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Enterobacterales (especially carbapenem-resistant) are considered an urgent threat to public health. The available antibiotic therapy is limited due to the increase of multidrug-resistant (MDR) strains. Tigecycline, a minocycline derivative, has emerged as a potential key agent in the treatment ofMDR isolates. The aim of the study was to evaluate the synergistic effect of tigecycline in combination with nine antimicrobial agents--ceftazidime/avibactam, colistin, ertapenem, gentamicin, imipenem, levofloxacin, meropenem/vaborbactam, polymyxin B, and rifampicin. Eighty clinical Enterobacter cloacae strains were obtained frompatients of two University Hospitals in Bialystok, Poland. The E-test method was used to determine synergistic interactions. Among all combinations, synergy was reported in 61% of cases, addition in 32%, and indifference in 7%. The highest synergy rates were observed in tigecycline combinations with: ceftazidime/avibactam(60/80; 75%), imipenem(60/80; 75%), polymyxin B (55/80; 68.75%) and rifampicin (55/80; 68.75%), while the lowest synergy rate was noted in tigecycline-levofloxacin (26/80; 32.5%). The tigecycline-gentamicin showed the highest rate of indifference; antagonism, was not observed in any combination. In conclusion, tigecycline appears more suitable for use in combination therapy rather than as monotherapy and can be effectively paired with various antimicrobial agents against MDR E. cloacae. Further research will be necessary to confirm these results. [ABSTRACT FROM AUTHOR]

Tigecycline (TGC) is currently used to treat carbapenem-resistant Acinetobacter baumannii (CRAB) infections, while eravacycline (ERV), a new-generation tetracycline, holds promise as a novel therapeutic option for these infections. However, differences in resistance mechanism between ERV and TGC against A. baumannii remain unclear. This study sought to compare the characteristics and mechanisms of ERV and TGC resistance among clinical A. baumannii isolates. A total of 492 isolates, including 253 CRAB and 239 carbapenemsensitive A. baumannii (CSAB) isolates, were collected from hospitalized patients in China. The MICs of ERV and TGC against A. baumannii were determined by broth microdilution. Genetic mutations and expressions of adeB, adeG, adeJ, adeS, adeL, and adeN in resistant strains were examined by PCR and qPCR, respectively. The in vitro recombination experiments were used to verify the resistance mechanism of ERV and TGC in A. baumannii. The MIC90 of ERV in CRAB and CSAB isolates were lower than those of TGC. A total of 24 strains resistant to ERV and/or TGC were categorized into three groups: only ERVresistant (n = 2), both ERV- and TGC-resistant (n = 7), and only TGC-resistant (n = 15). ST208 (75%, n = 18) was a major clone that has disseminated in all three groups. The ISAba1 insertion in adeS was identified in 66.7% (6/9) of strains in the only ERV-resistant and both ERV- and TGC-resistant groups, while the ISAba1 insertion in adeN was found in 53.3% (8/15) of strains in the only TGC-resistant group. The adeABC and adeRS expressions were significantly increased in the only ERV-resistant and both ERV- and TGC-resistant groups, while the adeABC and adeIJK expressions were significantly increased and adeN was significantly decreased in the only TGC-resistant group. Expression of adeS with the ISAba1 insertion in ERV- and TGC-sensitive strains significantly increased the ERV and TGC MICs and upregulated adeABC and adeRS expressions. Complementation of the wildtype adeN in TGC-resistant strains with the ISAba1 insertion in adeN restored TGC sensitivity and significantly downregulated adeIJK expression. In conclusion, our data illustrates that ERV is more effective against A. baumannii clinical isolates than TGC. ERV resistance is correlated with the ISAba1 insertion in adeS, while TGC resistance is associated with the ISAba1 insertion in adeN or adeS in A. baumannii. [ABSTRACT FROM AUTHOR]

Introduction: The dissemination of strains producing tetracyclines monooxygenase Tet(X) from breeding farms to the natural environment poses a potential threat to public health. Methods: Antimicrobial susceptibility testing and WGS were performed to identify resistance phenotypes and genotypes. Cloning experiments, sequence alignment, and homology modeling were used to characterize the function and formation mechanisms of the recombinant variant. The mobilization potential of Tet(X) was assessed by collinearity analysis, conjugation experiments, and phylogenetic analysis. Results: Three tet(X)-producing Elizabethkingia meningoseptica strains were isolated from bullfrog breeding ponds, the sewage outlet, and downstream river in Zhejiang Province, China. These strains carry a novel Tet(X) variant, differing from Tet(X6) by seven residues, and possess the ability to degrade tetracyclines. Interestingly, the novel Tet(X) is a recombinant variant formed by homologous recombination of Tet(X6) and the C-terminal of Tet(X2). Further analysis revealed that Tet(X6) formed several Tet(X) variants, including Tet(X5), through homologous recombination. The novel tet(X) gene is located on a circularizable integrative and conjugative element (ICEEmeChn3), with ISwz1 participating in the recombination of its multi-drug resistance region, potentially facilitating the mobilization and recombination of tet(X) in early hosts. These three strains were clonally transmitted and shared a close genetic relationship (SNP < 62) with a clinically-sourced strain isolated from the same province. Discussion: To our knowledge, this is the first report of homologous recombination between Tet(X) variants with differing activities. These clonal strains provide evidence of the transmission of tet(X)-positive strains from aquaculture sewage to the natural environment, highlighting the need to strengthen the monitoring and management of this emerging farming model. [ABSTRACT FROM AUTHOR]

The emergence and widespread of tigecycline resistance undoubtedly poses a serious threat to public health globally. The exploration of combination therapies has become preferred antibacterial strategies to alleviate this global burden. In this study, tigecycline-resistant tet(X4)-positive Escherichia coli were selected for adjuvant screening. Interestingly, 9-aminominocycline (9-AMC), one of the tigecycline metabolites, exhibits synergistic antibacterial activity with tigecycline using checkerboard assay. The efficacy in vitro and in vivo was evaluated, and the synergistic mechanism was further explored. The results suggested that 9-AMC combined with tigecycline could inhibit the growth of antibiotic resistant bacteria, efficiently retard the evolution of tet(X4) gene and narrow the drug mutant selection window. In addition, the combination of tigecycline and 9-AMC could destroy the normal membrane structure of bacteria, inhibit the formation of biofilm, remarkably reduce the level of intracellular ATP level, and accelerate the oxidative damage of bacteria. Furthermore, 9-AMC is more stable in the bind of Tet(X4) inactivating enzyme. The transcriptomics analysis revealed that the genes related to the 9-AMC and tigecycline were mainly enriched in ABC transporters. Collectively, the results reveal the potentiation effects on tigecycline and the probability of 9-AMC as a novel tigecycline adjuvant against tet(X4)-positive Escherichia coli, which provides new insights for adjuvant screening. [ABSTRACT FROM AUTHOR]

Introduction: The rapid spread of plasmid-mediated tet(X4) conferring high tigecycline resistance poses a significant threat to public health. Escherichia coli as the most common pathogen which carries tet(X4) has been widely disseminated in China. Thus, comprehensive investigations are required to understand the mechanism of transmission of tet(X4)-positive E. coli. Methods: In this study, a total of 775 nonduplicate samples were collected in Guangdong, China from 2019 to 2020. We screened for tet(X4)-positive E. coli by PCR amplification and species identification. Furthermore, we analyzed the phylogenetics and genetic context of tet(X4)-positive E. coli through wholegenome sequencing and long-reads sequencing. Results: Overall, 146 (18.84%) tet(X4)-positive E. coli were isolated, comprising 2 isolates from humans and 144 isolates from pigs. The majority of tet(X4)-positive E. coli exhibited resistance to multiple antibiotics but all of them were susceptible to amikacin and colistin. Phylogenetic analysis showed that ST877, ST871, and ST195 emerged as the predominant sequence types in tet(X4)-positive E. coli. Further analysis revealed various genetic environments associated with the horizontal transfer of tet(X4). Notably, a 100-kbp large fragment insertion was discovered downstream of tet(X4), containing a replicon and a 40-kbp gene cluster for the bacterial type IV secretion system. Discussion: The high colonization rate of tet(X4)-positive E. coli in animals suggests that colonization as a key factor in its dissemination to humans. Diverse genetic context may contribute to the transfer of tet(X4). Our findings underline the urgent need for controlling the spread of plasmid-mediated tigecycline resistance. [ABSTRACT FROM AUTHOR]

Enterobacterales (especially carbapenem-resistant) are considered an urgent threat to public health. The available antibiotic therapy is limited due to the increase of multidrug-resistant (MDR) strains. Tigecycline, a minocycline derivative, has emerged as a potential key agent in the treatment of MDR isolates. The aim of the study was to evaluate the synergistic effect of tigecycline in combination with nine antimicrobial agents—ceftazidime/avibactam, colistin, ertapenem, gentamicin, imipenem, levofloxacin, meropenem/vaborbactam, polymyxin B, and rifampicin. Eighty clinical Enterobacter cloacae strains were obtained from patients of two University Hospitals in Bialystok, Poland. The E-test method was used to determine synergistic interactions. Among all combinations, synergy was reported in 61% of cases, addition in 32%, and indifference in 7%. The highest synergy rates were observed in tigecycline combinations with: ceftazidime/avibactam (60/80; 75%), imipenem (60/80; 75%), polymyxin B (55/80; 68.75%) and rifampicin (55/80; 68.75%), while the lowest synergy rate was noted in tigecycline-levofloxacin (26/80; 32.5%). The tigecycline-gentamicin showed the highest rate of indifference; antagonism, was not observed in any combination. In conclusion, tigecycline appears more suitable for use in combination therapy rather than as monotherapy and can be effectively paired with various antimicrobial agents against MDR E. cloacae. Further research will be necessary to confirm these results.

