Collateral sensitivity increases the efficacy of a rationally designed bacteriophage combination to control Salmonella enterica.

The ability of virulent bacteriophages to lyse bacteria influences bacterial evolution, fitness, and population structure. Knowledge of both host susceptibility and resistance factors is crucial for the successful application of bacteriophages as biological control agents in clinical therapy, food processing, and agriculture. In this study, we isolated 12 bacteriophages termed SPLA phage which infect the foodborne pathogen Salmonella enterica. To determine phage host range, a diverse collection of Enterobacteriaceae and Salmonella enterica was used and genes involved in infection by six SPLA phages were identified using Salmonella Typhimurium strain ST4/74. Candidate host receptors included lipopolysaccharide (LPS), cellulose, and BtuB. Lipopolysaccharide was identified as a susceptibility factor for phage SPLA1a and mutations in LPS biosynthesis genes spontaneously emerged during culture with S. Typhimurium. Conversely, LPS was a resistance factor for phage SPLA5b which suggested that emergence of LPS mutations in culture with SPLA1a represented collateral sensitivity to SPLA5b. We show that bacteria-phage co-culture with SPLA1a and SPLA5b was more successful in limiting the emergence of phage resistance compared to single phage co-culture. Identification of host susceptibility and resistance genes and understanding infection dynamics are critical steps in the rationale design of phage cocktails against specific bacterial pathogens.IMPORTANCEAs antibiotic resistance continues to emerge in bacterial pathogens, bacterial viruses (phage) represent a potential alternative or adjunct to antibiotics. One challenge for their implementation is the predisposition of bacteria to rapidly acquire resistance to phages. We describe a functional genomics approach to identify mechanisms of susceptibility and resistance for newly isolated phages that infect and lyse Salmonella enterica and use this information to identify phage combinations that exploit collateral sensitivity, thus increasing efficacy. Collateral sensitivity is a phenomenon where resistance to one class of antibiotics increases sensitivity to a second class of antibiotics. We report a functional genomics approach to rationally design a phage combination with a collateral sensitivity dynamic which resulted in increased efficacy. Considering such evolutionary trade-offs has the potential to manipulate the outcome of phage therapy in favor of resolving infection without selecting for escape mutants and is applicable to other virus-host interactions.

ABCB1-dependent collateral sensitivity of multidrug-resistant colorectal cancer cells to the survivin inhibitor MX106-4C.

AIMS: To investigate the collateral sensitivity (CS) of ABCB1-positive multidrug resistant (MDR) colorectal cancer cells to the survivin inhibitor MX106-4C and the mechanism. METHODS: Biochemical assays (MTT, ATPase, drug accumulation/efflux, Western blot, RT-qPCR, immunofluorescence, flow cytometry) and bioinformatic analyses (mRNA-sequencing, reversed-phase protein array) were performed to investigate the hypersensitivity of ABCB1 overexpressing colorectal cancer cells to MX106-4C and the mechanisms. Synergism assay, long-term selection, and 3D tumor spheroid test were used to evaluate the anti-cancer efficacy of MX106-4C. RESULTS: MX106-4C selectively killed ABCB1-positive colorectal cancer cells, which could be reversed by an ABCB1 inhibitor, knockout of ABCB1, or loss-of-function ABCB1 mutation, indicating an ABCB1 expression and function-dependent mechanism. MX106-4C's selective toxicity was associated with cell cycle arrest and apoptosis through ABCB1-dependent survivin inhibition and activation on caspases-3/7 as well as modulation on p21-CDK4/6-pRb pathway. MX106-4C had good selectivity against ABCB1-positive colorectal cancer cells and retained this in multicellular tumor spheroids. In addition, MX106-4C could exert a synergistic anti-cancer effect with doxorubicin or re-sensitize ABCB1-positive cancer cells to doxorubicin by reducing ABCB1 expression in the cell population via long-term exposure. CONCLUSIONS: MX106-4C selectively kills ABCB1-positive MDR colorectal cancer cells via a novel ABCB1-dependent survivin inhibition mechanism, providing a clue for designing CS compound as an alternative strategy to overcome ABCB1-mediated colorectal cancer MDR.

