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.

Mutational background influences P. aeruginosa ciprofloxacin resistance evolution but preserves collateral sensitivity robustness.

Collateral sensitivity is an evolutionary trade-off whereby acquisition of the adaptive phenotype of resistance to an antibiotic leads to the nonadaptive increased susceptibility to another. The feasibility of harnessing such a trade-off to design evolutionary-based approaches for treating bacterial infections has been studied using model strains. However, clinical application of collateral sensitivity requires its conservation among strains presenting different mutational backgrounds. Particularly relevant is studying collateral sensitivity robustness of already-antibiotic-resistant mutants when challenged with a new antimicrobial, a common situation in clinics that has hardly been addressed. We submitted a set of diverse Pseudomonas aeruginosa antibiotic-resistant mutants to short-term evolution in the presence of different antimicrobials. Ciprofloxacin selects different clinically relevant resistance mutations in the preexisting resistant mutants, which gave rise to the same, robust, collateral sensitivity to aztreonam and tobramycin. We then experimentally determined that alternation of ciprofloxacin with aztreonam is more efficient than ciprofloxacin­tobramycin alternation in driving the extinction of the analyzed antibiotic-resistant mutants. Also, we show that the combinations ciprofloxacin­aztreonam or ciprofloxacin­tobramycin are the most effective strategies for eliminating the tested P. aeruginosa antibiotic-resistant mutants. These findings support that the identification of conserved collateral sensitivity patterns may guide the design of evolution-based strategies to treat bacterial infections, including those due to antibiotic-resistant mutants. Besides, this is an example of phenotypic convergence in the absence of parallel evolution that, beyond the antibiotic-resistance field, could facilitate the understanding of evolution processes, where the selective forces giving rise to new, not clearly adaptive phenotypes remain unclear.

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.

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.

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.

Molecular mechanisms of collateral sensitivity to the antibiotic nitrofurantoin.

Antibiotic resistance increasingly limits the success of antibiotic treatments, and physicians require new ways to achieve efficient treatment despite resistance. Resistance mechanisms against a specific antibiotic class frequently confer increased susceptibility to other antibiotic classes, a phenomenon designated collateral sensitivity (CS). An informed switch of antibiotic may thus enable the efficient treatment of resistant strains. CS occurs in many pathogens, but the mechanisms that generate hypersusceptibility are largely unknown. We identified several molecular mechanisms of CS against the antibiotic nitrofurantoin (NIT). Mutants that are resistant against tigecycline (tetracycline), mecillinam (ß-lactam), and protamine (antimicrobial peptide) all show CS against NIT. Their hypersusceptibility is explained by the overexpression of nitroreductase enzymes combined with increased drug uptake rates, or increased drug toxicity. Increased toxicity occurs through interference of the native drug-response system for NIT, the SOS response, with growth. A mechanistic understanding of CS will help to develop drug switches that combat resistance.

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.

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.

Knockout of P-glycoprotein abolish the collateral sensitivity of CHORC5 multidrug resistant cells.

Multidrug resistant tumor cells show collaterally sensitive to a range of non-toxic drugs. In this report, we describe the isolation of several P-glycoprotein-knockout cell clones, using CRISPR/Cas9, from Chinese hamster multidrug resistant model cell line and its parental cells (e.g., CHORC5 and AuxB1, respectively). All three P-glycoprotein-knockout clones of CHORC5 cells show complete loss of resistance to anti-cancer drugs (e.g., colchicine and doxorubicin), while gaining resistance to well characterized collateral sensitivity drugs (e.g., verapamil, progesterone and NSC73306). A correlation between P-glycoprotein and Sorcin expression levels and a possible role for the latter in low grade resistance to colchicine and doxorubicin was observed. Furthermore, we show that P-glycoprotein expression is necessary for the ROS-mediated mechanism of collateral sensitivity. However, expectantly, P-glycoprotein-knockout clones of CHORC5 cells revealed a dramatic increase in the accumulation of Rhodamine 123, Mito tracker red and doxorubicin, but not Hoechst 33342. The latter findings and their significance to P-glycoprotein collateral sensitivity remain to be determined.