Background: Due to the growing resistance to routinely used antibiotics, the search for new antibiotics or their combinations with effective inhibitors against multidrug-resistant microorganisms is ongoing. In our study, we assessed the in vitro drug susceptibility of Klebsiella pneumoniae strains producing New Delhi metallo-ß-lactamases (NDM) to antibiotics included in the Infectious Diseases Society of America (IDSA) and European Society of Clinical Microbiology and Infectious Diseases (ESCMID) recommendations. Methods: A total of 60 strains of NDM-producing K. pneumoniae were obtained from different patients hospitalized at the 4th Military Hospital in Wroclaw between 2019 and 2022 and subjected to drug susceptibility to selected antibiotics, including the effects of drug combinations. Results: Among the tested antibiotics, the highest sensitivity (100%) was observed for cefiderocol, eravacycline (interpreted according to the European Committee on Antimicrobial Susceptibility Testing [EUCAST]), and tigecycline. Sensitivity to intravenous fosfomycin varied depending on the method used. Using the "strip stacking" method, determining cumulative sensitivity to ceftazidime/avibactam and aztreonam demonstrated 100% in vitro sensitivity to this combination among the tested strains. Conclusion: The in vitro susceptibility assessment demonstrated that, the best therapeutic option for treating infections caused by carbapenemase-producing strains seems to be a combination of ceftazidime/avibactam with aztreonam. Due to the safety of using both drugs, cost effectiveness, and the broadest indications for use among the tested antibiotics, this therapy should be the first-line treatment for carbapenemase-producing Enterobacterales infections. Nevertheless, a comprehensive evaluation of the efficacy of treating infections caused by NDM-producing K. pneumoniae strains should include not only in vitro susceptibility assessment but also an analysis of clinical cases. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii is one of the most common multidrug-resistant pathogens causing nosocomial infections. The prevalence of multidrugresistant A. baumannii infections is increasing because of several factors, including unregulated antibiotic use. A. baumannii drug resistance rate is high; in particular, its resistance rates for tigecycline and polymyxin--the drugs of last resort for extensively drug-resistant A. baumannii--has been increasing annually. Patients with a severe infection of extensively antibiotic-resistant A. baumannii demonstrate a high mortality rate along with a poor prognosis, which makes treating them challenging. Through carbapenem enzyme production and other relevant mechanisms, A. baumannii has rapidly acquired a strong resistance to carbapenem antibiotics--once considered a class of strong antibacterials for A. baumannii infection treatment. Therefore, understanding the resistance mechanism of A. baumannii is particularly crucial. This review summarizes mechanisms underlying common antimicrobial resistance in A. baumannii, particularly those underlying tigecycline and polymyxin resistance. This review will serve as a reference for reasonable antibiotic use at clinics, as well as new antibiotic development. [ABSTRACT FROM AUTHOR]

Objective: The co-occurrence of colistin and tigecycline resistance genes in Klebsiella pneumoniae poses a serious public health problem. This study aimed to characterize a K. pneumoniae strain, K82, co-harboring a colistin resistance gene (CoRG) and tigecycline resistance gene (TRG), and, importantly, investigate the genetic characteristics of the plasmid with CoRG or TRG in GenBank. Methods: K. pneumoniae strain K82 was subjected to antimicrobial susceptibility testing, conjugation assay, and whole-genome sequencing (WGS). In addition, comparative genomic analysis of CoRG or TRG-harboring plasmids from K82 and GenBank was conducted. K. pneumoniae strain K82 was resistant to all the tested antimicrobials including colistin and tigecycline, except for carbapenems. Results: WGS and bioinformatic analysis showed that K82 belonged to the ST656 sequence type and carried multiple drug resistance genes, including mcr-1 and tmexCD1-toprJ1, which located on IncFIA/IncHI2/IncHI2A/IncN/IncRtype plasmid pK82-mcr-1 and IncFIB/IncFII-type plasmid pK82-tmexCD-toprJ, respectively. The pK82-mcr-1 plasmid was capable of conjugation. Analysis of the CoRG/TRG-harboring plasmid showed that mcr-8 and tmexCD1-toprJ1 were the most common CoRG and TRG of Klebsiella spp., respectively. These TRG/CoRGharboring plasmids could be divided into two categories based on mash distance. Moreover, we found an IncFIB/IncHI1B-type plasmid, pSYCC1_tmex_287k, coharboring mcr-1 and tmexCD1-toprJ1. To the best of our knowledge, this is the first report on the co-occurrence of mcr-1 and tmexCD1-toprJ1 on a single plasmid. Conclusion: Our research expands the known diversity of CoRG and TRG-harboring plasmids in K. pneumoniae. Effective surveillance should be implemented to assess the prevalence of co-harboring CoRG and TRG in a single K. pneumoniae isolate or even a single plasmid. [ABSTRACT FROM AUTHOR]

Objectives: To investigate the in vitro activity of antibiotic combinations against Stenotrophomonas maltophilia isolates and their associated biofilms. Methods: Thirty-two S. maltophilia clinical isolates with at least twenty-five different pulsotypes were tested. The antibacterial activity of various antibiotic combinations against seven randomly selected planktonic and biofilm-embedded S. maltophilia strains with strong biofilm formation was assessed using broth methods. Extraction of bacterial genomic DNA and PCR detection of antibiotic resistance and biofilm-related genes were also performed. Results: The susceptibility rates of levofloxacin (LVX), fosfomycin (FOS), tigecycline (TGC) and sulfamethoxazole-trimethoprim (SXT) against 32 S. maltophilia isolates were 56.3, 71.9, 71.9 and 90.6%, respectively. Twenty-eight isolates were detected with strong biofilm formation. Antibiotic combinations, including aztreonamclavulanic (ATM-CLA) with LVX, ceftazidime-avibactam (CZA) with LVX and SXT with TGC, exhibited potent inhibitory activity against these isolates with strong biofilm formation. The antibiotic resistance phenotype might not be fully caused by the common antibiotic-resistance or biofilm-formation gene. Conclusion: S. maltophilia remained resistant to most antibiotics, including LVX and ß-lactam/ß-lactamases; however, TGC, FOS and SXT still exhibited potent activity. Although all tested S. maltophilia isolates exhibited moderate-to-strong biofilm formation, combination therapies, especially ATM-CLA with LVX, CZA with LVX and SXT with TGC, exhibited a higher inhibitory activity for these isolates. [ABSTRACT FROM AUTHOR]

Introduction: This study aimed to investigate the genetic factors promoting widespread Q6 dissemination of tet(X4) between Escherichia coli and to characterize the genetic contexts of tet(X4). Methods: We isolated E. coli from feces, water, soil and flies collected across a largescale chicken farm in China in 2020. Antimicrobial susceptibility testing and PFGE typing were used to identify tigecycline resistance and assess clonal relationships among isolates. Plasmids present and genome sequences were analyzed by conjugation, S1 pulsed-field gel electrophoresis (PFGE), plasmid stability testing and whole-genome sequencing. Results: A total of 204 tigecycline-resistant E. coli were isolated from 662 samples. Of these, we identified 165 tet(X4)-carrying E. coli and these strains exhibited a high degree of multidrug resistance. Based on the geographical location distribution of the sampled areas, number of samples in each area and isolation rate of tigecyclineresistant strains and tet(X4)-carrying isolates, 72 tet(X4)-positive isolates were selected for further investigation. Tigecycline resistance was shown to be mobile in 72 isolates and three types of tet(X4)-carrying plasmids were identified, they were IncHI1 (n= 67), IncX1 (n= 3) and pO111-like/IncFIA(HI1) (n= 2). The pO111-like/IncFIA(HI1) is a novel plasmid capable of transferring tet(X4). The transfer efficiency of IncHI1 plasmids was extremely high in most cases and IncHI1 plasmids were stable when transferred into common recipient strains. The genetic structures flanked by IS1, IS26 and ISCR2 containing tet(X4) were complex and varied in different plasmids. Discussion: The widespread dissemination of tigecycline-resistant E. coli is a major threat to public health. This data suggests careful use of tetracycline on farms is important to limit spread of resistance to tigecycline. Multiple mobile elements carrying tet(X4) are in circulation with IncHI1 plasmids the dominant vector in this setting. [ABSTRACT FROM AUTHOR]

The emergence and spread of carbapenemase genes, colistin resistance genes mcr-1, and tigecycline resistance gene tet(X) represent a significant threat to clinical therapy and public health. In this study, we investigated the presence of carbapenemase genes, mcr-1, and tet(X) in 298 Escherichia coli strains obtained froma teaching hospital inChina. In total, eight (2.68%), six (2.01%), and one (0.34%) E. coli isolates carried blaNDM, mcr-1, and tet(X4), respectively. The blaNDM gene was located on IncX3 (n = 4), F2:A-:B- (n = 3), and F2:A1:B1 (n = 1) plasmids, with high similarity to multiple plasmids belonging to the same incompatibility type from Enterobacteriaceae. Six MCR-producing strains contained mcr-1-carrying IncI2 plasmids, organized similarly to other mcr-1-bearing IncI2 plasmids from animals in China. The blaCTX--M--55/64/132/199 gene located within a typical transposition unit (ISEcp1-blaCTX--M-orf477Δ) was inserted near dnaJ to generate 5-bp direct repeats in four mcr-1-positive plasmids. The tet(X) and another four resistance genes [aadA2, tet(A), floR, and Δlnu(F)] were co-located on an IncX1 plasmid, highly similar to other tet(X4)-carrying IncX1 plasmids from Escherichia and Klebsiella of animal or food origin, except that the conjugative transfer region of IncX1 plasmids was absent in our plasmid. Although a low prevalence of blaNDM, mcr-1, and tet(X) was observed in E. coli from patients in this study, their dissemination associated with some successful pandemic plasmids is of great concern. The continued surveillance of these crucial resistance genes in patients should be strengthened. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii (A. baumannii) is now considered a highly resistant pathogen to various types of antibiotics. Therefore, tracking the source of its prevalence and continuous control is crucial. This study aimed to determine antibiotic resistance and perform various molecular typing methods on clinical isolates of A. baumannii isolated from hospitalized burn patients in Shahid Motahari Burn Hospital, Tehran, Iran. Hospital isolates were confirmed by phenotypic and molecular methods. Then the sensitivity to different antibiotics was determined using the minimum inhibitory concentration (MIC) method. In order to perform molecular typing, three-locus dual assay multiplex polymerase chain reaction (PCR), multiple-locus variable-number tandem repeat analysis (MLVA), and multilocus sequence typing (MLST) methods were used. Among the 60 isolates collected, the frequencies of multidrugresistant (MDR) and extensively drug-resistant (XDR) isolates were 90 and 10%, respectively. The most effective antibiotics were colistin with 100% and tigecycline with 83.33% sensitivity. Isolates were 100% resistant to piperacillin/ tazobactam and cephalosporins, and 68.3% were resistant to carbapenem. The results of multiplex PCR showed five groups that international clone I (IC I) and IC II were the most common. The MLVA method identified 34 MLVA types (MTs), 5 clusters, and 25 singletons. Multilocus sequence typing results for tigecycline-resistant isolates showed seven different sequence types (STs). Increasing antibiotic resistance in A. baumannii isolates requires careful management to control and prevent the occurrence of the pre-antibiotic era. The results of this study confirm that the population structure of A. baumannii isolates has a high diversity. More extensive studies are needed in Iran to better understand the epidemiology of A. baumannii. [ABSTRACT FROM AUTHOR]

