Tigecycline Resistance Mechanism Research Articles

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

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

Molecular mechanisms of tigecycline-resistance among Enterobacterales.

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

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

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

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

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

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

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

Tigecycline-resistance mechanisms and biological characteristics of drug-resistant Salmonella Typhimurium strains in vitro

Increased drug resistance of Gram-negative bacteria to tetracycline caused by the unreasonable overuse of tigecycline has attracted extensive attention to reveal potential mechanisms. Here, we identified a tigecycline-resistant strain called TR16, derived from Salmonella Typhimurium ATCC13311 (AT), and examined its biological characteristics. Compared with AT, the TR16 strain showed significantly higher resistance to amoxicillin but lower resistance to gentamicin. Although the growth curves of TR16 and AT were similar, TR16 showed a significantly increased capacity for biofilm formation and a notably decreased motility compared to AT. Furthermore, transcriptome sequencing and reverse transcription–quantitative PCR (RT-qPCR) were implemented to evaluate the genetic difference between AT and TR16. Whole genome sequencing (WGS) analysis was also conducted to identify single nucleotide polymorphism (SNPs) and screened out two genetic mutations (lptD and rpsJ). The acrB gene of TR16 was knocked out through CRISPR/Cas9 system to further elucidate underlying mechanisms of tigecycline resistance in Salmonella Typhimurium. The up-regulation of acrB in TR16 was verified by RNA-seq and RT-qPCR, and the lack of acrB resulted in a 16-fold reduction in tigecycline resistance in TR16. Collectively, these results implied that AcrB efflux pump plays a key role in the tigecycline resistance of Salmonella, shedding light on the potential of AcrB efflux pump as a novel target for the discovery and development of new antibiotics.

Study on the Genome and Mechanism of Tigecycline Resistance of a Clinical Chryseobacterium indologenes Strain.

Purpose: Chryseobacterium indologenes is a clinically relevant microorganism that has been on the rise, with multidrug-resistant (MDR) strains being reported. C. indologenes carrying tet(X2) has been demonstrated to be resistant to the antibiotic tigecycline, yet, sensitive to all other members of the tetracycline family. This inconsistency in resistance prompts an inquiry into the contribution of tet(X2) to tigecycline resistance in C. indologenes. Materials and Methods: In this study, we report on a comprehensive analysis of the genomic mechanisms underlying tigecycline resistance in a MDR C. indologenes strain (CI3125) that was resistant to tigecycline but sensitive to tetracycline, doxycycline, and minocycline. We used whole-genome sequencing, quantitative reverse transcription PCR, Western blot, antibiotic-degrading tests, and efflux pump inhibiting tests to reveal the mechanism of tigecycline resistance in C. indologenes and elucidate the inconsistency in the antibiotic resistance mechanism for the tetracycline family. Results: Our findings demonstrate that CI3125 carries 60 antibiotic resistance genes distributed on 6 different genetic islands (GIs), with the potential for horizontal transfer. Notably, the tet(X2) gene is located on GI06 of CI3125. Genetic environment analysis of tet(X2) showed that all tet(X2) genes in Flavobacterium and Bacteroides share a conservative and functional ribosome-binding site upstream. Contrary to expectation, our RT-qPCR showed that tet(X2) was not transcribed in CI3125, and Western blot suggested the absence of tet(X2) protein in CI3125. Rather, we demonstrate that minimum inhibitory concentration values for tigecycline decreased two- to eight-folds in the presence of five different efflux pump inhibitors [1-(1-naphthyl- methyl)-piperazine, phenyl-arginine-β-naphthylamide, verapamil, reserpine, and carbonyl cyanide 3-chlorophenylhydrazone]. This finding provides evidence for the involvement of efflux pumps in tigecycline resistance, which is likely to be a universal mechanism among C. indologenes. Our study proposes that the inconsistency in resistance to the tetracycline family in CI3125 may be ascribed to the silence of tet(X2) and the functions of efflux pumps for tigecycline. Conclusions: Overall, our results highlight the importance of genomic approaches in understanding the underlying mechanisms of antibiotic resistance in clinically relevant microorganisms. While tet(X2) in CI3125 is silent, our findings suggest that it may be horizontally spread through GIs. Hence, our findings have significant implications for the management of C. indologenes infections in clinical settings.