Characterization of dabrafenib-induced drug insensitivity via cellular barcoding and collateral sensitivity to second-line therapeutics.

Drug insensitivity is arguably one of the biggest challenges in cancer therapeutics. Although effective therapeutic solutions in cancer are limited due to the emergence of drug insensitivity, exploiting evolutionary understanding in this context can provide potential second-line therapeutics sensitizing the drug insensitive populations. Targeted therapeutic agent dabrafenib is used to treat CRC patients with BRAF V600E genotype and insensitivity to dabrafenib is often observed. Understanding underlying clonal architecture of dabrafenib-induced drug insensitivity and identification of potential second-line therapeutics that could sensitize dabrafenib insensitive populations remain to be elucidated. For this purpose, we utilized cellular barcoding technology to decipher dabrafenib-induced clonal evolution in BRAF V600E mutant HT-29 cells. This approach revealed the detection of both pre-existing and de novo barcodes with increased frequencies as a result of dabrafenib insensitivity. Furthermore, our longitudinal monitoring of drug insensitivity based on barcode detection from floating DNA within used medium enabled to identify temporal dynamics of pre-existing and de novo barcodes in relation to dabrafenib insensitivity in HT-29 cells. Moreover, whole-exome sequencing analysis exhibited possible somatic CNVs and SNVs contributing to dabrafenib insensitivity in HT-29 cells. Last, collateral drug sensitivity testing demonstrated oxaliplatin and capecitabine, alone or in combination, as successful second-like therapeutics in inducing collateral sensitivity in dabrafenib-insensitive HT-29 cells. Overall, our findings demonstrate clonal dynamics of dabrafenib-insensitivity in HT-29 cells. In addition, oxaliplatin and capecitabine, alone or in combination, were successful second-line therapeutics in inducing collateral sensitivity in dabrafenib-insensitive HT-29 cells.

The role of hypermutation and collateral sensitivity in antimicrobial resistance diversity of Pseudomonas aeruginosa populations in cystic fibrosis lung infection.

Pseudomonas aeruginosa is an opportunistic pathogen which causes chronic, drug-resistant lung infections in cystic fibrosis (CF) patients. In this study, we explore the role of genomic diversification and evolutionary trade-offs in antimicrobial resistance (AMR) diversity within P. aeruginosa populations sourced from CF lung infections. We analyzed 300 clinical isolates from four CF patients (75 per patient) and found that genomic diversity is not a consistent indicator of phenotypic AMR diversity. Remarkably, some genetically less diverse populations showed AMR diversity comparable to those with significantly more genetic variation. We also observed that hypermutator strains frequently exhibited increased sensitivity to antimicrobials, contradicting expectations from their treatment histories. Investigating potential evolutionary trade-offs, we found no substantial evidence of collateral sensitivity among aminoglycoside, beta-lactam, or fluoroquinolone antibiotics, nor did we observe trade-offs between AMR and growth in conditions mimicking CF sputum. Our findings suggest that (i) genomic diversity is not a prerequisite for phenotypic AMR diversity, (ii) hypermutator populations may develop increased antimicrobial sensitivity under selection pressure, (iii) collateral sensitivity is not a prominent feature in CF strains, and (iv) resistance to a single antibiotic does not necessarily lead to significant fitness costs. These insights challenge prevailing assumptions about AMR evolution in chronic infections, emphasizing the complexity of bacterial adaptation during infection.IMPORTANCEUpon infection in the cystic fibrosis (CF) lung, Pseudomonas aeruginosa rapidly acquires genetic mutations, especially in genes involved in antimicrobial resistance (AMR), often resulting in diverse, treatment-resistant populations. However, the role of bacterial population diversity within the context of chronic infection is still poorly understood. In this study, we found that hypermutator strains of P. aeruginosa in the CF lung undergoing treatment with tobramycin evolved increased sensitivity to tobramycin relative to non-hypermutators within the same population. This finding suggests that antimicrobial treatment may only exert weak selection pressure on P. aeruginosa populations in the CF lung. We further found no evidence for collateral sensitivity in these clinical populations, suggesting that collateral sensitivity may not be a robust, naturally occurring phenomenon for this microbe.