The prevalence of plasmid-mediated tigecycline resistance gene tet(X4) is presenting an increasing trend. Once tet(X4)-bearing plasmids are captured by multidrug-resistant bacteria, such as blaNDM and mcr-coharboring bacteria, it will promote bacteria to develop an ultra-broad resistance spectrum, limiting clinical treatment options. However, little is known about the destiny of such bacteria or how they will evolve in the future. Herein, we constructed a multidrug-resistant bacteria coharboring tet(X4), blaNDM-5, and mcr-1 by introducing a tet(X4)-bearing plasmid into a blaNDM-5 and mcr-1 positive E. coli strain. Subsequently, the stability of tet(X4) and the plasmid was measured after being evolved under tigecycline or antibiotic-free circumstance. Interestingly, we observed both tet(X4)-bearing plasmids in tigecycline treated strains and non-tigecycline treated strains were stable, which might be jointly affected by the increased conjugation frequency and the structural alterations of the tet(X4)-positive plasmid. However, the stability of tet(X4) gene showed different scenarios in the two types of evolved strains. The tet(X4) gene in nontigecycline treated strains was stable whereas the tet(X4) gene was discarded rapidly in tigecycline treated strains. Accordingly, we found the expression levels of tet(X4) gene in tigecycline-treated strains were several times higher than in non-tigecycline treated strains and ancestral strains, which might in turn impose a stronger burden on the host bacteria. SNPs analysis revealed that a myriad of mutations occurred in genes involving in conjugation transfer, and the missense mutation of marR gene in chromosome of tigecycline treated strains might account for the completely different stability of tet(X4)-bearing plasmid and tet(X4) gene. Collectively, these findings shed a light on the possibility of the emergence of multidrug resistant bacteria due to the transmission of tet(X4)-bearing plasmid, and highlighted that the antibiotic residues may be critical to the development of such bacteria. [ABSTRACT FROM AUTHOR]

With the large-scale use of antibiotics, antibiotic resistant bacteria (ARB) continue to rise, and antibiotic resistance genes (ARGs) are regarded as emerging environmental pollutants. The new tetracycline-class antibiotic, tigecycline is the last resort for treating multidrug-resistant (MDR) bacteria. Plasmid-mediated horizontal transfer enables the sharing of genetic information among different bacteria. The tigecycline resistance gene tet(X) threatens the efficacy of tigecycline, and the adjacent ISCR2 or IS26 are often detected upstream and downstream of the tet(X) gene, which may play a crucial driving role in the transmission of the tet(X) gene. Since the first discovery of the plasmid-mediated high-level tigecycline resistance gene tet(X4) in China in 2019, the tet(X) genes, especially tet(X4), have been reported within various reservoirs worldwide, such as ducks, geese, migratory birds, chickens, pigs, cattle, aquatic animals, agricultural field, meat, and humans. Further, our current researches also mentioned viruses as novel environmental reservoirs of antibiotic resistance, which will probably become a focus of studying the transmission of ARGs. Overall, this article mainly aims to discuss the current status of plasmid-mediated transmission of different tet(X) genes, in particular tet(X4), as environmental pollutants, which will risk to public health for the "One Health" concept. [ABSTRACT FROM AUTHOR]

Tigecycline (TGC) is currently used to treat carbapenem-resistant Acinetobacter baumannii (CRAB) infections, while eravacycline (ERV), a new-generation tetracycline, holds promise as a novel therapeutic option for these infections. However, differences in resistance mechanism between ERV and TGC against A. baumannii remain unclear. This study sought to compare the characteristics and mechanisms of ERV and TGC resistance among clinical A. baumannii isolates. A total of 492 isolates, including 253 CRAB and 239 carbapenem-sensitive A. baumannii (CSAB) isolates, were collected from hospitalized patients in China. The MICs of ERV and TGC against A. baumannii were determined by broth microdilution. Genetic mutations and expressions of adeB, adeG, adeJ, adeS, adeL , and adeN in resistant strains were examined by PCR and qPCR, respectively. The in vitro recombination experiments were used to verify the resistance mechanism of ERV and TGC in A. baumannii . The MIC 90 of ERV in CRAB and CSAB isolates were lower than those of TGC. A total of 24 strains resistant to ERV and/or TGC were categorized into three groups: only ERV-resistant ( n  = 2), both ERV- and TGC-resistant ( n  = 7), and only TGC-resistant ( n  = 15). ST208 (75%, n  = 18) was a major clone that has disseminated in all three groups. The IS Aba1 insertion in adeS was identified in 66.7% (6/9) of strains in the only ERV-resistant and both ERV- and TGC-resistant groups, while the IS Aba1 insertion in adeN was found in 53.3% (8/15) of strains in the only TGC-resistant group. The adeABC and adeRS expressions were significantly increased in the only ERV-resistant and both ERV- and TGC-resistant groups, while the adeABC and adeIJK expressions were significantly increased and adeN was significantly decreased in the only TGC-resistant group. Expression of adeS with the IS Aba1 insertion in ERV- and TGC-sensitive strains significantly increased the ERV and TGC MICs and upregulated adeABC and adeRS expressions. Complementation of the wildtype adeN in TGC-resistant strains with the IS Aba1 insertion in adeN restored TGC sensitivity and significantly downregulated adeIJK expression. In conclusion, our data illustrates that ERV is more effective against A. baumannii clinical isolates than TGC. ERV resistance is correlated with the IS Aba1 insertion in adeS , while TGC resistance is associated with the IS Aba1 insertion in adeN or adeS in A. baumannii., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Chen, Li, Lin, Guo, He, Wang, Wang, Xu, Song, Tan, Zhuo and Lin.)

In this study, we investigated the in vitro activity and resistance mechanisms of the new generation tetracycline agents, namely eravacycline, omadacycline, and tigecycline, against Staphylococcus aureus isolates. A total of 1,017 non-duplicate S. aureus isolates were collected and subjected to susceptibility testing against eravacycline, omadacycline, and tigecycline using the broth microdilution method. Tetracyclines-resistant (eravacycline/omadacycline/tigecycline-resistant) isolates were selected to elucidate the resistance mechanisms using polymerase chain reaction (PCR), cloning experiment, efflux pump inhibition, and quantitative real-time PCR. The results of the antibacterial susceptibility testing showed that compared with omadacycline, eravacycline and tigecycline had superior antibacterial activity against S. aureus isolates. Among 1,017 S. aureus, 41 tetracyclines-resistant isolates were identified. These resistant isolates possessed at least one tetracycline resistance gene and genetic mutation in the MepRAB efflux pump and 30S ribosome units. A frameshift mutation in mepB was detected in most tetracyclines-resistant strains (except for JP3349) compared with tetracyclines-susceptible (eravacycline/omadacycline/tigecycline-susceptible) strains. This was first shown to decrease susceptibility to omadacycline, but not to eravacycline and tigecycline. After treatment with eravacycline, omadacycline or tigecycline, overexpression of mepA, tet38, tet(K) and tet(L) was detected. Moreover, multi-locus sequence typing showed a major clonal dissemination type, ST5, and its variant ST764 were seen in most tetracyclines-resistant strains. To conclude, eravacycline and tigecycline exhibited better activity against S. aureus including tetracycline-resistant isolates than omadacycline. The resistance to these new generation tetracyclines due to an accumulation of many resistance mechanisms.

Acinetobacter baumannii (A. baumannii) is now considered a highly resistant pathogen to various types of antibiotics. Therefore, tracking the source of its prevalence and continuous control is crucial. This study aimed to determine antibiotic resistance and perform various molecular typing methods on clinical isolates of A. baumannii isolated from hospitalized burn patients in Shahid Motahari Burn Hospital, Tehran, Iran. Hospital isolates were confirmed by phenotypic and molecular methods. Then the sensitivity to different antibiotics was determined using the minimum inhibitory concentration (MIC) method. In order to perform molecular typing, three-locus dual assay multiplex polymerase chain reaction (PCR), multiple-locus variable-number tandem repeat analysis (MLVA), and multilocus sequence typing (MLST) methods were used. Among the 60 isolates collected, the frequencies of multidrug-resistant (MDR) and extensively drug-resistant (XDR) isolates were 90 and 10%, respectively. The most effective antibiotics were colistin with 100% and tigecycline with 83.33% sensitivity. Isolates were 100% resistant to piperacillin/tazobactam and cephalosporins, and 68.3% were resistant to carbapenem. The results of multiplex PCR showed five groups that international clone I (IC I) and IC II were the most common. The MLVA method identified 34 MLVA types (MTs), 5 clusters, and 25 singletons. Multilocus sequence typing results for tigecycline-resistant isolates showed seven different sequence types (STs). Increasing antibiotic resistance in A. baumannii isolates requires careful management to control and prevent the occurrence of the pre-antibiotic era. The results of this study confirm that the population structure of A. baumannii isolates has a high diversity. More extensive studies are needed in Iran to better understand the epidemiology of A. baumannii.

The rapid dissemination of plasmid-mediated tet(X) genes in Acinetobacter species has compromised the clinical effectiveness of tigecycline, one of the last-resort antibiotics. However, the classification strategy and homology group of tet(X)-positive Acinetobacter spp. plasmids remain largely unknown. In this study, we classified them by genome-based replicon typing, followed by analyses of structural characteristics, transferability and in vivo effect. A total of 34 plasmids distributed in at least nine Acinetobacter species were collected, including three tet(X3)-positive plasmids and one tet(X6)- positive plasmid from our genome sequencing results. Among them, there were 28 plasmids carrying Rep_3 superfamily replicase genes and classified into six homology groups, consisting of GR31 (82.1%), GR26 (3.6%), GR41 (3.6%), GR59 (3.6%), and novel groups GR60 (3.6%) and GR61 (3.6%). Our tet(X3)-positive plasmids pYH16040-1, pYH16056-1, and pYH12068-1 belonged to the dominant GR31 group, whereas the tet(X6)- positive plasmid pYH12068-2 was unclassified. Structurally, all tet(X)-positive GR31 plasmids shared similar plasmid replication (repB), stability (parA and parB) and accessory modules [tet(X) and sul2], and 97.6% of plasmidmediated tet(X) genes in Acinetobacter species were adjacent to ISCR2. Conjugation and susceptibility testing revealed pYH16040-1, pYH16056-1, and pYH12068-2, carrying plasmid transfer modules, were able to mediate the mobilization of multiple antibiotic resistance. Under the treatment of tigecycline, the mortality rate of Galleria mellonella infected by pYH16040- 1-mediated tet(X3)-positive Acinetobacter spp. isolate significantly increased when compared with its plasmid-cured strain (p < 0.0001). The spread of such plasmids is of great clinical concern, more effects are needed and will facilitate the future analysis of tet(X)-positive Acinetobacter spp. plasmids. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the prevalence and characterization of tet(X4) in Escherichia coli isolates from a pig farm in Shanghai, China, and to elucidate tet(X4) dissemination mechanism in this swine farm. Forty-nine (80.33%) E. coli strains were isolated from 61 samples from a pig farm and were screened for the presence of tet(X). Among them, six (12.24%) strains were positive for tet(X4) and exhibited resistance to tigecycline (MIC = 16 mg/L). They were further sequenced by Illumina Hiseq. Six tet(X4)-positive strains belonged to ST761 with identical resistance genes, resistance profiles, plasmid replicons, and cgMLST type except that additional ColE10 plasmid was present in isolate SH21PTE35. Isolate SH21PTE31, as a representative ST761 E. coli strain, was further sequenced using Nanopore MinION. The tet(X4) in SH21PTE31 was located on IncFIA18/IncFIB(K)/IncX1 hybrid plasmid pYUSHP31-1, highly similar to other tet(X4)-carrying IncFIA18/IncFIB(K)/IncX1 plasmids from ST761 E. coli and other E. coli lineages in China. These IncFIA18/IncFIB(K)/IncX1 plasmids shared closely related multidrug resistance regions, and could reorganize, acquire or lose resistance modules mediated by mobile elements such as ISCR2 and IS26. Phylogenetic analysis were performed including all tet(X4)-positive isolates obtained in this pig farm combined with 43 tet(X4)-positive E. coli from pigs, cow, pork, wastewater, and patients with the same ST from NCBI. The 50 tet(X4)-carrying E. coli ST761 isolates from different areas in China shared a close phylogenetic relationship (0-49 SNPs). In conclusion, clonal transmission of tet(X4)-positive E. coli ST761 has occurred in this swine farm. E. coli ST761 has the potential to become a high-risk clone for tet(X4) dissemination in China. [ABSTRACT FROM AUTHOR]

