ResearchPad - streptomycin https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Substantial improvement of tetraene macrolide production in <i>Streptomyces diastatochromogenes</i> by cumulative drug resistance mutations]]> https://www.researchpad.co/article/elastic_article_7861 Tetraene macrolides remain one of the most reliable fungicidal agents as resistance of fungal pathogens to these antibiotics is relatively rare. The modes of action and biosynthesis of polyene macrolides had been the focus of research over the past few years. However, few studies have been carried out on the overproduction of polyene macrolides. In the present study, cumulative drug-resistance mutation was used to obtain a quintuple mutant G5-59 with huge tetraene macrolide overproduction from the starting strain Streptomyces diastatochromogenes 1628. Through DNA sequence analysis, the mutation points in the genes of rsmG, rpsL and rpoB were identified. Additionally, the growth characteristic and expression level of tetrRI gene (belonging to the large ATP binding regulator of LuxR family) involved in the biosynthesis of tetraene macrolides were analyzed. As examined with 5L fermentor, the quintuple mutant G5-59 grew very well and the maximum productivity of tetramycin A, tetramycin P and tetrin B was as high as 1735, 2811 and 1500 mg/L, which was 8.7-, 16- and 25-fold higher than that of the wild-type strain 1628, respectively. The quintuple mutant G5-59 could be useful for further improvement of tetraene macrolides production at industrial level.

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<![CDATA[A novel cell-free method to culture Schistosoma mansoni from cercariae to juvenile worm stages for in vitro drug testing]]> https://www.researchpad.co/article/5c58d63ed5eed0c484031973

Background

The arsenal in anthelminthic treatment against schistosomiasis is limited and relies almost exclusively on a single drug, praziquantel (PZQ). Thus, resistance to PZQ could constitute a major threat. Even though PZQ is potent in killing adult worms, its activity against earlier stages is limited. Current in vitro drug screening strategies depend on newly transformed schistosomula (NTS) for initial hit identification, thereby limiting sensitivity to new compounds predominantly active in later developmental stages. Therefore, the aim of this study was to establish a highly standardized, straightforward and reliable culture method to generate and maintain advanced larval stages in vitro. We present here how this method can be a valuable tool to test drug efficacy at each intermediate larval stage, reducing the reliance on animal use (3Rs).

Methodology/Principal findings

Cercariae were mechanically transformed into skin-stage (SkS) schistosomula and successfully cultured for up to four weeks with no loss in viability in a commercially available medium. Under these serum- and cell-free conditions, development halted at the lung-stage (LuS). However, the addition of human serum (HSe) propelled further development into liver stage (LiS) worms within eight weeks. Skin and lung stages, as well as LiS, were submitted to 96-well drug screening assays using known anti-schistosomal compounds such as PZQ, oxamniquine (OXM), mefloquine (MFQ) and artemether (ART). Our findings showed stage-dependent differences in larval susceptibility to these compounds.

Conclusion

With this robust and highly standardized in vitro assay, important developmental stages of S. mansoni up to LiS worms can be generated and maintained over prolonged periods of time. The phenotype of LiS worms, when exposed to reference drugs, was comparable to most previously published works for ex vivo harvested adult worms. Therefore, this in vitro assay can help reduce reliance on animal experiments in search for new anti-schistosomal drugs.

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<![CDATA[Triple oral beta-lactam containing therapy for Buruli ulcer treatment shortening]]> https://www.researchpad.co/article/5c58d647d5eed0c484031a67

The potential use of clinically approved beta-lactams for Buruli ulcer (BU) treatment was investigated with representative classes analyzed in vitro for activity against Mycobacterium ulcerans. Beta-lactams tested were effective alone and displayed a strong synergistic profile in combination with antibiotics currently used to treat BU, i.e. rifampicin and clarithromycin; this activity was further potentiated in the presence of the beta-lactamase inhibitor clavulanate. In addition, quadruple combinations of rifampicin, clarithromycin, clavulanate and beta-lactams resulted in multiplicative reductions in their minimal inhibitory concentration (MIC) values. The MIC of amoxicillin against a panel of clinical isolates decreased more than 200-fold within this quadruple combination. Amoxicillin/clavulanate formulations are readily available with clinical pedigree, low toxicity, and orally and pediatric available; thus, supporting its potential inclusion as a new anti-BU drug in current combination therapies.