Structures revealing mechanisms of resistance and collateral sensitivity of Plasmodium falciparum to proteasome inhibitors.

The proteasome of the malaria parasite Plasmodium falciparum (Pf20S) is an advantageous drug target because its inhibition kills P. falciparum in multiple stages of its life cycle and synergizes with artemisinins. We recently developed a macrocyclic peptide, TDI-8304, that is highly selective for Pf20S over human proteasomes and is potent in vitro and in vivo against P. falciparum. A mutation in the Pf20S ß6 subunit, A117D, confers resistance to TDI-8304, yet enhances both enzyme inhibition and anti-parasite activity of a tripeptide vinyl sulfone ß2 inhibitor, WLW-vs. Here we present the high-resolution cryo-EM structures of Pf20S with TDI-8304, of human constitutive proteasome with TDI-8304, and of Pf20Sß6A117D with WLW-vs that give insights into the species selectivity of TDI-8304, resistance to it, and the collateral sensitivity associated with resistance, including that TDI-8304 binds ß2 and ß5 in wild type Pf20S as well as WLW-vs binds ß2 and ß5 in Pf20Sß6A117D. We further show that TDI-8304 kills P. falciparum as quickly as chloroquine and artemisinin and is active against P. cynomolgi at the liver stage. This increases interest in using these structures to facilitate the development of Pf20S inhibitors that target multiple proteasome subunits and limit the emergence of resistance.

Diverse evolutionary pathways challenge the use of collateral sensitivity as a strategy to suppress resistance.

Drug resistance remains a major obstacle to malaria control and eradication efforts, necessitating the development of novel therapeutic strategies to treat this disease. Drug combinations based on collateral sensitivity, wherein resistance to one drug causes increased sensitivity to the partner drug, have been proposed as an evolutionary strategy to suppress the emergence of resistance in pathogen populations. In this study, we explore collateral sensitivity between compounds targeting the Plasmodium dihydroorotate dehydrogenase (DHODH). We profiled the cross-resistance and collateral sensitivity phenotypes of several DHODH mutant lines to a diverse panel of DHODH inhibitors. We focus on one compound, TCMDC-125334, which was active against all mutant lines tested, including the DHODH C276Y line, which arose in selections with the clinical candidate DSM265. In six selections with TCMDC-125334, the most common mechanism of resistance to this compound was copy number variation of the dhodh locus, although we did identify one mutation, DHODH I263S, which conferred resistance to TCMDC-125334 but not DSM265. We found that selection of the DHODH C276Y mutant with TCMDC-125334 yielded additional genetic changes in the dhodh locus. These double mutant parasites exhibited decreased sensitivity to TCMDC-125334 and were highly resistant to DSM265. Finally, we tested whether collateral sensitivity could be exploited to suppress the emergence of resistance in the context of combination treatment by exposing wildtype parasites to both DSM265 and TCMDC-125334 simultaneously. This selected for parasites with a DHODH V532A mutation which were cross-resistant to both compounds and were as fit as the wildtype parent in vitro. The emergence of these cross-resistant, evolutionarily fit parasites highlights the mutational flexibility of the DHODH enzyme.

Malaria affects around 240 million people around the world every year. The microscopic parasite responsible for the disease are carried by certain mosquitoes and gets transmitted to humans through bites. These parasites are increasingly acquiring genetic mutations that make anti-malaria medication less effective, creating an urgent need for alternative treatment approaches. Several new malaria drugs being explored in preclinical research work by binding to an enzyme known as DHODH and preventing it from performing its usual role in the parasite. Previous work found that, in some cases, malaria parasites that evolved resistance to one type of DHODH inhibitor (by acquiring mutations in their DHODH enzyme) then became more vulnerable to another kind. It may be possible to leverage this 'collateral sensitivity' by designing treatments which combine two DHODH inhibitors and therefore make it harder for the parasites to evolve resistance. To investigate this possibility, Mandt et al. first tested several DHODH inhibitors to find the one that was most potent against drug-resistant parasites. In subsequent experiments, they combined TCMDC-125334, the best candidate that emerged from these tests, with a DHODH inhibitor that works well against vulnerable parasites. However, the parasites still rapidly evolved resistance. Further work identified a new DHODH mutation that allowed the parasites to evade both drugs simultaneously. Together, these findings suggest that the DHODH enzyme may not be the best target for new malaria drugs because many it can acquire many possible mutations that confer resistance. Such results may inform other studies that aim to harness collateral sensitivity to fight against a range of harmful agents.