Mobile tigecycline resistance (MTR) threatens the clinical efficacy of the salvage antibiotic, tigecycline (TIG) used in treating deadly infections in humans caused by superbugs (multidrug-, extensively drug-, and pandrug-resistant bacteria), including carbapenem- and colistin-resistant bacteria. Currently, non-mobile tet(X) and mobile plasmid-mediated transmissible tet(X) and resistance-nodulation-division (RND) efflux pump tmexCD-toprJ genes, conferring high-level TIG (HLT) resistance have been detected in humans, animals, and environmental ecosystems. Given the increasing rate of development and spread of plasmid-mediated resistance against the two last-resort antibiotics, colistin (COL) and TIG, there is a need to alert the global community on the emergence and spread of plasmid-mediated HLT resistance and the need for nations, especially developing countries, to increase their antimicrobial stewardship. Justifiably, MTR spread projects One Health ramifications and portends a monumental threat to global public and animal health, which could lead to outrageous health and economic impact due to limited options for therapy. To delve more into this very important subject matter, this current work will discuss why MTR is an emerging health catastrophe requiring urgent One Health global intervention, which has been constructed as follows: (a) antimicrobial activity of TIG; (b) mechanism of TIG resistance; (c) distribution, reservoirs, and traits of MTR gene-harboring isolates; (d) causes of MTR development; (e) possible MTR gene transfer mode and One Health implication; and (f) MTR spread and mitigating strategies. [ABSTRACT FROM AUTHOR]

Emergence of bla NDM–1 and bla KPC–2 co-producing Klebsiella pneumoniae strains is currently attracting widespread attention, but little information is available about their tigecycline resistance, virulence, and prevalence in Southwest China. In July 2021, an extensively drug-resistant K. pneumoniae strain AHSWKP25 whose genome contained both bla NDM–1 and bla KPC–2 genes was isolated from the blood of a patient with the malignant hematological disease in Luzhou, China. We investigated the resistance profiles of AHSWKP25 using microbroth dilution, agar dilution, modified carbapenemase inactivation (mCIM), and EDTA-modified carbapenemase inactivation methods (eCIM). The virulence of AHSWKP25 was assessed through string tests, serum killing assays, and a Galleria mellonella larval infection model. Conjugation and plasmid stability experiments were conducted to determine the horizontal transfer capacity of plasmids. And efflux pump phenotype test and real-time quantitative reverse transcription-PCR (RT-PCR) were used to determine its efflux pump activity. Sequencing of AHSWKP25 determined that AHSWKP25 belonged to ST464, which is resistant to antibiotics such as carbapenems, tetracycline, fluoroquinolones, tigecycline, and fosfomycin. The efflux pump phenotype tests and RT-PCR results demonstrated that efflux pumps were overexpressed in the AHSWKP25, which promoted the tigecycline resistance of the bacteria. AHSWKP25 also showed hypervirulence and serum resistance in vitro model. AHSWKP25 carried several different plasmids that contained bla NDM–1, bla KPC–2, and mutated tet(A) genes. Sequence alignment revealed that the plasmids carrying bla NDM–1 and bla KPC–2 underwent recombination and insertion events, respectively. We demonstrated that an X3 plasmid carrying bla NDM–1 was transferred from pSW25NDM1 to E. coli J53. We also identified missense mutations in the ramR , rcsA , lon , and csrD genes of AHSWKP25. Our results highlighted the potential of bla NDM–1 and bla KPC–2 co-producing K. pneumoniae strains to further develop antimicrobial resistance and hypervirulent phenotypes, but measures should be taken to closely monitor and control the spread of superbugs with multidrug-resistant phenotypes and hypervirulence. [ABSTRACT FROM AUTHOR]

CG258 is the dominant carbapenemase-producing Klebsiella pneumoniae clone worldwide and treatment of infections caused by this clone relies largely on the last-line antibiotics, colistin, and tigecycline. However, the emergence and global dissemination of mcr and tmexCD1 - toprJ1 genes have significantly compromised their clinical applications. CG258 K. pneumoniae carrying both mcr and tmexCD1 - toprJ1 have not been reported. A colistin-resistant strain T698-1 belonging to ST1326, a member of CG258, was isolated from the intestinal sample of a patient and characterized by the antimicrobial susceptibility testing, conjugation assay, WGS and bioinformatics analysis. It was resistant to colistin, tetracycline, aminoglycoside, fluoroqinolone, phenicols, sulfonamide, and some β-lactams, and positive for mcr-8.2 , tmexCD1 - toprJ1 , and ESBL genes (bla DHA– 1 and bla CTX–M– 15). The tmexCD1 - toprJ1 gene cluster was located in an multi-drug resistant (MDR) region flanked by Tn As1 elements on an IncHI1B/FIB plasmid. The genetic context of tmexCD1 - toprJ1 was slightly distinct from previously reported Tn 5393 -like structures, with an IS 26 element disrupting the upstream Tn 5393 and its adjacent genetic elements. The mcr-8.2 gene was inserted into the backbone of an IncFII/FIA plasmid with the genetic context of IS Ecl1 - mcr-8.2 - orf -IS Kpn26. To our knowledge, this is the first report of co-occurrence of mcr-8.2 and tmexCD1 - toprJ1 in a CG258 K. pneumoniae strain. Though this strain is tigecycline sensitive, the acquisition of colistin and tigecycline resistance determinants by the endemic CG258 K. pneumoniae clone still poses a serious public health concern. CG258, which became resistant to multiple last resort antibiotics, would be the next emerging superbug. [ABSTRACT FROM AUTHOR]

The rise and global dissemination of extensively drug-resistant (XDR) bacteria are often related to plasmid-borne mobile antimicrobial resistance genes. Notably, isolates having multiple plasmids are often highly resistant to almost all the antibiotics available. In this study, we characterized an extensively drug-resistant Klebsiella pneumoniae 1678, which exhibited high-level resistance to almost all the available antibiotics. Through whole-genome sequencing (WGS), more than 20 resistant elements and 5 resistant plasmids were observed. Notably, the tigecycline resistance of K. pneumoniae 1678 was not related to the plasmid-borne tetA gene but associated with the overexpression of AcrAB and OqxAB efflux pumps, according to the susceptibility results of tetA -transformant and the related mRNA quantification of RND efflux pumps. Except for tigecycline resistance, three plasmids, mediating resistance to colistin, Fosfomycin, and ceftazidime–avibactam, respectively, were focused. Detailed comparative genetic analysis showed that all these plasmids belonged to dominated epidemic plasmids, and harbored completed conjugation systems. Results of conjugation assay indicated that these three plasmids not only could transfer to E. coli J53 with high conjugation frequencies, respectively, but also could co-transfer to E. coli J53 effectively, which was additionally confirmed by the S1-PFGE plasmids profile. Moreover, multiple insertion sequences (IS) and transposons (Tn) were also found surrounding the vital resistant genes, which may form several novel mechanisms involved in the resistant determinants' mobilization. Overall, we characterized and reported the uncommon co-existence and co-transferring of FosA3-, NDM-5, and MCR-1-encoding plasmids in a K. pneumoniae isolate, which may increase the risk of spread of these resistant phenotypes and needing great concern. [ABSTRACT FROM AUTHOR]

Efflux pumps contribute to multidrug resistance in Acinetobacter baumannii due to their ability to expel a wide variety of structurally unrelated compounds. This study aimed to characterize the effect of subinhibitory concentrations of clinically-relevant antibiotics and disinfectants on the promoter activity of members of the Resistance-Nodulation-Division (RND) family in A. baumannii. The promoter regions from three RND efflux pumps (AdeABC, AdeFGH and AdeIJK) and the AdeRS regulatory system from three different A. baumannii strains (ATCC 17961, ATCC 17978, and ATCC 19606) were cloned into a luciferase reporter system (pLPV1Z). Promoter activity was quantitatively assessed in both exponential and stationary phase cultures after exposure to subinhibitory concentrations of four antibiotics from different classes (rifampicin, meropenem, tigecycline and colistin) and two disinfectants (ethanol and chlorhexidine). Subinhibitory concentrations of the compounds tested had variable effects on promoter activity that were highly dependent on the A. baumannii strain, the compound tested and the growth phase. Fold changes in AdeABC promoter activity ranged from 1.97 to 113.7, in AdeFGH from −5.6 to 1.13, in AdeIJK from −2.5 to 2, and in AdeRS from −36.2 to −1.32. Taken together, these results indicate that subinhibitory concentrations of clinically-relevant antibiotics and disinfectants affect the promoter activity of RND family members in A. baumannii in a strain and growth phase dependent manner. These results may have important implications for the treatment of infections caused by A. baumannii. [ABSTRACT FROM AUTHOR]

Colistin and tigecycline are the last options against carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP). Intersecting resistance determinants have been detected between these antibiotics; however, there is only limited evidence of such association. Here, we describe a colistin-resistant CR-hvKP isolated from a patient with severe neonatal bacteremia treated with tigecycline as opposed to colistin before isolation of this strain, providing a clinical clue to colistin resistance under tigecycline pressure. Furthermore, an ST11-K64 KPC-2–producing, colistin-susceptible CR-hvKP strain was subjected to experimental evolution toward colistin resistance under tigecycline and colistin pressure to verify this phenomenon in vitro. The biological impact of acquiring colistin resistance on fitness and virulence was also studied. As expected, the parental strain rapidly developed colistin resistance under both tigecycline and colistin selection. However, different from the colistin resistance mechanism in the clinical strain that was due to an IS Kpn26 insertion in the mgrB gene, the mutants in this study developed colistin resistance through a ∼4.4 or ∼4.6 kb deletion including the mgrB locus as well as the kdgR , yobH , yebO , yobF , cspC , ftsI , and rlmA genes. Although the virulence of the colistin-resistant mutants, as determined in the Galleria mellonella model, decreased compared with that of the parent strain, it was still higher than that of NTUH-K2044. This suggests a slight virulence cost when CR-hvKP develops colistin resistance under tigecycline or colistin pressure. Together, our results provide clinical and experimental evidence for the association between colistin resistance and tigecycline pressure in CR-hvKP, highlighting a critical issue in the clinical setting. [ABSTRACT FROM AUTHOR]