Antimicrobial resistance tendency and collateral sensitivity of Staphylococcus aureus adapted to antibiotics or extracts of medicinal plants grown in Viet Nam.

Antimicrobial resistance has silently turned into one of the biggest threats to global health, marking the fall of the Golden age of antibiotics. In the search for antibiotic replacement or enhancement, plant-derived natural compounds have attracted lots of interest. Even though firmly believed, the low-resistance tendency of pathogenic bacteria against plant extracts has been scarcely demonstrated. In this study, we investigated the antibacterial activities of diethyl ether extracts from six medicinal plants grown in Viet Nam against Staphylococcus aureus and its variants, which were in vitro adapted to the same extracts. After 30 passages of S. aureus growing under sub-lethal concentrations of plant extracts or antibiotics, the bacteria slowly adapted to the extracts while rapidly resisting the antibiotics. Most of the resulting strains obtained from the adaptation to plant extracts were collaterally sensitive to antibiotics. In contrast, antibiotic-adapted strains showed cross-resistance to both antibiotics and extracts. The findings provided evidence of the low-resistance tendency of S. aureus to antimicrobial plant extracts. It is the first time a collateral antibiotic sensitivity of S. aureus adapted to natural compounds has been observed, suggesting an alternative approach to fight antibiotic resistance.

Using Selection by Nonantibiotic Stressors to Sensitize Bacteria to Antibiotics.

Evolutionary adaptation of bacteria to nonantibiotic selective forces, such as osmotic stress, has been previously associated with increased antibiotic resistance, but much less is known about potentially sensitizing effects of nonantibiotic stressors. In this study, we use laboratory evolution to investigate adaptation of Enterococcus faecalis, an opportunistic bacterial pathogen, to a broad collection of environmental agents, ranging from antibiotics and biocides to extreme pH and osmotic stress. We find that nonantibiotic selection frequently leads to increased sensitivity to other conditions, including multiple antibiotics. Using population sequencing and whole-genome sequencing of single isolates from the evolved populations, we identify multiple mutations in genes previously linked with resistance to the selecting conditions, including genes corresponding to known drug targets or multidrug efflux systems previously tied to collateral sensitivity. Finally, we hypothesized based on the measured sensitivity profiles that sequential rounds of antibiotic and nonantibiotic selection may lead to hypersensitive populations by harnessing the orthogonal collateral effects of particular pairs of selective forces. To test this hypothesis, we show experimentally that populations evolved to a sequence of linezolid (an oxazolidinone antibiotic) and sodium benzoate (a common preservative) exhibit increased sensitivity to more stressors than adaptation to either condition alone. The results demonstrate how sequential adaptation to drug and nondrug environments can be used to sensitize bacteria to antibiotics and highlight new potential strategies for exploiting shared constraints governing adaptation to diverse environmental challenges.