Epigenetic suppression of PGC1α (PPARGC1A) causes collateral sensitivity to HMGCR-inhibitors within BRAF-treatment resistant melanomas.

While targeted treatment against BRAF(V600E) improve survival for melanoma patients, many will see their cancer recur. Here we provide data indicating that epigenetic suppression of PGC1α defines an aggressive subset of chronic BRAF-inhibitor treated melanomas. A metabolism-centered pharmacological screen further identifies statins (HMGCR inhibitors) as a collateral vulnerability within PGC1α-suppressed BRAF-inhibitor resistant melanomas. Lower PGC1α levels mechanistically causes reduced RAB6B and RAB27A expression, whereby their combined re-expression reverses statin vulnerability. BRAF-inhibitor resistant cells with reduced PGC1α have increased integrin-FAK signaling and improved extracellular matrix detached survival cues that helps explain their increased metastatic ability. Statin treatment blocks cell growth by lowering RAB6B and RAB27A prenylation that reduces their membrane association and affects integrin localization and downstream signaling required for growth. These results suggest that chronic adaptation to BRAF-targeted treatments drive novel collateral metabolic vulnerabilities, and that HMGCR inhibitors may offer a strategy to treat melanomas recurring with suppressed PGC1α expression.

Assessment of the potential of phage-antibiotic synergy to induce collateral sensitivity in Salmonella Typhimurium.

This study was designed to evaluate the synergistic effect of phage and antibiotic on the induction of collateral sensitivity in Salmonella Typhimurium. The synergistic effects of Salmonella phage PBST32 combined with ciprofloxacin (CIP) against S. Typhimurium KCCM 40253 (STKCCM) were evaluated using a fractional inhibitory concentration (FIC) assay. The CIP susceptibility of STKCCM was increased when combined with PBST32, showing 16-fold decrease at 7 log PFU/mL. The combination of 1/2 × MIC of CIP and PBST32 (CIP[1/2]+PBST32) effectively inhibited the growth of STKCCM up to below the detection limit (1.3 log CFU/mL) after 12 h of incubation at 37 °C. The significant reduction in bacterial swimming motility was observed for PBST32 and CIP[1/4]+PBST32. The CIP[1/4]+PBST32 increased the fitness cost (relative fitness = 0.57) and decreased the cross-resistance to different classes of antibiotics. STKCCM treated with PBST32 alone treatment exhibited the highest coefficient of variation (90%), followed by CIP[1/4]+PBST32 (75%). These results suggest that the combination of PBST32 and CIP can be used to control bacterial pathogens.

Novel hybrids of sclareol and 1,2,4-triazolo[1,5-a]pyrimidine show collateral sensitivity in multidrug-resistant glioblastoma cells.