Emergence of high-level tigecycline resistance due to the amplification of a tet(A) gene variant in clinical carbapenem-resistant Klebsiella pneumoniae

ObjectiveTo investigate the prevalence of a tet(A) gene variant and its role in developing high-level tigecycline resistance among carbapenem-resistant Klebsiella pneumoniae (CRKP) clinical isolates. MethodsThe mechanism of high-level tigecycline resistance in CRKP mediated by a tet(A) variant was explored by induction experiments, antimicrobial susceptibility testing, whole-genome sequencing and bioinformatics analysis. The amplification and overexpression of the tet(A) variant were measured by the determination of sequencing depth, gene copy numbers, and qRT-PCR. ResultsA high rate (62.1%, 998/1607) of tet(A) variant carriage was observed among 1607 CRKP clinical isolates from Henan Province, China. High-level tigecycline resistance could rapidly develop by the amplification of the tet(A) variant in these isolates. The analysis of the raw sequencing data and the plasmid mapping depth revealed that the ΔtnpA homologous sequence of Tn1721 supports the amplification of the region that harbours the tet(A) variant by forming a large number of repeat arrays through translocatable units (TUs). Moreover, the epidemiological analysis of tet(A) variant-carrying structures among 1607 clinical CRKPs showed that the TU structure is widely present. ConclusionThe presence of a tigecycline resistance-mediating tet(A) variant in CRKP clinical isolates represents a greater health concern than initially thought and should be monitored consistently.

The Mechanism of Tigecycline Resistance in Acinetobacter baumannii Revealed by Proteomic and Genomic Analysis.

The mechanism of tigecycline resistance in A. baumannii remains largely unclear. In this study, we selected a tigecycline-resistant and a tigecycline-susceptible strain from a tigecycline-susceptible and a resistant strain, respectively. Proteomic and genomic analyses were performed to elucidate the variations associated with tigecycline resistance. Our study showed proteins associated with efflux pump, biofilm formation, iron acquisition, stress response, and metabolic ability are upregulated in tigecycline resistant strains, and efflux pump should be the key mechanism for tigecycline resistance. By genomic analysis, we found several changes in the genome that can explain the increased level of efflux pump, including the loss of the global negative regulator hns in the plasmid and the disruption of the hns gene and acrR gene on the chromosome by the insertion of IS5. Collectively, we not only revealed the phenomenon that the efflux pump is mainly responsible for tigecycline resistance, but also highlighted the mechanism at the genomic level, which will help in understanding the resistance mechanism in detail and provide clues for the treatment of clinical multiple drug-resistant A. baumannii.

Resistance mechanisms of tigecycline in Acinetobacter baumannii.

Acinetobacter baumannii is widely distributed in nature and in hospital settings and is a common pathogen causing various infectious diseases. Currently, the drug resistance rate of A. baumannii has been persistently high, showing a worryingly high resistance rate to various antibiotics commonly used in clinical practice, which greatly limits antibiotic treatment options. Tigecycline and polymyxins show rapid and effective bactericidal activity against CRAB, and they are both widely considered to be the last clinical line of defense against multidrug resistant A. baumannii. This review focuses with interest on the mechanisms of tigecycline resistance in A. baumannii. With the explosive increase in the incidence of tigecycline-resistant A. baumannii, controlling and treating such resistance events has been considered a global challenge. Accordingly, there is a need to systematically investigate the mechanisms of tigecycline resistance in A. baumannii. Currently, the resistance mechanism of A. baumannii to tigecycline is complex and not completely clear. This article reviews the proposed resistance mechanisms of A. baumannii to tigecycline, with a view to providing references for the rational clinical application of tigecycline and the development of new candidate antibiotics.