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<![CDATA[Inferring fitness landscapes and selection on phenotypic states from single-cell genealogical data]]> https://www.researchpad.co/article/5989db53ab0ee8fa60bdce55

Recent advances in single-cell time-lapse microscopy have revealed non-genetic heterogeneity and temporal fluctuations of cellular phenotypes. While different phenotypic traits such as abundance of growth-related proteins in single cells may have differential effects on the reproductive success of cells, rigorous experimental quantification of this process has remained elusive due to the complexity of single cell physiology within the context of a proliferating population. We introduce and apply a practical empirical method to quantify the fitness landscapes of arbitrary phenotypic traits, using genealogical data in the form of population lineage trees which can include phenotypic data of various kinds. Our inference methodology for fitness landscapes determines how reproductivity is correlated to cellular phenotypes, and provides a natural generalization of bulk growth rate measures for single-cell histories. Using this technique, we quantify the strength of selection acting on different cellular phenotypic traits within populations, which allows us to determine whether a change in population growth is caused by individual cells’ response, selection within a population, or by a mixture of these two processes. By applying these methods to single-cell time-lapse data of growing bacterial populations that express a resistance-conferring protein under antibiotic stress, we show how the distributions, fitness landscapes, and selection strength of single-cell phenotypes are affected by the drug. Our work provides a unified and practical framework for quantitative measurements of fitness landscapes and selection strength for any statistical quantities definable on lineages, and thus elucidates the adaptive significance of phenotypic states in time series data. The method is applicable in diverse fields, from single cell biology to stem cell differentiation and viral evolution.

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<![CDATA[The Role of Adherence and Retreatment in De Novo Emergence of MDR-TB]]> https://www.researchpad.co/article/5989db0aab0ee8fa60bc9f19

Treatment failure after therapy of pulmonary tuberculosis (TB) infections is an important challenge, especially when it coincides with de novo emergence of multi-drug-resistant TB (MDR-TB). We seek to explore possible causes why MDR-TB has been found to occur much more often in patients with a history of previous treatment. We develop a mathematical model of the replication of Mycobacterium tuberculosis within a patient reflecting the compartments of macrophages, granulomas, and open cavities as well as parameterizing the effects of drugs on the pathogen dynamics in these compartments. We use this model to study the influence of patient adherence to therapy and of common retreatment regimens on treatment outcome. As expected, the simulations show that treatment success increases with increasing adherence. However, treatment occasionally fails even under perfect adherence due to interpatient variability in pharmacological parameters. The risk of generating MDR de novo is highest between 40% and 80% adherence. Importantly, our simulations highlight the double-edged effect of retreatment: On the one hand, the recommended retreatment regimen increases the overall success rate compared to re-treating with the initial regimen. On the other hand, it increases the probability to accumulate more resistant genotypes. We conclude that treatment adherence is a key factor for a positive outcome, and that screening for resistant strains is advisable after treatment failure or relapse.

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<![CDATA[Genetic Susceptibility and Predictors of Paradoxical Reactions in Buruli Ulcer]]> https://www.researchpad.co/article/5989daffab0ee8fa60bc5f19

Introduction

Buruli ulcer (BU) is the third most frequent mycobacterial disease in immunocompetent persons after tuberculosis and leprosy. During the last decade, eight weeks of antimicrobial treatment has become the standard of care. This treatment may be accompanied by transient clinical deterioration, known as paradoxical reaction. We investigate the incidence and the risks factors associated with paradoxical reaction in BU.

Methods

The lesion size of participants was assessed by careful palpation and recorded by serial acetate sheet tracings. For every time point, surface area was compared with the previous assessment. All patients received antimicrobial treatment for 8 weeks. Serum concentration of 25-hydroxyvitamin D, the primary indicator of vitamin D status, was determined in duplex for blood samples at baseline by a radioimmunoassay. We genotyped four polymorphisms in the SLC11A1 gene, previously associated with susceptibility to BU. For testing the association of genetic variants with paradoxical responses, we used a binary logistic regression analysis with the occurrence of a paradoxical response as the dependent variable.