The synthesis of 24 hybrid molecules, consisting of naturally occurring sclareol (SCL) and synthetic 1,2,4-triazolo[1,5-a]pyrimidines (TPs), is described. New compounds were designed with the aim of improving the cytotoxic properties, activity, and selectivity of the parent compounds. Six analogs (12a-f) contained 4-benzylpiperazine linkage, while 4-benzyldiamine linkage was present in eighteen derivatives (12g-r and 13a-f). Hybrids 13a-f consist of two TP units. After purification, all hybrids (12a-r and 13a-f), as well as their precursors (9a-e and 11a-c), were tested on human glioblastoma U87 cells. More than half of the tested synthesized molecules, 16 out of 31, caused a significant reduction of U87 cell viability (more than 75% reduction) at 30 µM. The concentration-dependent cytotoxicity of these 16 compounds was also examined on U87 cells, corresponding multidrug-resistant (MDR) U87-TxR cells with increased P-glycoprotein (P-gp) expression and activity, and normal lung fibroblasts MRC-5. Importantly, 12l and 12r were active in the nanomolar range, while seven compounds (11b, 11c, 12i, 12l, 12n, 12q, and 12r) were more selective towards glioblastoma cells than SCL. All compounds except 12r evaded MDR, showing even better cytotoxicity in U87-TxR cells. In particular, 11c, 12a, 12g, 12j, 12k, 12m, 12n, and SCL showed collateral sensitivity. Hybrid compounds 12l, 12q, and 12r decreased P-gp activity to the same extent as a well-known P-gp inhibitor - tariquidar (TQ). Hybrid compound 12l and its precursor 11c affected different cellular processes including the cell cycle, cell death, and mitochondrial membrane potential, and changed the levels of reactive oxygen and nitrogen species (ROS/RNS) in glioblastoma cells. Collateral sensitivity towards MDR glioblastoma cells was caused by the modulation of oxidative stress accompanied by inhibition of mitochondria.

Alteration of adeS Contributes to Tigecycline Resistance and Collateral Sensitivity to Sulbactam in Acinetobacter baumannii.

The treatment of extensively drug-resistant (XDR) A. baumannii has emerged as a major problem. Tigecycline (TGC) and sulbactam (SUL) are both effective antibiotics against XDR A. baumannii. Here, we investigated the in-host evolution and mechanism of collateral sensitivity (CS) phenomenon in development of tigecycline resistance accompanied by a concomitant increase of sulbactam susceptibility. A total of four XDR A. baumannii strains were sequentially isolated from the same patient suffering from bacteremia. Core-genome multilocus sequence typing separated all the strains into two clusters. Comparative analysis of isolate pair 1 revealed that multiplication of blaOXA-23 within Tn2006 on the chromosome contributed to the change in the antimicrobial susceptibility phenotype of isolate pair 1. Additionally, we observed the emergence of CS to sulbactam in isolate pair 2, as demonstrated by an 8-fold increase in the TGC MIC with a simultaneous 4-fold decrease in the SUL MIC. Compared to the parental strain Ab-3557, YZM-0406 showed partial deletion in the two-component system sensor adeS. Reconstruction of the adeS mutant in Ab-3557 in situ suggested that TGC resistance and CS to SUL were mainly caused by the mutation of adeS. Overall, our study reported a novel CS combination of TGC and SUL in A. baumannii and further revealed a mechanism of CS attributed to the mutation of adeS. This study provides a valuable foundation for developing effective regimens and sequential combinations of tigecycline and sulbactam against XDR A. baumannii. IMPORTANCE Collateral sensitivity (CS) has become an increasingly common evolutionary trade-off during adaptive bacterial evolution. Here, we report a novel combination of tigecycline (TGC) resistance and CS to sulbactam (SUL) in A. baumannii. TGC and SUL are both effective antibiotics against XDR A. baumannii, and it is essential to reveal the mechanism of CS between TGC and SUL. In our study, the partial deletion of adeS, a two-component system sensor, was confirmed to be the key factor contributing to this CS phenomenon. This study provides a valuable foundation for developing effective regimens and sequential combinations of tigecycline and sulbactam against XDR A. baumannii.

Collateral sensitivity profiling in drug-resistant Escherichia coli identifies natural products suppressing cephalosporin resistance.

The rapid emergence of antimicrobial resistance presents serious health challenges to the management of infectious diseases, a problem that is further exacerbated by slowing rates of antimicrobial drug discovery in recent years. The phenomenon of collateral sensitivity (CS), whereby resistance to one drug is accompanied by increased sensitivity to another, provides new opportunities to address both these challenges. Here, we present a high-throughput screening platform termed Collateral Sensitivity Profiling (CSP) to map the difference in bioactivity of large chemical libraries across 29 drug-resistant strains of E. coli. CSP screening of 80 commercial antimicrobials demonstrated multiple CS interactions. Further screening of a 6195-member natural product library revealed extensive CS relationships in nature. In particular, we report the isolation of known and new analogues of borrelidin A with potent CS activities against cephalosporin-resistant strains. Co-dosing ceftazidime with borrelidin A slows broader cephalosporin resistance with no recognizable resistance to borrelidin A itself.