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is becoming increasingly problematic due to the limited effectiveness of new antimicrobials or other factors such as treatment cost. Thus, combination therapy remains a suitable treatment option. We aimed to evaluate the in vitro bactericidal activity of various antibiotic combinations against CRKP with different carbapenemase genotypes and sequence types (STs). Thirty-seven CRKP with various STs and carbapenemases were exposed to 11 antibiotic combinations (polymyxin B or tigecycline in combination with β-lactams including aztreonam, cefepime, piperacillin/tazobactam, doripenem, meropenem, and polymyxin B with tigecycline) in static time-kill studies (TKS) using clinically achievable concentrations. Out of the 407 isolate-combination pairs, only 146 (35.8%) were bactericidal (≥3 log10CFU/mL decrease from initial inoculum). Polymyxin B in combination with doripenem, meropenem, or cefepime was the most active, each demonstrating bactericidal activity in 27, 24, and 24 out of 37 isolates, respectively. Tigecycline in combination with β-lactams was rarely bactericidal. Aside from the lower frequency of bactericidal activity in the dual-carbapenemase producers, there was no apparent difference in combination activity among the strains with other carbapenemase types. In addition, bactericidal combinations were varied even in strains with similar STs, carbapenemases, and other genomic characteristics. Our findings demonstrate that the bactericidal activity of antibiotic combinations is highly strain-specific likely owing to the complex interplay of carbapenem-resistance mechanisms, i.e., carbapenemase genotype alone cannot predict in vitro bactericidal activity. The availability of WGS information can help rationalize the activity of certain combinations. Further studies should explore the use of genomic markers with phenotypic information to predict combination activity.

Introduction: The dissemination of strains producing tetracyclines monooxygenase Tet(X) from breeding farms to the natural environment poses a potential threat to public health., Methods: Antimicrobial susceptibility testing and WGS were performed to identify resistance phenotypes and genotypes. Cloning experiments, sequence alignment, and homology modeling were used to characterize the function and formation mechanisms of the recombinant variant. The mobilization potential of Tet(X) was assessed by collinearity analysis, conjugation experiments, and phylogenetic analysis., Results: Three tet (X)-producing Elizabethkingia meningoseptica strains were isolated from bullfrog breeding ponds, the sewage outlet, and downstream river in Zhejiang Province, China. These strains carry a novel Tet(X) variant, differing from Tet(X6) by seven residues, and possess the ability to degrade tetracyclines. Interestingly, the novel Tet(X) is a recombinant variant formed by homologous recombination of Tet(X6) and the C-terminal of Tet(X2). Further analysis revealed that Tet(X6) formed several Tet(X) variants, including Tet(X5), through homologous recombination. The novel tet (X) gene is located on a circularizable integrative and conjugative element (ICE EmeChn3 ), with IS wz1 participating in the recombination of its multi-drug resistance region, potentially facilitating the mobilization and recombination of tet (X) in early hosts. These three strains were clonally transmitted and shared a close genetic relationship (SNP < 62) with a clinically-sourced strain isolated from the same province., Discussion: To our knowledge, this is the first report of homologous recombination between Tet(X) variants with differing activities. These clonal strains provide evidence of the transmission of tet (X)-positive strains from aquaculture sewage to the natural environment, highlighting the need to strengthen the monitoring and management of this emerging farming model., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Jin, Jia, Jin, Zhu, Wang, Sun and Cui.)

The emergence and widespread of tigecycline resistance undoubtedly poses a serious threat to public health globally. The exploration of combination therapies has become preferred antibacterial strategies to alleviate this global burden. In this study, tigecycline-resistant tet (X4)-positive Escherichia coli were selected for adjuvant screening. Interestingly, 9-aminominocycline (9-AMC), one of the tigecycline metabolites, exhibits synergistic antibacterial activity with tigecycline using checkerboard assay. The efficacy in vitro and in vivo was evaluated, and the synergistic mechanism was further explored. The results suggested that 9-AMC combined with tigecycline could inhibit the growth of antibiotic resistant bacteria, efficiently retard the evolution of tet (X4) gene and narrow the drug mutant selection window. In addition, the combination of tigecycline and 9-AMC could destroy the normal membrane structure of bacteria, inhibit the formation of biofilm, remarkably reduce the level of intracellular ATP level, and accelerate the oxidative damage of bacteria. Furthermore, 9-AMC is more stable in the bind of Tet(X4) inactivating enzyme. The transcriptomics analysis revealed that the genes related to the 9-AMC and tigecycline were mainly enriched in ABC transporters. Collectively, the results reveal the potentiation effects on tigecycline and the probability of 9-AMC as a novel tigecycline adjuvant against tet (X4)-positive Escherichia coli, which provides new insights for adjuvant screening., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Sun, Zhang, Ma, Ali, Wu and Li.)

Introduction: The aim of this study was to predict and evaluate three antimicrobials for treatment of adult bloodstream infections (BSI) with carbapenem-resistant Enterobacterales (CRE) in China, so as to optimize the clinical dosing regimen further. Methods: Antimicrobial susceptibility data of blood isolates were obtained from the Blood Bacterial Resistance Investigation Collaborative Systems in China. Monte Carlo simulation was conducted to estimate the probability target attainment (PTA) and cumulative fraction of response (CFR) of tigecycline, polymyxin B, and ceftazidime/avibactam against CRE. Results: For the results of PTAs, tigecycline following administration of 50 mg every 12 h, 75 mg every 12 h, and 100 mg every 12 h achieved > 90% PTAs when minimum inhibitory concentration (MIC) was 0.25, 0.5, and 0.5 μg/mL, respectively; polymyxin B following administration of all tested regimens achieved > 90% PTAs when MIC was 1 μg/mL with CRE; ceftazidime/avibactam following administration of 1.25 g every 8 h, 2.5 g every 8 h achieved > 90% PTAs when MIC was 4 μg/mL, 8 μg/mL with CRE, respectively. As for CFR values of three antimicrobials, ceftazidime/avibactam achieved the lowest CFR values. The highest CFR value of ceftazidime/avibactam was 77.42%. For tigecycline and ceftazidime/avibactam, with simulated regimens daily dosing increase, the CFR values were both increased; the highest CFR of tigecycline values was 91.88%. For polymyxin B, the most aggressive dosage of 1.5 mg/kg every 12 h could provide the highest CFR values (82.69%) against CRE. Conclusion: This study suggested that measurement of MICs and individualized therapy should be considered together to achieve the optimal drug exposure. In particular, pharmacokinetic and pharmacodynamic modeling based on local antimicrobial resistance data can provide valuable guidance for clinicians for the administration of empirical antibiotic treatments for BSIs. [ABSTRACT FROM AUTHOR]

The emergence of extensive drug-resistant (XDR) Salmonella in livestock animals especially in poultry represents a serious public health and therapeutic challenge. Despite the wealth of information available on Salmonella resistance to various antimicrobials, there have been limited data on the genetic determinants of XDR Salmonella exhibiting co-resistance to ciprofloxacin (CIP) and tigecycline (TIG). This study aimed to determine the prevalence and serotype diversity of XDR Salmonella in poultry flocks and contact workers and to elucidate the genetic determinants involved in the co-resistance to CIP and TIG. Herein, 115 Salmonella enterica isolates of 35 serotypes were identified from sampled poultry (100/1210, 8.26%) and humans (15/375, 4.00%), with the most frequent serotype being Salmonella Typhimurium (26.96%). Twenty-nine (25.22%) Salmonella enterica isolates exhibited XDR patterns; 25 out of them (86.21%) showed CIP/TIG co-resistance. Exposure of CIP- and TIG-resistant isolates to the carbonyl cyanide 3-chlorophenylhydrazone (CCCP) efflux pump inhibitor resulted in an obvious reduction in their minimum inhibitory concentrations (MICs) values and restored the susceptibility to CIP and TIG in 17.24% (5/29) and 92% (23/25) of the isolates, respectively. Molecular analysis revealed that 89.66% of the isolates contained two to six plasmid-mediated quinolone resistance genes with the predominance of qepA gene (89.66%). Mutations in the gyrA gene were detected at codon S83 (34.62%) or D87 (30.77%) or both (34.62%) in 89.66% of XDR Salmonella. The tet (A) and tet (X4) genes were detected in 100% and 3.45% of the XDR isolates, respectively. Twelve TIG-resistant XDR Salmonella had point mutations at codons 120, 121, and 181 in the tet(A) interdomain loop region. All CIP and TIG co-resistant XDR Salmonella overexpressed ramA gene; 17 (68%) out of them harbored 4-bp deletion in the ramR binding region (T-288/A-285). However, four CIP/TIG co-resistant isolates overexpressed the oqxB gene. In conclusion, the emergence of XDR S. enterica exhibiting CIP/TIG co-resistance in poultry and humans with no previous exposure to TIG warrants an urgent need to reduce the unnecessary antimicrobial use in poultry farms in Egypt. [ABSTRACT FROM AUTHOR]

Background: Escherichia coli is the most common pathogens in patients with community-onset blood stream infections (COBSI). Knowledge of the epidemiology of this disease is crucial to improve allocation of health resources, formulate isolation strategies that prevent transmission, and guide empirical antibiotic therapy. Methods: This retrospective observational study examined patients with E. coli COBSI (EC-COBSI) at a non-tertiary hospital in China. Whole-genome sequencing and analysis of the isolates was performed. The relationships of clinical variables with antimicrobial resistance and the genetic background of the isolates were examined. Results: There were 148 isolates in patients with EC-COBSI. All isolates were susceptible to ceftazidime/avibactam, carbapenems, and tigecycline; 35.1% were positive for extended spectrum β-lactamase (ESBL+); and bla CTX – M –14 was the most common ESBL gene. Patients with ESBL- isolates were more likely to receive appropriate empiric treatment than those with ESBL+ isolates (61.5% vs. 91.4%, p < 0.001), but these two groups had similar mortality rates. The overall 30-day mortality rate was 9.5%. Phylogenetic analysis showed that the isolates were diverse, and that the main sequence types (STs) were ST95, ST131, and ST69. Intra-abdominal infection was the primary source of disease, and isolates from these patients had lower frequencies of virulence genes. Conclusion: The mortality rate of patients with EC-COBSI was unrelated to ESBL status of the isolates. Most isolates had low resistance to most of the tested antimicrobial agents. The isolates were diverse, and multiple strains were related. Prevention and control of EC-COBSI should target prevention of patient colonization and the living environment. [ABSTRACT FROM AUTHOR]