Results

Paradoxical reaction occurred in 22% of the patients; the reaction was significantly associated with trunk localization (p = .039 by Χ2), larger lesions (p = .021 by Χ2) and genetic factors. The polymorphisms 3’UTR TGTG ins/ins (OR 7.19, p < .001) had a higher risk for developing paradoxical reaction compared to ins/del or del/del polymorphisms.

Conclusions

Paradoxical reactions are common in BU. They are associated with trunk localization, larger lesions and polymorphisms in the SLC11A1 gene.

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<![CDATA[Susceptibility to Aminoglycosides and Distribution of aph and aac(3)-XI Genes among Corynebacterium striatum Clinical Isolates]]> https://www.researchpad.co/article/5989da03ab0ee8fa60b74b84

Corynebacterium striatum is an opportunistic pathogen, often multidrug-resistant, which has been associated with serious infections in humans. Aminoglycosides are second-line or complementary antibiotics used for the treatment of Corynebacterium infections. We investigated the susceptibility to six aminoglycosides and the molecular mechanisms involved in aminoglycoside resistance in a collection of 64 Corynebacterium striatum isolated in our laboratory during the period 2005–2009. Antimicrobial susceptibility was determined using E-test. The mechanisms of aminoglycoside resistance were investigated by PCR and sequencing. The 64 C. striatum were assessed for the possibility of clonal spreading by Pulsed-field Gel Electrophoresis (PFGE). Netilmicin and amikacin were active against the 64 C. striatum isolates (MICs90 = 0.38 and 0.5 mg/L, respectively). Twenty-seven of the 64 C. striatum strains showed a MIC90 for kanamycin > 256 mg/L, and 26 out the 27 were positive for the aph(3’)-Ic gene. Thirty-six out of our 64 C. striatum were streptomycin resistant, and 23 out of the 36 carried both the aph(3”)-Ib and aph(6)-Id genes. The gene aac(3)-XI encoding a new aminoglycoside 3-N acetyl transferase from C. striatum was present in 44 out of the 64 isolates, all of them showing MICs of gentamicin and tobramycin > 1 mg/L. CS4933, a C. striatum showing very low susceptibility to kanamycin and streptomycin, contains an aminoglycoside resistance region that includes the aph(3’)-Ic gene, and the tandem of genes aph(3”)-Ib and aph(6)-Id. Forty-six major PFGE types were identified among the 64 C. striatum isolates, indicating that they were mainly not clonal. Our results showed that the 64 clinical C. striatum were highly resistant to aminoglycosides and mostly unrelated.

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<![CDATA[Molecular Evolutionary Pathways toward Two Successful Community-Associated but Multidrug-Resistant ST59 Methicillin-Resistant Staphylococcus aureus Lineages in Taiwan: Dynamic Modes of Mobile Genetic Element Salvages]]> https://www.researchpad.co/article/5989d9e3ab0ee8fa60b6a337