Collateral sensitivity between tetracyclines and aminoglycosides constrains resistance evolution in carbapenem-resistant Klebsiella pneumoniae.

AIMS: The acquisition of resistance to one antibiotic may confer an increased sensitivity to another antibiotic in bacteria, which is an evolutionary trade-off between different resistance mechanisms, defined as collateral sensitivity (CS). Exploiting the role of CS in treatment design could be an effective method to suppress or even reverse resistance evolution. METHODS: Using experimental evolution, we systematically studied the CS between aminoglycosides and tetracyclines in carbapenem-resistant Klebsiella pneumoniae (CRKP) and explored the underlying mechanisms through genomic and transcriptome analyses. The application of CS-based therapies for resistance suppression, including combination therapy and alternating antibiotic therapy, was further evaluated in vitro and in vivo. RESULTS: Reciprocal CS existed between tetracyclines and aminoglycosides in CRKP. The increased sensitivity of aminoglycoside-resistant strains to tetracyclines was associated with the alteration of bacterial membrane potential, whereas the unbalanced oxidation-reduction process of tetracycline-resistant strains may lead to an increased bacterial sensitivity to aminoglycosides. CS-based combination therapy could efficiently constrain the evolution of CRKP resistance in vitro and in vivo. In addition, alternating antibiotic therapy can re-sensitize CRKP to previously resistant drugs, thereby maintaining the trade-off. CONCLUSIONS: These results provide new insights into constraining the evolution of CRKP resistance through CS-based therapies.

Collateral Sensitivity to Fosfomycin of Tobramycin-Resistant Mutants of Pseudomonas aeruginosa Is Contingent on Bacterial Genomic Background.

Understanding the consequences in bacterial physiology of the acquisition of drug resistance is needed to identify and exploit the weaknesses derived from it. One of them is collateral sensitivity, a potentially exploitable phenotype that, unfortunately, is not always conserved among different isolates. The identification of robust, conserved collateral sensitivity patterns is then relevant for the translation of this knowledge into clinical practice. We have previously identified a robust fosfomycin collateral sensitivity pattern of Pseudomonas aeruginosa that emerged in different tobramycin-resistant clones. To go one step further, here, we studied if the acquisition of resistance to tobramycin is associated with robust collateral sensitivity to fosfomycin among P. aeruginosa isolates. To that aim, we analyzed, using adaptive laboratory evolution approaches, 23 different clinical isolates of P. aeruginosa presenting diverse mutational resistomes. Nine of them showed collateral sensitivity to fosfomycin, indicating that this phenotype is contingent on the genetic background. Interestingly, collateral sensitivity to fosfomycin was linked to a larger increase in tobramycin minimal inhibitory concentration. Further, we unveiled that fosA low expression, rendering a higher intracellular accumulation of fosfomycin, and a reduction in the expression of the P. aeruginosa alternative peptidoglycan-recycling pathway enzymes, might be on the basis of the collateral sensitivity phenotype.

Tackling antibiotic resistance by inducing transient and robust collateral sensitivity.

Collateral sensitivity (CS) is an evolutionary trade-off traditionally linked to the mutational acquisition of antibiotic resistance (AR). However, AR can be temporally induced, and the possibility that this causes transient, non-inherited CS, has not been addressed. Mutational acquisition of ciprofloxacin resistance leads to robust CS to tobramycin in pre-existing antibiotic-resistant mutants of Pseudomonas aeruginosa. Further, the strength of this phenotype is higher when nfxB mutants, over-producing the efflux pump MexCD-OprJ, are selected. Here, we induce transient nfxB-mediated ciprofloxacin resistance by using the antiseptic dequalinium chloride. Notably, non-inherited induction of AR renders transient tobramycin CS in the analyzed antibiotic-resistant mutants and clinical isolates, including tobramycin-resistant isolates. Further, by combining tobramycin with dequalinium chloride we drive these strains to extinction. Our results support that transient CS could allow the design of new evolutionary strategies to tackle antibiotic-resistant infections, avoiding the acquisition of AR mutations on which inherited CS depends.