Tigecycline serves as one of the last-resort antibiotics to treat severe infections caused by carbapenem-resistant Enterobacterales. Recently, a novel plasmid-mediated resistance-nodulation-division (RND)-type efflux pump gene cluster, TmexCD1-ToprJ1 , and its variants, TmexCD2-ToprJ2 and TmexCD3-ToprJ3 , encoding tetracyclines and tigecycline resistance, were revealed. In this study, we reported three TmexCD2-ToprJ2 -harboring Klebsiella species strains, collected from two teaching tertiary hospitals in China, including one K. quasipneumoniae , one K. variicola , and one K. michiganensis. The three strains were characterized by antimicrobial susceptibility testing (AST), conjugation assay, WGS, and bioinformatics analysis. AST showed that K. variicola and K. quasipneumoniae strains were resistant to tigecycline with MIC values of 4μg/ml, whereas the K. michiganensis was susceptible to tigecycline with an MIC value of 1μg/ml. The TmexCD2-ToprJ2 clusters were located on three similar IncHI1B plasmids, of which two co-harbored the metallo-β-lactamase gene bla NDM-1. Conjugation experiments showed that all three plasmids were capable of self-transfer via conjugation. Our results showed, for the first time, that this novel plasmid-mediated tigecycline resistance mechanism TmexCD2-ToprJ2 has spread into different Klebsiella species, and clinical susceptibility testing may fail to detect. The co-occurrence of bla NDM-1 and TmexCD2-ToprJ2 in the same plasmid is of particular public health concern as the convergence of "mosaic" plasmids can confer both tigecycline and carbapenem resistance. Its further spread into other clinical high-risk Klebsiella clones will likely exacerbate the antimicrobial resistance crisis. A close monitoring of the dissemination of TmexCD-ToprJ encoding resistance should be considered. [ABSTRACT FROM AUTHOR]

Three carbapenem-resistant Klebsiella pneumoniae (CRKP; strains KP-426, KP-C76, and KP-CT77) were isolated from a patient with severe burns during the treatment of colistin and tigecycline. Single-nucleotide polymorphism typing showed that three ST11 CRKP were clonally related. Three isolates harbored the same set of antimicrobial resistance genes. bla KPC-2, bla SHV-12, bla TEM-1, and rmtB genes were located on the same 128,928-bp IncFII/IncR plasmid. Tet (A), catA2 , sul2 , and dfrA14 genes were located on a plasmid with an unknown Inc-type. bla SHV-11, fosA , and aadA2 were chromosomal genes. An IS 1 and an IS Kpn14 were found in the promoter region of the mgrB gene of two colistin-resistant CRKP, K. pneumoniae KP-C76, and KP-CT77, respectively. A novel amino acid substitution, G300E, was identified in the type 1 Tet(A) variant of K. pneumoniae KP-CT77 which exhibited high-level tigecycline resistance compared to strains KP-426 and KP-C76 (MIC of 32, 4, and 4mg/l, respectively). Conjugation and cloning experiments confirmed that the mutated Tet(A) resulted in a 4-fold increase in tigecycline minimal inhibitory concentration (MIC) of Escherichia coli. Three CRKP belonged to the K64 serotype and possessed a similar IncHI1B/repB virulence plasmid carrying rmpA , rmpA2 , and iucABCDiutA. The survival rates of Galleria Mellonella injected with K. pneumoniae KP-426, KP-C76, and KP-CT77 were 4.2, 20.8, and 8.3%, respectively. The emergence of colistin and tigecycline resistance in carbapenem-resistant hypervirulent K. pneumoniae posed a serious threat to clinical anti-infective therapy. The type 1 Tet(A) variant carrying G300E mutation, which conferred significantly elevated tigecycline MIC and was located on a conjugative plasmid, needs attention. [ABSTRACT FROM AUTHOR]

Intracranial infections caused by multidrug-resistant Gram-negative bacterium have led to considerable mortality due to extremely limited treatment options. Herein, we firstly reported a clinical carbapenem-resistant Escherichia coli isolate coharboring bla NDM –5 and bla CTX – M –65 from a patient with post-craniotomy meningitis. The carbapenem-resistant Escherichia coli strain CNEC001 belonging to Sequence Type 410 was only susceptible to amikacin and tigecycline, both of which have poor penetration through the blood-brain barrier (BBB). The bla CTX – M –65 gene was expressed on a 135,794 bp IncY plasmid. The bla NDM –5 gene was located on a genomic island region of an IncX3-type plasmid pNDM5-CNEC001. Based on the characteristics of the strain, we presented the successful treatment protocol of intravenous (IV) tigecycline and amikacin combined with intrathecal (ITH) amikacin in this study. Intracranial infection caused by Escherichia coli coharboring bla NDM –5 and bla CTX – M –65 is rare and fatal. Continuous surveillance and infection control measures for such strain need critical attention in clinical settings. [ABSTRACT FROM AUTHOR]

The emergence of extensive drug-resistant (XDR) Salmonella in livestock animals especially in poultry represents a serious public health and therapeutic challenge. Despite the wealth of information available on Salmonella resistance to various antimicrobials, there have been limited data on the genetic determinants of XDR Salmonella exhibiting co-resistance to ciprofloxacin (CIP) and tigecycline (TIG). This study aimed to determine the prevalence and serotype diversity of XDR Salmonella in poultry flocks and contact workers and to elucidate the genetic determinants involved in the co-resistance to CIP and TIG. Herein, 115 Salmonella enterica isolates of 35 serotypes were identified from sampled poultry (100/1210, 8.26%) and humans (15/375, 4.00%), with the most frequent serotype being Salmonella Typhimurium (26.96%). Twenty-nine (25.22%) Salmonella enterica isolates exhibited XDR patterns; 25 out of them (86.21%) showed CIP/TIG co-resistance. Exposure of CIP- and TIG-resistant isolates to the carbonyl cyanide 3-chlorophenylhydrazone (CCCP) efflux pump inhibitor resulted in an obvious reduction in their minimum inhibitory concentrations (MICs) values and restored the susceptibility to CIP and TIG in 17.24% (5/29) and 92% (23/25) of the isolates, respectively. Molecular analysis revealed that 89.66% of the isolates contained two to six plasmid-mediated quinolone resistance genes with the predominance of qepA gene (89.66%). Mutations in the gyrA gene were detected at codon S83 (34.62%) or D87 (30.77%) or both (34.62%) in 89.66% of XDR Salmonella. The tet(A) and tet(X4) genes were detected in 100% and 3.45% of the XDR isolates, respectively. Twelve TIG-resistant XDR Salmonella had point mutations at codons 120, 121, and 181 in the tet(A) interdomain loop region. All CIP and TIG co-resistant XDR Salmonella overexpressed ramA gene; 17 (68%) out of them harbored 4-bp deletion in the ramR binding region (T-288/A-285). However, four CIP/TIG co-resistant isolates overexpressed the oqxB gene. In conclusion, the emergence of XDR S. enterica exhibiting CIP/TIG co-resistance in poultry and humans with no previous exposure to TIG warrants an urgent need to reduce the unnecessary antimicrobial use in poultry farms in Egypt.

Introduction: The aim of this study was to predict and evaluate three antimicrobials for treatment of adult bloodstream infections (BSI) with carbapenem-resistant Enterobacterales (CRE) in China, so as to optimize the clinical dosing regimen further.Methods: Antimicrobial susceptibility data of blood isolates were obtained from the Blood Bacterial Resistance Investigation Collaborative Systems in China. Monte Carlo simulation was conducted to estimate the probability target attainment (PTA) and cumulative fraction of response (CFR) of tigecycline, polymyxin B, and ceftazidime/avibactam against CRE.Results: For the results of PTAs, tigecycline following administration of 50 mg every 12 h, 75 mg every 12 h, and 100 mg every 12 h achieved > 90% PTAs when minimum inhibitory concentration (MIC) was 0.25, 0.5, and 0.5 μg/mL, respectively; polymyxin B following administration of all tested regimens achieved > 90% PTAs when MIC was 1 μg/mL with CRE; ceftazidime/avibactam following administration of 1.25 g every 8 h, 2.5 g every 8 h achieved > 90% PTAs when MIC was 4 μg/mL, 8 μg/mL with CRE, respectively. As for CFR values of three antimicrobials, ceftazidime/avibactam achieved the lowest CFR values. The highest CFR value of ceftazidime/avibactam was 77.42%. For tigecycline and ceftazidime/avibactam, with simulated regimens daily dosing increase, the CFR values were both increased; the highest CFR of tigecycline values was 91.88%. For polymyxin B, the most aggressive dosage of 1.5 mg/kg every 12 h could provide the highest CFR values (82.69%) against CRE.Conclusion: This study suggested that measurement of MICs and individualized therapy should be considered together to achieve the optimal drug exposure. In particular, pharmacokinetic and pharmacodynamic modeling based on local antimicrobial resistance data can provide valuable guidance for clinicians for the administration of empirical antibiotic treatments for BSIs.

Tigecycline has been used as one of the therapeutic choices for the treatment of infections caused by multidrug-resistant Klebsiella pneumoniae. However, the emergence of tigecycline heteroresistance has led to great challenges in treating these infections. The purpose of this study was to investigate whether tigecycline-heteroresistant K. pneumoniae (TGCHR-Kp) exists in clinical isolates, and to further characterize the underlying molecular mechanisms involved in the development of tigecycline-resistant subpopulations. Of the 268 tigecycline-susceptible clinical K. pneumoniae isolates, 69 isolates were selected as tigecycline-heteroresistant candidates in the preliminary heteroresistant phenotypic selection by a modified disk diffusion method, and only 21 strains were confirmed as TGCHR-Kp by the population analysis profile (PAP). Pulsed-field gel electrophoresis (PFGE) analysis demonstrated that all the parental TGCHR-Kp isolates were clonally unrelated, and colonies confirmed as the heteroresistant subpopulation showed no significant differences from their respective parental TGCHR-Kp isolates. Efflux pump inhibitors reversed the tigecycline susceptibility in heteroresistant subpopulations. Mutations in the ramR and soxR genes lead to upregulation of the ramA and soxS transcriptional regulators, which in turn induced overexpression of the AcrAB-TolC efflux pump genes in TGCHR-Kps-resistant subpopulations. Moreover, mutations of rpsJ were also found in resistant subpopulations, which suggested that the rpsJ mutation may also lead to tigecycline resistance. Time-kill assays showed that the efficacy of tigecycline against TGCHR-Kps was weakened, whereas the number of resistant subpopulations was enriched by the presence of tigecycline. Our findings imply that the presence of TGCHR-Kps in clinical strains causes severe challenges for tigecycline therapy in clinical practice.