Efflux Pumps and Different Genetic Contexts of tet(X4) Contribute to High Tigecycline Resistance in Escherichia fergusonii from Pigs.

Tigecycline is a last-resort antibiotic for the treatment of infections caused by multidrug-resistant bacteria. The emergence of plasmid-mediated tigecycline resistance genes is posing a serious threat to food safety and human health and has attracted worldwide attention. In this study, we characterized six tigecycline-resistant Escherichia fergusonii strains from porcine nasal swab samples collected from 50 swine farms in China. All the E. fergusonii isolates were highly resistant to tigecycline with minimal inhibitory concentration (MIC) values of 16-32 mg/L, and all contained the tet(X4) gene. In addition, 13-19 multiple resistance genes were identified in these isolates, revealed by whole-genome sequencing analysis. The tet(X4) gene was identified as being located in two different genetic structures, hp-abh-tet(X4)-ISCR2 in five isolates and hp-abh-tet(X4)-ΔISCR2-ISEc57-IS26 in one isolate. The role of efflux pumps in tigecycline resistance was evaluated by using inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The MIC values of tigecycline showed a 2- to 4-fold reduction in the presence of CCCP, indicating the involvement of active efflux pumps in tigecycline resistance in E. fergusonii. The tet(X4) gene was found to be transferable to Escherichia coli J53 by conjugation and resulted in the acquisition of tigcycline resistances in the transconjugants. Whole-genome multilocus sequence typing (wgMLST) and phylogenetic analysis showed a close relationship of five isolates originating from different pig farms, suggesting the transmission of tet(X4)-positive E. fergusonii between farms. In conclusion, our findings suggest that E. fergusonii strains in pigs are reservoirs of a transferable tet(X4) gene and provide insights into the tigecycline resistance mechanism as well as the diversity and complexity of the genetic context of tet(X4) in E. fergusonii.

Fitness Costs of Tigecycline Resistance in Acinetobacter baumannii and the Resistance Mechanism Revealed by a Transposon Mutation Library.

Acinetobacter baumannii is one of the main pathogens causing nosocomial and community-acquired infections. Tigecycline is an important antibiotic for the treatment of multidrug-resistant A. baumannii infections, but strains resistant to tigecycline have also emerged. There are still many unclear questions concerning the mechanism of tigecycline resistance in A. baumannii. In this study, tigecycline-susceptible and tigecycline-intermediate strains were gradually cultured with sub-minimum inhibitory concentrations of tigecycline to select for tigecycline-resistant mutants, and a tigecycline-resistant strain was cultured under 42 °C to select for tigecycline-susceptible mutants. We found that the acquisition of tigecycline resistance affected the susceptibility of the strains to other antibiotics. Resistance to ampicillin–sulbactam is negatively correlated with tigecycline resistance. The strains will experience fitness costs along with the acquisition of tigecycline resistance. Tigecycline resistance in the strains was not related to 16S rRNA target variation or outer membrane integrity alteration. By constructing a transposon mutation library, we found that transposon insertion of the adeL gene reduced the sensitivity of A. baumannii to tigecycline. This study provides important clues for understanding the mechanism of tigecycline resistance in A. baumannii.

Mobile Tigecycline Resistance: An Emerging Health Catastrophe Requiring Urgent One Health Global Intervention.

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.

Molecular mechanisms and genomic basis of tigecycline-resistant Enterobacterales from swine slaughterhouses