Clonal complex 59 (CC59) Staphylococcus aureus in Taiwan includes both methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA). As the most prominent community-associated MRSA (CA-MRSA) in Taiwan, CC59 has two major clones characterized as PVL-negative SCCmec IV (carrying the staphylococcal cassette chromosome mec IV but Panton-Valentine leukocidin-negative) and PVL-positive SCCmec V (5C2&5). We investigated the drug resistance, phylogeny and the distribution and sequence variation of SCCmec, staphylococcal bacteriophage φSA3, genomic island νSaβ and MES (an enterococcal mobile genetic element conferring multidrug resistance) in 195 CC59 S. aureus. Sequencing and PCR mapping revealed that all of the CC59/SCCmec V (5C2&5) MRSA strains had acquired MESPM1 or its segregants, and obtained a φSA3-related fragment in νSaβ. In contrast, MES6272-2 and MES4578, which showed gentamicin resistance that was not encoded by MESPM1, were dominant in SCCmec IVg MRSA. Translocation of a whole φSA3 into νSaβ instead of only a φSA3-related fragment was common in SCCmec IVg MRSA. However, the non-subtype-g SCCmec IV MRSA (SCCmec IVa is the major) still carried MES and νSaβ structures similar to those in SCCmec V (5C2&5) MRSA. A minimum spanning tree constructed by multiple-locus variable-number tandem repeat analysis revealed that SCCmec IVg MRSA and SCCmec V (5C2&5) MRSA grouped respectively in two major clades. The CC59 MSSA was equally distributed among the two clades, while the non-subtype-g SCCmec IV MRSA mostly clustered with SCCmec V (5C2&5) MRSA. Our findings strongly suggest that CC59 MSSA acquired divergent mobile genetic elements and evolved to SCCmec IVg MRSA and SCCmec V (5C2&5) MRSA/non-subtype-g SCCmec IV MRSA independently. The evolutionary history of CC59 S. aureus explains how mobile genetic elements increase the antimicrobial resistance and virulence and contribute to the success of CA-MRSA in Taiwan.

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<![CDATA[Discordance across Phenotypic and Molecular Methods for Drug Susceptibility Testing of Drug-Resistant Mycobacterium tuberculosis Isolates in a Low TB Incidence Country]]> https://www.researchpad.co/article/5989da83ab0ee8fa60b9b3b7

With increasing incidence of multidrug-resistant tuberculosis (MDR-TB), accurate drug susceptibility testing (DST) of Mycobacterium tuberculosis to first-line drugs has become crucial for proper patient management. We evaluated concordance of DST results for 70 M. tuberculosis isolates across two phenotypic and two molecular methods: BACTEC 460TB, MGIT 960 system, GenoType MTBDRplus and DNA sequencing of gene segments most commonly implicated in conferring resistance to anti-TB drugs. Most (84%) M. tuberculosis isolates were multidrug-resistant. Twenty-four isolates yielded discrepant DST results. For rifampicin, isoniazid and streptomycin, 96%, 97% and 93% of isolates, respectively, were susceptible or resistant by all four methods, whereas for ethambutol, this agreement was observed for only 76% of isolates (P <0.05 for rifampicin or isoniazid or streptomycin versus ethambutol). Occurrence of rare mutations in three isolates that confer low-level resistance caused lower agreement for rifampicin among the four methods (kappa coefficient (κ) range, 0.84 to 0.95). For isoniazid, there was perfect agreement among phenotypic methods and molecular methods (κ, 1.00) but lower agreement between phenotypic and molecular methods. Three isolates were detected as polydrug-resistant by MGIT 960 system but as multidrug-resistant by DNA sequence-based method. The agreement was higher for streptomycin among the two phenotypic methods (κ, 0.97) while targeted sequencing yielded lower agreement (κ range, 0.86 to 0.89). The discrepancy for ethambutol resulted largely due to lower concordance of MGIT 960 results (κ range, 0.53 to 0.64). The MGIT 960 system is an accurate method for DST of M. tuberculosis against isoniazid and streptomycin while the results of rifampicin susceptibility should be complemented with DNA sequencing-based method when the suspicion for resistance is high. The possibility of false susceptibility to ethambutol with MGIT 960 system suggests that molecular or other phenotypic methods may be more useful when accurate ethambutol susceptibility results are warranted.

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<![CDATA[Association between genotype and drug resistance profiles of Mycobacterium tuberculosis strains circulating in China in a national drug resistance survey]]> https://www.researchpad.co/article/5989db50ab0ee8fa60bdc184