Tigecycline is a last-resort antibiotic for infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP). This study aimed to broaden our understanding of the acquisition of collateral hypersensitivity by CRKP, as an evolutionary trade-off of developing resistance to tigecycline. Experimental induction of tigecycline resistance was conducted with tigecycline-sensitive CRKP clinical isolates. Antimicrobial susceptibility testing, microbial fitness assessment, genotypic analysis and full-genome sequencing were carried out for these clinical isolates and their resistance-induced descendants. We found that tigecycline resistance was successfully induced after exposing CRKP clinical isolates to tigecycline at gradually increased concentrations, at a minor fitness cost of bacterial cells. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) found higher expression of the efflux pump gene acrB (5.3–64.5-fold) and its regulatory gene ramA (7.4–65.8-fold) in resistance-induced strains compared to that in the tigecycline-sensitive clinical isolates. Stable hypersensitivities to aminoglycosides and other antibiotics were noticed in resistance-induced strains, showing significantly lowered MICs (X 4 – >500 times). Full genome sequencing and plasmid analysis suggested the induced collateral hypersensitivity might be multifaceted, with the loss of an antimicrobial resistance (AMR) plasmid being a possible major player. This study rationalized the sequential combination of tigecycline with aminoglycosides for the treatment of CRKP infections.

BackgroundThe widespread use of antibiotics has led to the emergence of multidrug-resistant (MDR) bacteria such as multidrug-resistant Acinetobacter baumannii (AB). Tigecycline (TGC), as the first glycylcycline antibiotic approved by FDA, is a broad-spectrum antibiotic which remains highly effective to treat AB infections.ObjectiveTo confirm the TGC treatment dosage and effectiveness to treat AB infections in the Chinese population by performing therapeutic drug monitoring (TDM).MethodsThis study was performed from October 2018 through March 2019 at the PLA General Hospital. A high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was validated and employed to determine the plasma concentrations of TGC in patients with infectious diseases. The minimum inhibitory concentration (MIC) of TGC to clinically isolated AB was determined by broth microdilution method, agar dilution method, and disk diffusion method. Moreover, a model of population pharmacokinetics/pharmacodynamics (PPK/PD) was constructed.ResultsA total of 186 plasma samples from 67 patients were detected by the validated HPLC-MS/MS method. The MIC values determined by the broth microdilution method were more sensitive and accurate than the other two methods. The microbial and clinical PK/PD breakpoints were reached when the maintenance dose of TGC was 100 mg.ConclusionOur study established a validated HPLC-MS/MS method to monitor the plasma concentrations of TGC. In view of the MIC range to AB isolates in our hospital and the PPK/PD modeling results, we recommend a relatively high dose of 100 mg q12h regimen to achieve the optimal clinical efficacy and antimicrobial response.

Tigecycline is a tetracycline derivative that is being used as an antibiotic of last resort. Both tigecycline and tetracycline bind to the small (30S) ribosomal subunit and inhibit translation. Target mutations leading to resistance to these antibiotics have been identified both in the 16S ribosomal RNA and in ribosomal proteins S3 and S10 (encoded by the rpsJ gene). Several different mutations in the S10 flexible loop tip residue valine 57 (V57) have been observed in tigecycline-resistant Escherichia coli isolates. However, the role of these mutations in E. coli has not yet been characterized in a defined genetic background. In this study, we chromosomally integrated 10 different rpsJ mutations into E. coli, resulting in different exchanges or a deletion of S10 V57, and investigated the effects of the mutations on growth and tigecycline/tetracycline resistance. While one exchange, V57K, decreased the minimal inhibitory concentration (MIC) (Etest) to tetracycline to 0.75 μg/ml (compared to 2 μg/ml in the parent strain) and hence resulted in hypersensitivity to tetracycline, most exchanges, including the ones reported previously in resistant isolates (V57L, V57D, and V57I) resulted in slightly increased MICs to tigecycline and tetracycline. The strongest increase was observed for the V57L mutant, with a MIC (Etest) to tigecycline of 0.5 μg/ml (compared to 0.125 μg/ml in the parent strain) and a MIC to tetracycline of 4.0 μg/ml. Nevertheless, none of these exchanges increased the MIC to the extent observed in previously described clinical tigecycline-resistant isolates. We conclude that, next to S10 mutations, additional mutations are necessary in order to reach high-level tigecycline resistance in E. coli. In addition, our data reveal that mutants carrying S10 V57 exchanges or deletion display growth defects and, in most cases, also thermosensitivity. The defects are particularly strong in the V57 deletion mutant, which is additionally cold-sensitive. We hypothesize that the S10 loop tip residue is critical for the correct functioning of S10. Both the S10 flexible loop and tigecycline are in contact with helix h31 of the 16S rRNA. We speculate that exchanges or deletion of V57 alter the positioning of h31, thereby influencing both tigecycline binding and S10 function.

Tetracycline has positively impacted human health as well as the farming and animal industries. Its extensive usage and versatility led to the spread of resistance mechanisms followed by the development of new variants of the antibiotic. Tetracyclines inhibit bacterial growth by impeding the binding of elongator tRNAs to the ribosome. However, a small number of reports indicated that Tetracyclines could also inhibit translation initiation, yet the molecular mechanism remained unknown. Here, we use biochemical and computational methods to study how Oxytetracycline (Otc), Demeclocycline (Dem), and Tigecycline (Tig) affect the translation initiation phase of protein synthesis. Our results show that all three Tetracyclines induce Initiation Factor IF3 to adopt a compact conformation on the 30S ribosomal subunit, similar to that induced by Initiation Factor IF1. This compaction was faster for Tig than Dem or Otc. Furthermore, all three tested tetracyclines affected IF1-bound 30S complexes. The dissociation rate constant of IF1 in early 30S complexes was 14-fold slower for Tig than Dem or Otc. Late 30S initiation complexes (30S pre-IC or IC) exhibited greater IF1 stabilization by Tig than for Dem and Otc. Tig and Otc delayed 50S joining to 30S initiation complexes (30S ICs). Remarkably, the presence of Tig considerably slowed the progression to translation elongation and retained IF1 in the resulting 70S initiation complex (70S IC). Molecular modeling of Tetracyclines bound to the 30S pre-IC and 30S IC indicated that the antibiotics binding site topography fluctuates along the initiation pathway. Mainly, 30S complexes show potential contacts between Dem or Tig with IF1, providing a structural rationale for the enhanced affinity of the antibiotics in the presence of the factor. Altogether, our data indicate that Tetracyclines inhibit translation initiation by allosterically perturbing the IF3 layout on the 30S, retaining IF1 during 70S IC formation, and slowing the transition toward translation elongation. Thus, this study describes a new complementary mechanism by which Tetracyclines may inhibit bacterial protein synthesis.

Objectives: Carbapenems, colistin, and tigecycline are critically important antibiotics in clinics. After the global appearance of bla NDM and mcr mediating the resistance to carbapenems and colistin, respectively, tigecycline becomes the last-resort drug against severe human infections caused by multidrug-resistant bacteria. Recently, a mobile tigecycline resistance gene tet (X4) has been identified in Escherichia coli , Klebsiella pneumoniae , and Acinetobacter baumannii that causes high resistance to tigecycline and other tetracyclines. In this study, the prevalence of tet (X4) in E. coli isolates from duck and goose farms in Southeast China was identified and characterized. Methods: Feces, soil, sewage, and dust samples were collected from duck and goose farms along with the southeast coast provinces of China. Antimicrobial susceptibility testing and polymerase chain reaction screening were performed to investigate the phenotype and genotype of tigecycline resistance. Conjugation, S1 pulsed-field gel electrophoresis (PFGE), and whole-genome sequencing were used to determine the transferability, genetic location, and the genomic characteristics of tet (X4). Results: In total, 1,716 samples were collected, and 16 isolates (0.9%) recovered from Guangdong, Shandong, and Jiangsu were positive for tet (X4) gene with tigecycline minimum inhibitory concentrations ≥16 mg/L. Notably, among these tet (X4)-positive E. coil isolates, seven of them were from the environment samples (soil and sewage). PFGE and multilocus sequence typing demonstrated that ST3997 was the most prevalent sequence type (eight isolates, 50%) in Jiangsu province. By conjugation assays, 11 isolates were able to transfer tet (X4) plasmid to E. coli C600 recipient, and these plasmids belonged to IncHI1 and IncX1 detected by sequence analysis. tet (X4) was found adjacent to an insertion sequence IS CR2 downstream and a cat D gene upstream for all isolates. In addition, multiple-drug resistance to tigecycline, chlortetracycline, ampicillin, florfenicol, ciprofloxacin, gentamicin, trimethoprim/sulfamethoxazole, and fosfomycin was profiled in most of the tet (X4)-positive isolates. Conclusion: The identification of tet (X4) harboring E. coli strains in duck farms and their surrounding environment enlarges our knowledge of the variety and prevalence of tigecycline resistance. The prevalence of tet (X4) raises concern for the use of tetracyclines in animal farming, and the tet (X4) gene should be listed as primary gene for resistance surveillance. [ABSTRACT FROM AUTHOR]

Emergence of extensively drug-resistant isolates of Klebsiella pneumoniae has prompted increased reliance on the last-resort antibiotics such as tigecycline (TGC) for treating infections caused by these pathogens. Consumption of human antibiotics in the food production industry has been found to contribute to the current antibiotic resistance crisis. In the current study, we aimed to investigate the mechanisms of TGC resistance among 18 TGC-non-susceptible (resistant or intermediate) K. pneumoniae (TGC-NSKP) isolates obtained from human (n = 5), food animals (n = 7), and in vitro selection experiment (n = 6). Isolates were genotyped by multilocus sequence typing (MLST). ramR , acrR , rpsJ , tetA , and mgrB (for colistin resistance) genes were sequenced. The presence of tetX , tetX1 , and carbapenemase genes was examined by PCR. Susceptibility to different classes of antibiotics was evaluated by disc diffusion and broth macrodilution methods. The expression level of acrB was quantified by RT-qPCR assay. The 12 TGC-NSKP isolates [minimum inhibitory concentrations (MICs) = 4–32 mg/l] belonged to 10 distinct sequence types including ST37 (n = 2), ST11, ST15, ST45, ST1326 (animal isolates); ST147 (n = 2, human and animal isolates); and ST16, ST377, ST893, and ST2935 (human isolates). Co-resistance to TGC and colistin was identified among 57 and 40% of animal and human isolates, respectively. All human TGC-NSKP isolates carried carbapenemase genes (bla OXA–48, bla NDM–1, and bla NDM–5). tetX / X1 genes were not detected in any isolates. About 83% of TGC-NSKP isolates (n = 15) carried ramR and/or acrR alterations including missense/nonsense mutations (A19V, L44Q, I141T, G180D, A28T, R114L, T119S, Y59stop, and Q122stop), insertions (positions +205 and +343), or deletions (position +205) for ramR , and R90G substitution or frameshift mutations for acrR. In one isolate ramR amplicon was not detected using all primers used in this study. Among seven colistin-resistant isolates, five harbored inactivated/mutated MgrB due to premature termination by nonsense mutations, insertion of IS elements, and frameshift mutations. All isolates revealed wild-type RpsJ and TetA (if present). Increased expression of acrB gene was detected among all resistant isolates, with the in vitro selected mutants showing the highest values. A combination of RamR and AcrR alterations was involved in TGC non-susceptibility in the majority of studied isolates. [ABSTRACT FROM AUTHOR]