Rapid Phenotypic Convergence towards Collateral Sensitivity in Clinical Isolates of Pseudomonas aeruginosa Presenting Different Genomic Backgrounds.

Collateral sensitivity (CS) is an evolutionary trade-off by which acquisition of resistance to an antibiotic leads to increased susceptibility to another. This Achilles' heel of antibiotic resistance could be exploited to design evolution-based strategies for treating bacterial infections. To date, most studies in the field have focused on the identification of CS patterns in model strains. However, one of the main requirements for the clinical application of this trade-off is that it must be robust and has to emerge in different genomic backgrounds, including preexisting drug-resistant isolates, since infections are frequently caused by pathogens already resistant to antibiotics. Here, we report the first analysis of CS robustness in clinical strains of Pseudomonas aeruginosa presenting different ab initio mutational resistomes. We identified a robust CS pattern associated with short-term evolution in the presence of ciprofloxacin of clinical P. aeruginosa isolates, including representatives of high-risk epidemic clones belonging to sequence type (ST) 111, ST175, and ST244. We observed the acquisition of different ciprofloxacin resistance mutations in strains presenting varied STs and different preexisting mutational resistomes. Importantly, despite these genetic differences, the use of ciprofloxacin led to a robust CS to aztreonam and tobramycin. In addition, we describe the possible application of this evolutionary trade-off to drive P. aeruginosa infections to extinction by using the combination of ciprofloxacin-tobramycin or ciprofloxacin-aztreonam. Our results support the notion that the identification of robust patterns of CS may establish the basis for developing evolution-informed treatment strategies to tackle bacterial infections, including those due to antibiotic-resistant pathogens. IMPORTANCE Collateral sensitivity (CS) is a trade-off of antibiotic resistance evolution that could be exploited to design strategies for treating bacterial infections. Clinical application of CS requires it to robustly emerge in different genomic backgrounds. In this study, we performed an analysis to identify robust patterns of CS associated with the use of ciprofloxacin in clinical isolates of P. aeruginosa presenting different mutational resistomes and including high-risk epidemic clones (ST111, ST175, and ST244). We demonstrate the robustness of CS to tobramycin and aztreonam and the potential application of this evolutionary observation to drive P. aeruginosa infections to extinction. Our results support the notion that the identification of robust CS patterns may establish the basis for developing evolutionary strategies to tackle bacterial infections, including those due to antibiotic-resistant pathogens.

Antibiotic Cycling Affects Resistance Evolution Independently of Collateral Sensitivity.

Antibiotic cycling has been proposed as a promising approach to slow down resistance evolution against currently employed antibiotics. It remains unclear, however, to which extent the decreased resistance evolution is the result of collateral sensitivity, an evolutionary trade-off where resistance to one antibiotic enhances the sensitivity to the second, or due to additional effects of the evolved genetic background, in which mutations accumulated during treatment with a first antibiotic alter the emergence and spread of resistance against a second antibiotic via other mechanisms. Also, the influence of antibiotic exposure patterns on the outcome of drug cycling is unknown. Here, we systematically assessed the effects of the evolved genetic background by focusing on the first switch between two antibiotics against Salmonella Typhimurium, with cefotaxime fixed as the first and a broad variety of other drugs as the second antibiotic. By normalizing the antibiotic concentrations to eliminate the effects of collateral sensitivity, we demonstrated a clear contribution of the evolved genetic background beyond collateral sensitivity, which either enhanced or reduced the adaptive potential depending on the specific drug combination. We further demonstrated that the gradient strength with which cefotaxime was applied affected both cefotaxime resistance evolution and adaptation to second antibiotics, an effect that was associated with higher levels of clonal interference and reduced cost of resistance in populations evolved under weaker cefotaxime gradients. Overall, our work highlights that drug cycling can affect resistance evolution independently of collateral sensitivity, in a manner that is contingent on the antibiotic exposure pattern.