We describe the population structure of a representative collection of 3,133 Mycobacterium tuberculosis isolates, collected within the framework of a national resistance survey from 2007 in China. Genotyping data indicate that the epidemic strains in China can be divided into seven major complexes, of which 92% belonged to the East Asian (mainly Beijing strains) or the Euro-American lineage. The epidemic Beijing strains in China are closely related to the Beijing B0/W148 strain earlier described in Russia and a large cluster of these strains has spread national wide. The density of Beijing strains is high in the whole of China (average 70%), but the highest prevalence was found North of the Yellow river. The Euro-American lineage consists of three sublineages (sublineage_1, 2, and 3) and is more prevalent in the South. Beijing lineage showed the highest cluster rate of 48% and a significantly higher level of resistance to rifampicin (14%, p<0.001), ethambutol (9%, p = 0.001), and ofloxacin (5%, p = 0.011). Within the Euro-American Lineage, sublineage_3 revealed the highest cluster rate (28%) and presented a significantly elevated level of resistance to streptomycin (44%, p<0.001). Our findings suggest that standardised treatment in this region may have contributed to the successful spread of certain strains: sublineage_3 in the Euro-American lineage may have thrived when streptomycin was used without rifampicin for treatment, while later under DOTS based treatment, in which rifampicin plays a key role, Beijing lineage appears to be spreading.

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<![CDATA[Construction of novel pJRD215-derived plasmids using chloramphenicol acetyltransferase (cat) gene as a selection marker for Acidithiobacillus caldus]]> https://www.researchpad.co/article/5aafcccf463d7e7f05234530

Background

Acidithiobacillus caldus, a Gram-negative, chemolithotrophic sulfur-oxidizing bacterium, is widely applied in bioleaching. The absence of an ideal selection marker has become a major obstacle to achieve high efficiency of the gene transfer system for A. caldus. Plasmid pJRD215, widely used in Acidithiobacillus spp., has severe drawbacks in molecular manipulations and potential biosafety issues due to its mobility. Therefore, finding a new selection marker and constructing new plasmids have become an urgent and fundamental work for A. caldus.

Results

Effective inhibitory effect of chloramphenicol on the growth of A. caldus was elucidated for the first time. The P2-cat gene cassette, including a chloramphenicol acetyltransferase gene (cat) from plasmid pACBSR and a promoter (P2) upstream of the tetracycline resistance gene on pBR322, was designed, chloramphenicol acetyltransferase was expressed in A. caldus, and the enzyme activity was assessed. A new vector pSDU1 carrying the replication and mobilization regions derived from pJRD215, the P2-cat gene cassette and a multiple cloning site from pUC19 was successfully constructed. Compared with pJRD215, pSDU1 had a 27-fold increase in electrotransformation efficiency (30.43±0.88×104 CFU/μg DNA for pSDU1 and 1.09±0.11×104 CFU/μg DNA for pJRD215), better carrying capacity and could offer more convenience for the restriction enzyme digestion. In addition, the generated plasmid pSDU1Δmob, a novel non-mobilizable derivative of pSDU1 lacking some DNA sequences involved in the mobilization process, had increased copy number in A. caldus and lost its mobility for biosafety considerations. Both pSDU1 and pSDU1Δmob exhibited stable maintenance in A. caldus within 50 passages. However, further deletion of orfEF region involved in regulating repAC operon resulted in a negative effect on transformation efficiency, copy number and stability of plasmid pSDU1ΔmobΔorfEF in A. caldus.

Conclusion

Chloramphenicol was proved to be an ideal selection marker for A. caldus. Novel plasmids carrying cat gene were constructed. The utilization of these vectors will undoubtedly facilitate efficient genetic manipulations and accelerate the research progress in A. caldus.

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<![CDATA[A new subclass of intrinsic aminoglycoside nucleotidyltransferases, ANT(3")-II, is horizontally transferred among Acinetobacter spp. by homologous recombination]]> https://www.researchpad.co/article/5989db53ab0ee8fa60bdcdb9

The emergence and spread of antibiotic resistance among Acinetobacter spp. have been investigated extensively. Most studies focused on the multiple antibiotic resistance genes located on plasmids or genomic resistance islands. On the other hand, the mechanisms controlling intrinsic resistance are still not well understood. In this study, we identified the novel subclass of aminoglycoside nucleotidyltransferase ANT(3")-II in Acinetobacter spp., which comprised numerous variants distributed among three main clades. All members of this subclass can inactivate streptomycin and spectinomycin. The three ant(3")-II genes, encoding for the three ANT(3")-II clades, are widely distributed in the genus Acinetobacter and always located in the same conserved genomic region. According to their prevalence, these genes are intrinsic in Acinetobacter baumannii, Acinetobacter pittii, and Acinetobacter gyllenbergii. We also demonstrated that the ant(3")-II genes are located in a homologous recombination hotspot and were recurrently transferred among Acinetobacter species. In conclusion, our findings demonstrated a novel mechanism of natural resistance in Acinetobacter spp., identified a novel subclass of aminoglycoside nucleotidyltransferase and provided new insight into the evolutionary history of intrinsic resistance genes.