The continuous emergence of tigecycline-resistant bacteria is undermining the effectiveness of clinical tigecycline. Environmental tigecycline-resistant bacteria have the potential to infect humans through human-environment interactions. Furthermore, the mechanisms of tigecycline resistance in Enterobacterales are complicated. In this study, we aimed to investigate the additional pathways of tigecycline resistance in environmental Enterobacterales besides tet(X) and tmexCD-toprJ. During the years 2019-2020, tigecycline-resistant Enterobacterales (n = 45) negative for tet(X) and tmexCD-toprJ were recovered from 328 different samples from two slaughterhouses. Five distinct bacteria species were identified, of which Klebsiella pneumoniae (n = 37) was the most common, with K. pneumoniae ST45 and ST35 being the predominant clones. Tigecycline resistance determinants analysis showed that tet(A) mutations and ramR inactivation were the most prevalent mechanisms for tigecycline resistance in the 45 strains. Two known tet(A) variants (type 1 and tet(A)-v) and one novel tet(A) variant (type 3) were identified. Cloning experiments confirmed that the novel type 3 tet(A) could enhance the 4-fold MIC for tigecycline. Inactivation of ramR was induced by either point mutations or indels of sequences, which could result in the overexpression of AcrAB pump genes leading to tigecycline resistance. In addition, all isolates were resistant to a wide range of antimicrobials and carried various resistance genes. These findings enriched the epidemiological and genomic characterizations of tigecycline-resistant Enterobacterales from slaughterhouses and contributed to a better understanding of the complex mechanisms of tigecycline resistance in environmental bacteria.

Characterization of IncHI1B Plasmids Encoding Efflux Pump TmexCD2-ToprJ2 in Carbapenem-Resistant Klebsiella variicola, Klebsiella quasipneumoniae, and Klebsiella michiganensis Strains.

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 blaNDM-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 blaNDM-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.

Tigecycline Heteroresistance and Resistance Mechanism in Clinical Isolates of Acinetobacter baumannii.

ABSTRACTTigecycline is regarded as a last-resort treatment for multidrug-resistant Acinetobacter baumannii. However, tigecycline resistance in A. baumannii has increased worldwide. In this study, we investigated tigecycline heteroresistance in A. baumannii isolates from South Korea. Antibiotic susceptibility testing was performed on 323 nonduplicated A. baumannii isolates. Among 260 and 37 tigecycline-susceptible and -intermediate-resistant A. baumannii isolates, 146 (56.2%) and 22 (59.5%) isolates were identified as heteroresistant to tigecycline through a disk diffusion assay and population analysis profiling. For selected isolates, an in vitro time-kill assay was performed, and survival rates were measured after preincubation with diverse concentrations of tigecycline. Heteroresistant isolates showed regrowth after 12 h of 2× MIC of tigecycline treatment, and resistant subpopulations were selected by preexposure to tigecycline. Furthermore, genetic alterations in adeABC, adeRS, and rpsJ were assessed, and the relative mRNA expression levels of adeB and adeS were compared. The tigecycline resistance in subpopulations might be due to the insertion of ISAba1 in adeS, leading to the overexpression of the AdeABC efflux pump. However, the tigecycline resistance of subpopulations was not stable during serial passages in antibiotic-free medium. The reversion of tigecycline susceptibility by antibiotic-free passages might occur by additional insertions of ISAba10 in adeR and nucleotide alterations in adeS in some mutants. Tigecycline heteroresistance is prevalent in A. baumannii isolates, which results in treatment failure. Tigecycline resistance is mainly due to the overexpression of the AdeABC efflux pump, which is associated with genetic mutations, but this resistance could be reversed into susceptibility by additional mutations in antibiotic-free environments.IMPORTANCE The evidence that antibiotic heteroresistance is responsible for treatment failure in clinical settings is increasing. Thus, detection and characterization of heteroresistance would be important for appropriate therapeutic guidance to treat bacterial infections. However, data on tigecycline heteroresistance in Gram-negative bacteria is currently limited, although tigecycline is regarded as a last-line antibiotic against infections caused by antibiotic-resistant pathogens. In this study, we investigated the tigecycline heteroresistance in Acinetobacter baumannii, which has been listed by the WHO as a priority for research and development of new antibiotics. We found very high prevalence of tigecycline-heteroresistant A. baumannii clinical isolates, which may result in treatment failure due to the selection of resistant subpopulations. We also identified the main resistance mechanism in tigecycline-resistant subpopulations, that is, upregulation of AdeABC efflux pumps due to ISAba1 insertion in adeS.