Tigecycline has been used as one of the therapeutic choices for the treatment of infections caused by multidrug-resistant Klebsiella pneumoniae. However, the emergence of tigecycline heteroresistance has led to great challenges in treating these infections. The purpose of this study was to investigate whether tigecycline-heteroresistant K. pneumoniae (TGCHR-Kp) exists in clinical isolates, and to further characterize the underlying molecular mechanisms involved in the development of tigecycline-resistant subpopulations. Of the 268 tigecycline-susceptible clinical K. pneumoniae isolates, 69 isolates were selected as tigecycline-heteroresistant candidates in the preliminary heteroresistant phenotypic selection by a modified disk diffusion method, and only 21 strains were confirmed as TGCHR-Kp by the population analysis profile (PAP). Pulsed-field gel electrophoresis (PFGE) analysis demonstrated that all the parental TGCHR-Kp isolates were clonally unrelated, and colonies confirmed as the heteroresistant subpopulation showed no significant differences from their respective parental TGCHR-Kp isolates. Efflux pump inhibitors reversed the tigecycline susceptibility in heteroresistant subpopulations. Mutations in the ramR and soxR genes lead to upregulation of the ramA and soxS transcriptional regulators, which in turn induced overexpression of the AcrAB-TolC efflux pump genes in TGCHR-Kps-resistant subpopulations. Moreover, mutations of rpsJ were also found in resistant subpopulations, which suggested that the rpsJ mutation may also lead to tigecycline resistance. Time-kill assays showed that the efficacy of tigecycline against TGCHR-Kps was weakened, whereas the number of resistant subpopulations was enriched by the presence of tigecycline. Our findings imply that the presence of TGCHR-Kps in clinical strains causes severe challenges for tigecycline therapy in clinical practice. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii is an important nosocomial pathogen that requires thoughtful consideration in the antibiotic prescription strategy due to its multidrug resistant phenotype. Tetracycline antibiotics have recently been re-administered as part of the combination antimicrobial regimens to treat infections caused by A. baumannii. We show that the TetA(G) efflux pump of A. baumannii AYE confers resistance to a variety of tetracyclines including the clinically important antibiotics doxycycline and minocycline, but not to tigecycline. Expression of tetA(G) gene is regulated by the TetR repressor of A. baumannii AYE (AbTetR). Thermal shift binding experiments revealed that AbTetR preferentially binds tetracyclines which carry a O-5H moiety in ring B, whereas tetracyclines with a 7-dimethylamino moiety in ring D are less well-recognized by AbTetR. Confoundingly, tigecycline binds to AbTetR even though it is not transported by TetA(G) efflux pump. Structural analysis of the minocycline-bound AbTetR-Gln116Ala variant suggested that the non-conserved Arg135 interacts with the ring D of minocycline by cation-π interaction, while the invariant Arg104 engages in H-bonding with the O-11H of minocycline. Interestingly, the Arg135Ala variant exhibited a binding preference for tetracyclines with an unmodified ring D. In contrast, the Arg104Ala variant preferred to bind tetracyclines which carry a O-6H moiety in ring C except for tigecycline. We propose that Arg104 and Arg135, which are embedded at the entrance of the AbTetR binding pocket, play important roles in the recognition of tetracyclines, and act as a barrier to prevent the release of tetracycline from its binding pocket upon AbTetR activation. The binding data and crystal structures obtained in this study might provide further insight for the development of new tetracycline antibiotics to evade the specific efflux resistance mechanism deployed by A. baumannii. [ABSTRACT FROM AUTHOR]

Tigecycline is a last-resort antibiotic for infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP). This study aimed to broaden our understanding of the acquisition of collateral hypersensitivity by CRKP, as an evolutionary trade-off of developing resistance to tigecycline. Experimental induction of tigecycline resistance was conducted with tigecycline-sensitive CRKP clinical isolates. Antimicrobial susceptibility testing, microbial fitness assessment, genotypic analysis and full-genome sequencing were carried out for these clinical isolates and their resistance-induced descendants. We found that tigecycline resistance was successfully induced after exposing CRKP clinical isolates to tigecycline at gradually increased concentrations, at a minor fitness cost of bacterial cells. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) found higher expression of the efflux pump gene acrB (5.3–64.5-fold) and its regulatory gene ramA (7.4–65.8-fold) in resistance-induced strains compared to that in the tigecycline-sensitive clinical isolates. Stable hypersensitivities to aminoglycosides and other antibiotics were noticed in resistance-induced strains, showing significantly lowered MICs (X 4 – >500 times). Full genome sequencing and plasmid analysis suggested the induced collateral hypersensitivity might be multifaceted, with the loss of an antimicrobial resistance (AMR) plasmid being a possible major player. This study rationalized the sequential combination of tigecycline with aminoglycosides for the treatment of CRKP infections. [ABSTRACT FROM AUTHOR]

Tetracycline has positively impacted human health as well as the farming and animal industries. Its extensive usage and versatility led to the spread of resistance mechanisms followed by the development of new variants of the antibiotic. Tetracyclines inhibit bacterial growth by impeding the binding of elongator tRNAs to the ribosome. However, a small number of reports indicated that Tetracyclines could also inhibit translation initiation, yet the molecular mechanism remained unknown. Here, we use biochemical and computational methods to study how Oxytetracycline (Otc), Demeclocycline (Dem), and Tigecycline (Tig) affect the translation initiation phase of protein synthesis. Our results show that all three Tetracyclines induce Initiation Factor IF3 to adopt a compact conformation on the 30S ribosomal subunit, similar to that induced by Initiation Factor IF1. This compaction was faster for Tig than Dem or Otc. Furthermore, all three tested tetracyclines affected IF1-bound 30S complexes. The dissociation rate constant of IF1 in early 30S complexes was 14-fold slower for Tig than Dem or Otc. Late 30S initiation complexes (30S pre-IC or IC) exhibited greater IF1 stabilization by Tig than for Dem and Otc. Tig and Otc delayed 50S joining to 30S initiation complexes (30S ICs). Remarkably, the presence of Tig considerably slowed the progression to translation elongation and retained IF1 in the resulting 70S initiation complex (70S IC). Molecular modeling of Tetracyclines bound to the 30S pre-IC and 30S IC indicated that the antibiotics binding site topography fluctuates along the initiation pathway. Mainly, 30S complexes show potential contacts between Dem or Tig with IF1, providing a structural rationale for the enhanced affinity of the antibiotics in the presence of the factor. Altogether, our data indicate that Tetracyclines inhibit translation initiation by allosterically perturbing the IF3 layout on the 30S, retaining IF1 during 70S IC formation, and slowing the transition toward translation elongation. Thus, this study describes a new complementary mechanism by which Tetracyclines may inhibit bacterial protein synthesis. [ABSTRACT FROM AUTHOR]

Tigecycline is a tetracycline derivative that is being used as an antibiotic of last resort. Both tigecycline and tetracycline bind to the small (30S) ribosomal subunit and inhibit translation. Target mutations leading to resistance to these antibiotics have been identified both in the 16S ribosomal RNA and in ribosomal proteins S3 and S10 (encoded by the rpsJ gene). Several different mutations in the S10 flexible loop tip residue valine 57 (V57) have been observed in tigecycline-resistant Escherichia coli isolates. However, the role of these mutations in E. coli has not yet been characterized in a defined genetic background. In this study, we chromosomally integrated 10 different rpsJ mutations into E. coli , resulting in different exchanges or a deletion of S10 V57, and investigated the effects of the mutations on growth and tigecycline/tetracycline resistance. While one exchange, V57K, decreased the minimal inhibitory concentration (MIC) (Etest) to tetracycline to 0.75 μg/ml (compared to 2 μg/ml in the parent strain) and hence resulted in hypersensitivity to tetracycline, most exchanges, including the ones reported previously in resistant isolates (V57L, V57D, and V57I) resulted in slightly increased MICs to tigecycline and tetracycline. The strongest increase was observed for the V57L mutant, with a MIC (Etest) to tigecycline of 0.5 μg/ml (compared to 0.125 μg/ml in the parent strain) and a MIC to tetracycline of 4.0 μg/ml. Nevertheless, none of these exchanges increased the MIC to the extent observed in previously described clinical tigecycline-resistant isolates. We conclude that, next to S10 mutations, additional mutations are necessary in order to reach high-level tigecycline resistance in E. coli. In addition, our data reveal that mutants carrying S10 V57 exchanges or deletion display growth defects and, in most cases, also thermosensitivity. The defects are particularly strong in the V57 deletion mutant, which is additionally cold-sensitive. We hypothesize that the S10 loop tip residue is critical for the correct functioning of S10. Both the S10 flexible loop and tigecycline are in contact with helix h31 of the 16S rRNA. We speculate that exchanges or deletion of V57 alter the positioning of h31, thereby influencing both tigecycline binding and S10 function. [ABSTRACT FROM AUTHOR]

Background: The widespread use of antibiotics has led to the emergence of multidrug-resistant (MDR) bacteria such as multidrug-resistant Acinetobacter baumannii (AB). Tigecycline (TGC), as the first glycylcycline antibiotic approved by FDA, is a broad-spectrum antibiotic which remains highly effective to treat AB infections. Objective: To confirm the TGC treatment dosage and effectiveness to treat AB infections in the Chinese population by performing therapeutic drug monitoring (TDM). Methods: This study was performed from October 2018 through March 2019 at the PLA General Hospital. A high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was validated and employed to determine the plasma concentrations of TGC in patients with infectious diseases. The minimum inhibitory concentration (MIC) of TGC to clinically isolated AB was determined by broth microdilution method, agar dilution method, and disk diffusion method. Moreover, a model of population pharmacokinetics/pharmacodynamics (PPK/PD) was constructed. Results: A total of 186 plasma samples from 67 patients were detected by the validated HPLC-MS/MS method. The MIC values determined by the broth microdilution method were more sensitive and accurate than the other two methods. The microbial and clinical PK/PD breakpoints were reached when the maintenance dose of TGC was 100 mg. Conclusion: Our study established a validated HPLC-MS/MS method to monitor the plasma concentrations of TGC. In view of the MIC range to AB isolates in our hospital and the PPK/PD modeling results, we recommend a relatively high dose of 100 mg q12h regimen to achieve the optimal clinical efficacy and antimicrobial response. [ABSTRACT FROM AUTHOR]