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<![CDATA[Characterization of Multidrug Resistant E. faecalis Strains from Pigs of Local Origin by ADSRRS-Fingerprinting and MALDI -TOF MS; Evaluation of the Compatibility of Methods Employed for Multidrug Resistance Analysis]]> https://www.researchpad.co/article/5989db4fab0ee8fa60bdb8ee

The aim of this study was to characterize multidrug resistant E. faecalis strains from pigs of local origin and to analyse the relationship between resistance and genotypic and proteomic profiles by amplification of DNA fragments surrounding rare restriction sites (ADSRRS-fingerprinting) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI -TOF MS). From the total pool of Enterococcus spp. isolated from 90 pigs, we selected 36 multidrug resistant E. faecalis strains, which represented three different phenotypic resistance profiles. Phenotypic resistance to tetracycline, macrolides, phenicols, and lincomycin and high-level resistance to aminoglycosides were confirmed by the occurrence of at least one corresponding resistance gene in each strain. Based on the analysis of the genotypic and phenotypic resistance of the strains tested, five distinct resistance profiles were generated. As a complement of this analysis, profiles of virulence genes were determined and these profiles corresponded to the phenotypic resistance profiles. The demonstration of resistance to a wide panel of antimicrobials by the strains tested in this study indicates the need of typing to determine the spread of resistance also at the local level. It seems that in the case of E. faecalis, type and scope of resistance strongly determines the genotypic pattern obtained with the ADSRRS-fingerprinting method. The ADSRRS-fingerprinting analysis showed consistency of the genetic profiles with the resistance profiles, while analysis of data with the use of the MALDI- TOF MS method did not demonstrate direct reproduction of the clustering pattern obtained with this method. Our observations were confirmed by statistical analysis (Simpson’s index of diversity, Rand and Wallace coefficients). Even though the MALDI -TOF MS method showed slightly higher discrimination power than ADSRRS-fingerprinting, only the latter method allowed reproduction of the clustering pattern of isolates based on phenotypic resistance and analysis of resistance and virulence genes (Wallace coefficient 1.0). This feature seems to be the most useful for epidemiological purposes and short-term analysis.

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<![CDATA[Multidrug-resistant bacteria compensate for the epistasis between resistances]]> https://www.researchpad.co/article/5989db5aab0ee8fa60bdf420

Mutations conferring resistance to antibiotics are typically costly in the absence of the drug, but bacteria can reduce this cost by acquiring compensatory mutations. Thus, the rate of acquisition of compensatory mutations and their effects are key for the maintenance and dissemination of antibiotic resistances. While compensation for single resistances has been extensively studied, compensatory evolution of multiresistant bacteria remains unexplored. Importantly, since resistance mutations often interact epistatically, compensation of multiresistant bacteria may significantly differ from that of single-resistant strains. We used experimental evolution, next-generation sequencing, in silico simulations, and genome editing to compare the compensatory process of a streptomycin and rifampicin double-resistant Escherichia coli with those of single-resistant clones. We demonstrate that low-fitness double-resistant bacteria compensate faster than single-resistant strains due to the acquisition of compensatory mutations with larger effects. Strikingly, we identified mutations that only compensate for double resistance, being neutral or deleterious in sensitive or single-resistant backgrounds. Moreover, we show that their beneficial effects strongly decrease or disappear in conditions where the epistatic interaction between resistance alleles is absent, demonstrating that these mutations compensate for the epistasis. In summary, our data indicate that epistatic interactions between antibiotic resistances, leading to large fitness costs, possibly open alternative paths for rapid compensatory evolution, thereby potentially stabilizing costly multiple resistances in bacterial populations.

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