Multiple Mechanisms of Tigecycline Resistance in Enterobacteriaceae from a Pig Farm, China.

ABSTRACTWe isolated eight tigecycline-resistant Enterobacteriaceae strains from a pig farm in Shanghai, China, including Escherichia coli (n = 1), Proteus cibarius (n = 1), and Enterobacter hormaechei (n = 6). Two of them (E. coli and P. cibarius) were positive for tet(X). E. coli SH19PTE6 contained an IncFIA18/IncFIB(K)/IncX1 hybrid plasmid pYUSHP6-tetX, highly similar to other tet(X)-bearing hybrid plasmids from E. coli in China. In P. cibarius SH19PTE4, tet(X) was located within a new chromosomal integrative and conjugative element (ICE), ICEPciChn2, belonging to the SXT/R391 ICE family. All tigecycline-resistant E. hormaechei isolates carried the tet(A) variant; cloning and transfer of this tet(A) variant into various hosts increased their MICs for tigecycline (4- to 8-fold). Tigecycline resistance observed on a pig farm is mediated by the tet(A) variant and tet(X) via a plasmid or ICE. The rational use of antibiotics such as doxycycline and surveillance of tigecycline resistance in livestock are warranted.IMPORTANCE As a last-resort antimicrobial agent to treat serious infections, the emergence and spread of tigecycline resistance in Enterobacteriaceae and Acinetobacter have raised global concerns. Multiple mechanisms mediate tigecycline resistance in Enterobacteriaceae, such as the monooxygenase Tet(X), mutations in Tet proteins, and overexpression of efflux pumps. Although tigecycline is not approved for animals, tigecycline resistance has been observed in Escherichia coli, Proteus cibarius, and Enterobacter hormaechei isolates on a pig farm, mediated by the tet(A) variant and tet(X) via a plasmid or ICE. The heavy use of tetracyclines such as doxycycline in food-producing animals in China may be the reason for the emergence and transmission of tigecycline resistance.

PGN-027 - Tigecycline heteroresistance and resistance mechanism in clinical isolates of Acinetobacter baumannii

Tigecycline is regarded as a last-resort treatment for multidrug-resistant Acinetobacter baumannii. However, tigecycline resistance in A. baumannii has increased worldwide. In this study, we investigated tigecycline heteroresistance in A. baumannii isolates from South Korea. Antibiotic susceptibility testing was performed on 323 nonduplicated A. baumannii isolates. Among 260 and 37 tigecycline-susceptible and -intermediate-resistant A. baumannii isolates, 146 (56.2%) and 22 (59.5%) isolates were identified as heteroresistant to tigecycline through a disk diffusion assay and population analysis profiling. For selected isolates, an in vitro time-kill assay was performed, and survival rates were measured after preincubation with diverse concentrations of tigecycline. Heteroresistant isolates showed regrowth after 12 h of 2× MIC of tigecycline treatment, and resistant subpopulations were selected by preexposure to tigecycline. Furthermore, genetic alterations in adeABC, adeRS, and rpsJ were assessed, and the relative mRNA expression levels of adeB and adeS were compared. The tigecycline resistance in subpopulations might be due to the insertion of ISAba1 in adeS, leading to the overexpression of the AdeABC efflux pump. However, the tigecycline resistance of subpopulations was not stable during serial passages in antibiotic-free medium. The reversion of tigecycline susceptibility by antibiotic-free passages might occur by additional insertions of ISAba10 in adeR and nucleotide alterations in adeS in some mutants. Tigecycline heteroresistance is prevalent in A. baumannii isolates, which results in treatment failure. Tigecycline resistance is mainly due to the overexpression of the AdeABC efflux pump, which is associated with genetic mutations, but this resistance could be reversed into susceptibility by additional mutations in antibiotic-free environments. IMPORTANCE The evidence that antibiotic heteroresistance is responsible for treatment failure in clinical settings is increasing. Thus, detection and characterization of heteroresistance would be important for appropriate therapeutic guidance to treat bacterial infections. However, data on tigecycline heteroresistance in Gram-negative bacteria is currently limited, although tigecycline is regarded as a last-line antibiotic against infections caused by antibiotic-resistant pathogens. In this study, we investigated the tigecycline heteroresistance in Acinetobacter baumannii, which has been listed by the WHO as a priority for research and development of new antibiotics. We found very high prevalence of tigecycline-heteroresistant A. baumannii clinical isolates, which may result in treatment failure due to the selection of resistant subpopulations. We also identified the main resistance mechanism in tigecycline-resistant subpopulations, that is, upregulation of AdeABC efflux pumps due to ISAba1 insertion in adeS.

