated colistin-resistant bacteria. In this study, we developed a glutamate-based, chromosomal, plasmid-balanced lethal conjugative CRISPR/Cas9 system, which can simultaneously resensitize tet(X4)-mediated tigecycline-resistant and mcr-1-mediated colistin-resistant Escherichia coli. The counts of tigecycline- and colistin-resistant bacteria decreased to 1% in vivo after the resensitization system was administered. This study opens up new pathways for the development of CRISPR-based tools for selective bacterial pathogen elimination and precise microbiome composition change.

Earliest observation of the tetracycline destructase tet(X3).

Tigecycline is an antibiotic of last resort for infections with carbapenem-resistant Acinetobacter baumannii. Plasmids harboring variants of the tetracycline destructase gene tetX promote rising tigecycline resistance rates. We report the earliest observation of tet(X3) in a clinical strain predating tigecycline's commercialization, suggesting selective pressures other than tigecycline contributed to its emergence. IMPORTANCE: We present the earliest observation of a tet(X3)-positive bacterial strain, predating by many years the earliest reports of this gene so far. This finding is significant as tigecycline is an antibiotic of last resort for carbapenem-resistant Acinetobacter baumannii (CRAB), which the World Health Organization ranks as one of its top three critical priority pathogens, and tet(X3) variants have become the most prevalent genes responsible for enabling CRAB to become tigecycline resistant. Moreover, the tet(X3)-positive strain we report is the first and only to be found that predates the commercialization of tigecycline, an antibiotic that was thought to have contributed to the emergence of this resistance gene. Understanding the factors contributing to the origin and spread of novel antibiotic resistance genes is crucial to addressing the major global public health issue, which is antimicrobial resistance.

The Mechanism of Tigecycline Resistance in Acinetobacter baumannii under Sub-Minimal Inhibitory Concentrations of Tigecycline.

The presence of sub-minimal inhibitory concentration (sub-MIC) antibiotics in our environment is widespread, and their ability to induce antibiotic resistance is inevitable. Acinetobacter baumannii, a pathogen known for its strong ability to acquire antibiotic resistance, has recently shown clinical resistance to the last-line antibiotic tigecycline. To unravel the complex mechanism of A. baumannii drug resistance, we subjected tigecycline-susceptible, -intermediate, and -mildly-resistant strains to successive increases in sub-MIC tigecycline and ultimately obtained tigecycline-resistant strains. The proteome of both key intermediate and final strains during the selection process was analyzed using nanoLC-MS/MS. Among the more than 2600 proteins detected in all strains, we found that RND efflux pump AdeABC was associated with the adaptability of A. baumannii to tigecycline under sub-MIC pressure. qRT-PCR analysis also revealed higher expression of AdeAB in strains that can quickly acquire tigecycline resistance compared with strains that displayed lower adaptability. To validate our findings, we added an efflux pump inhibitor, carbonyl cyanide m-chlorophenyl hydrazine (CCCP), to the medium and observed its ability to inhibit tigecycline resistance in A. baumannii strains with quick adaptability. This study contributes to a better understanding of the mechanisms underlying tigecycline resistance in A. baumannii under sub-MIC pressure.

Co-transfer of IncFII/IncFIB and IncFII plasmids mediated by IS26 facilitates the transmission of mcr-8.1 and tmexCD1-toprJ1.

PURPOSE: This study aimed to characterise the whole-genome structure of two clinical Klebsiella pneumoniae strains co-harbouring mcr-8.1 and tmexCD1-toprJ1, both resistant to colistin and tigecycline. METHODS: K. pneumoniae strains TGC-02 (ST656) and TGC-05 (ST273) were isolated from urine samples of different patients hospitalised at separate times in 2021. Characterisation involved antimicrobial susceptibility testing (AST), conjugation assays, whole-genome sequencing (WGS), and bioinformatics analysis. Comparative genomic analysis was conducted on mcr-8.1-carrying and tmexCD1-toprJ1-carrying plasmids. RESULTS: Both K. pneumoniae isolates displayed a multidrug-resistant phenotype, exhibiting resistance or reduced susceptibility to ampicillin, ampicillin/sulbactam, cefazolin, aztreonam, amikacin, gentamicin, tobramycin, ciprofloxacin, levofloxacin, nitrofurantoin, trimethoprim/sulfamethoxazole, apramycin, tigecycline and colistin. WGS analysis revealed that clinical strain TGC-02 carried the TmexCD1-toprJ1 gene on a 200-Kb IncFII/IncFIB-type plasmid, while mcr-8 was situated on a 146-Kb IncFII-type plasmid. In clinical strain TGC-05, TmexCD1-toprJ1 was found on a 300-Kb IncFIB/IncHI1B/IncR-type plasmid, and mcr-8 was identified on a 137-Kb IncFII/IncFIA-type plasmid. Conjugation experiments assessed the transferability of these plasmids. While transconjugants were not obtained for TGC-05 despite multiple screening with tigecycline or colistin, pTGC-02-tmex and pTGC-02-mcr8 from clinical K. pneumoniae TGC-02 demonstrated self-transferability through conjugation. Notably, the rearrangement of pTGC-02-tmex and pTGC-02-mcr8 via IS26-based homologous recombination was observed. Moreover, the conjugative and fusion plasmids of the transconjugant co-harboured the tmexCD1-toprJ1 gene cluster and mcr-8.1, potentially resulting from IS26-based homologous recombination. CONCLUSION: The emergence of colistin- and tigecycline-resistant K. pneumoniae strains is concerning, and effective surveillance measures should be implemented to prevent further dissemination.

High ceftazidime-avibactam resistance among carbapenem-resistant Enterobacter species: Data from the Antimicrobial Testing Leadership and Surveillance (ATLAS) programme, 2014-2021.

OBJECTIVES: Trends in the susceptibility to ceftazidime-avibactam (CZA) and tigecycline (TGC) among Enterobacter species from different geographic areas are unknown.This study aimed to analyse the trends in CZA and TGC susceptibility changes across different continents from 2014 to 2021 utilizing Antimicrobial Testing Leadership and Surveillance (ATLAS) data. METHODS: A total of 23 669 isolates of Enterobacter species were collected over an 8-y period. RESULTS: The overall non-susceptibility rate of Enterobacter isolates to both CZA and TGC was 3.2%. India (16.5%), Guatemala (15.4%), and the Philippines (13.1%) exhibited the highest resistance to CZA. The increase in CZA resistance rates was particularly evident in Asia, with an increase from 4.0% to 8.3%, and in Latin America, from 1.5% to 5%. The non-susceptibility rate for TGC mildly increased in Africa/Middle East but decreased in other continents during the study period. The overall rate of carbapenem resistance increased from 2.9% in 2014-2017 to 4.3% in 2018-2021. Among carbapenem-resistant Enterobacter isolates, the CZA resistance rate was highest in Asia (87.4%), followed by Europe (69.2%) and Africa/Middle East (60.8%). Among the 380 Enterobacter isolates resistant to CZA and carbapenem, the most common genotype of carbapenemase genes was blaNDM (59.2%), followed by blaVIM (24.2%), blaOXA (4.2%), blaIMP (1.1%), and blaKPC (1.1%). The susceptibility of carbapenem-resistant Enterobacter to TGC remained high, with an overall susceptibility rate of 90%. CONCLUSIONS: The heterogeneous distribution of CZA resistance rates among different geographical regions highlights the divergent therapeutic options for drug-resistant Enterobacter species.