Development of Resistance to Eravacycline by Klebsiella pneumoniae and Collateral Sensitivity-Guided Design of Combination Therapies.

The evolution of bacterial antibiotic resistance is exhausting the list of currently used antibiotics and endangers those in the pipeline. The combination of antibiotics is a promising strategy that may suppress resistance development and/or achieve synergistic therapeutic effects. Eravacycline is a newly approved antibiotic that is effective against a variety of multidrug-resistant (MDR) pathogens. However, the evolution of resistance to eravacycline and strategies to suppress the evolution remain unexplored. Here, we demonstrated that a carbapenem-resistant Klebsiella pneumoniae clinical isolate quickly developed resistance to eravacycline, which is mainly caused by mutations in the gene encoding the Lon protease. The evolved resistant mutants display collateral sensitivities to ß-lactam/ß-lactamase inhibitor (BLBLI) combinations aztreonam/avibactam and ceftazidime-avibactam. Proteomic analysis revealed upregulation of the multidrug efflux system AcrA-AcrB-TolC and porin proteins OmpA and OmpU, which contributed to the increased resistance to eravacycline and susceptibility to BLBLIs, respectively. The combination of eravacycline with aztreonam/avibactam or ceftazidime-avibactam suppresses resistance development. We further demonstrated that eravacycline-resistant mutants evolved from an NDM-1-containing K. pneumoniae strain display collateral sensitivity to aztreonam/avibactam, and the combination of eravacycline with aztreonam/avibactam suppresses resistance development. In addition, the combination of eravacycline with aztreonam/avibactam or ceftazidime-avibactam displayed synergistic therapeutic effects in a murine cutaneous abscess model. Overall, our results revealed mechanisms of resistance to eravacycline and collateral sensitivities to BLBLIs and provided promising antibiotic combinations in the treatment of multidrug-resistant K. pneumoniae infections. IMPORTANCE The increasing bacterial antibiotic resistance is a serious threat to global public health, which demands novel antimicrobial medicines and treatment strategies. Eravacycline is a newly approved antibiotic that belongs to the tetracycline antibiotics. Here, we found that a multidrug-resistant Klebsiella pneumoniae clinical isolate rapidly developed resistance to eravacycline and the evolved resistant mutants displayed collateral sensitivity to antibiotics aztreonam/avibactam and ceftazidime-avibactam. We demonstrated that the combination of eravacycline with aztreonam/avibactam or ceftazidime-avibactam repressed resistance development and improved the treatment efficacies. We also elucidated the mechanisms that contribute to the increased resistance to eravacycline and susceptibility to aztreonam/avibactam and ceftazidime-avibactam. This work demonstrated the mechanisms of antibiotic resistance and collateral sensitivity and provided a new therapeutically option for effective antibiotic combinations.

Novel Hybrids of 3-Substituted Coumarin and Phenylsulfonylfuroxan as Potent Antitumor Agents with Collateral Sensitivity against MCF-7/ADR.

Twenty-three new coumarin-furoxan hybrids were synthesized, which exhibited nanomole antiproliferation activities in A2780, A2780/CDDP, MCF-7/ADR, and MDA-MB-231. Among them, compound 9 showed the strongest collateral sensitivity to MCF-7/ADR with 499-fold potency compared with MCF-7. Notably, the solubility of compound 9 increased 70-fold compared with the lead 2. And preliminary pharmacological studies displayed that compound 9 obviously increased Rh123 accumulation in MCF-7/ADR and released NO to produce ROS in lysosomes, which were able to damage lysosomal membrane and induce apoptosis. These results reasonably explained that the collateral sensitivity of compound 9 to MCF-7/ADR was closely related to P-gp-mediated lysosome damage and apoptosis. Additionally, compound 9 showed a very weak cytotoxicity both in MCF-10A and hERG potassium channels and had a desirable safety in ion cyclotron resonance (ICR) mice. Hence, compound 9 was merited to further study for developing a desirable candidate against MDR MCF-7/ADR via a potential mechanism of collateral sensitivity in MDR cancer cell lines.

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