A stop-gain mutation in sigma factor SigH (MAB_3543c) may be associated with tigecycline resistance in Mycobacteroides abscessus.

Introduction. Tigecycline is currently acknowledged to be one of the most effective antibiotics against infections caused by Mycobacteroides abscessus.Gap statement. The genetic determinants of tigecycline resistance in M. abscessus are not well understood.Aim. In this study, we characterized a tigecycline-resistant M. abscessus mutant, designated CL7, to identify the potential resistance mechanism.Methodology. CL7 was characterized using antimicrobial susceptibility testing, whole-genome sequencing, PCR and RT-qPCR. For biological verification, gene overexpression assays were carried out.Results. Whole-genome sequencing and the subsequent gene overexpression assays showed that CL7 harboured a stop-gain mutation in MAB_3543 c, which may be responsible for the tigecycline resistance phenotype. This gene encodes an orthologue of SigH, which is involved in the positive regulation of physiological stress response and is negatively regulated by the RshA anti-sigma factor in Mycobacterium tuberculosis. We hypothesized that the MAB_3543 c mutation may disrupt the interaction between SigH and RshA (MAB_3542 c). RT-qPCR analyses revealed the upregulation of MAB_3543 c and other key stress response genes, which has previously been shown to be a hallmark of SigH-RshA bond disruption and tigecycline resistance.Conclusion. The MAB_3543c mutation may represent a novel determinant of tigecycline resistance in M. abscessus. The findings of this study will hopefully contribute to our knowledge of potential tigecycline resistance mechanisms in M. abscessus, which may lead to better diagnostics and treatment modalities in the future.

Prevalence of RND efflux pump regulator variants associated with tigecycline resistance in carbapenem-resistant Acinetobacter baumannii from a worldwide survey

To determine the most common tigecycline resistance mechanisms in carbapenem-resistant Acinetobacter baumannii isolates obtained during the global Tigecycline Evaluation Surveillance Trial (TEST). Tigecycline MICs were determined by broth microdilution. WGS was used to screen for the previously identified tigecycline resistance mechanisms, as well as mutations in resistance-nodulation-cell division (RND)-type efflux pump regulators. From a total 313 isolates, 113 genetically unique tigecycline-resistant isolates were analysed. The most frequent and worldwide distributed mechanism associated with tigecycline resistance was disruption of adeN, which encodes the repressor of the RND efflux pump AdeIJK, either by IS elements or nucleotide deletions causing premature stop codons. However, mutations leading to amino acid substitutions and disruption by IS elements within the two-component regulatory system adeRS, which regulates expression of the AdeABC efflux pump, correlate with higher tigecycline MICs, but these were found less frequently and were mainly restricted to Southern European countries. Furthermore, an altered version of tviB was identified in several tigecycline-resistant isolates that did not have putative resistance mutations within RND-type regulators. The resistance determinants tet(A) and tet(X), as well as resistance mutations in putative resistance determinants trm, plsC, rrf, msbA and genes encoding 30S ribosomal proteins, were not identified in any isolate. The most prevalent tigecycline resistance mechanisms were caused by alterations in the regulators of RND-type efflux pumps. These data provide the basis for further characterization of regulator alterations and their contribution to increased efflux and tigecycline resistance, and also should be taken into account in drug discovery programmes to overcome the contribution of efflux pumps.