CD40-mediated immune cell activation enhances response to anti-PD-1 in murine intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: While cholangiocarcinomas (CCAs) commonly express programmed cell death 1 (PD-1) and its ligand (PD-L1), they respond poorly to immune checkpoint inhibitors (ICIs). We aimed to determine whether stimulating antigen-presenting cells, including macrophages and dendritic cells, using a CD40 agonist could improve this response. METHODS: We compared treatment responses in subcutaneous, orthotopic, and 2 plasmid-based murine intrahepatic CCA (iCCA) models. Mice were treated for 4 weeks with weekly IgG control, a CD40 agonistic antibody, anti-PD-1, or the combination of both (anti-CD40/PD-1). Flow cytometric (FACS) analysis of lymphocytes and myeloid cell populations (including activation status) was performed. We used dendritic cell knockout mice, and macrophage, CD4+ and CD8+ T cell depletion models to identify effector cells. Anti-CD40/PD-1 was combined with chemotherapy (gemcitabine/cisplatin) to test for improved therapeutic efficacy. RESULTS: In all 4 models, anti-PD-1 alone was minimally efficacious. Mice exhibited a moderate response to CD40 agonist monotherapy. Combination anti-CD40/PD-1 therapy led to a significantly greater reduction in tumor burden. FACS demonstrated increased number and activation of CD4+ and CD8+ T cells, natural killer cells, and myeloid cells in tumor and non-tumor liver tissue of tumor-bearing mice treated with anti-CD40/PD-1. Depletion of macrophages, dendritic cells, CD4+ T cells, or CD8+ T cells abrogated treatment efficacy. Combining anti-CD40/PD-1 with gemcitabine/cisplatin resulted in a significant survival benefit compared to gemcitabine/cisplatin alone. CONCLUSION: CD40-mediated activation of macrophages and dendritic cells in iCCA significantly enhances response to anti-PD-1 therapy. This regimen may enhance the efficacy of first-line chemotherapy. LAY SUMMARY: Checkpoint inhibition, a common form of immune therapy, is generally ineffective for the treatment of cholangiocarcinoma. These tumors suppress the infiltration and function of surrounding immune cells. Stimulating immune cells such as macrophages and dendritic cells via the CD40 receptor activates downstream immune cells and enhances the response to checkpoint inhibitors.

Insights into collateral susceptibility and collateral resistance in Acinetobacter baumannii during antimicrobial adaptation.

The susceptibility of Acinetobacter baumannii exposed to primary antibiotic can be either increased or decreased when exposed to secondary antibiotic. This study was designed to assess the relative fitness, collateral susceptibility and collateral resistance of polymyxin B- (PMB-) adapted A. baumannii to ciprofloxacin (CIP), meropenem (MER), PMB, tetracycline (TET) and tobramycin (TOB). Strains of wild-type A. baumannii KACC 12454 (ABKACC ), wild-type A. baumannii CCARM 12088 (ABCCARM ), PMB-adapted ABKACC , PMB-adapted ABCCARM , stabilized ABKACC and stabilized ABCCARM were used in this study. Compared to the wild-type ABKACC , the MICs of PMB were increased from 2 to 128 µg ml-1 against PMB-adapted ABKACC , while MICs of CIP, MER, TET and TOB were decreased from 2 to 1 µg ml-1 , 16 to 1 µg ml-1 , 16 to 2 µg ml-1 and 64 to 16 µg ml-1 , respectively. The PMB-adapted ABCCARM was resistant to CIP (32 µg ml-1 ) and PMB (64 µg ml-1 ) compared to the wild-type ABCCARM . The resistance of stabilized ABKACC and ABCCARM to all antibiotics was lost after antibiotic-free culture in the exception of CIP and TET. The susceptibilities of wild-type, PMB-adapted and stabilized ABKACC and ABCCARM to CIP, MER, PMB, TET and TOB were increased in the presence of ß-lactamase and efflux pump inhibitors. The high levels of relative fitness were observed for stabilized ABKACC , PMB-adapted ABCCARM and stabilized ABCCARM . The stabilized ABKACC and PMB-adapted ABCCARM were highly heteroresistance to PMB and TET, respectively. The PMB-adapted ABKACC and ABCCARM showed various antibiotic patterns, known as collateral susceptibility and collateral resistance. The results provide useful information for designing effective antibiotic regimens that can enhance the antibiotic activity against A. baumannii infections.

MRP1-Collateral Sensitizers as a Novel Therapeutic Approach in Resistant Cancer Therapy: An In Vitro and In Vivo Study in Lung Resistant Tumor.

Multidrug resistance (MDR) is the main obstacle to current chemotherapy and it is mainly due to the overexpression of some efflux transporters such as MRP1. One of the most studied strategies to overcome MDR has been the inhibition of MDR pumps through small molecules, but its translation into the clinic unfortunately failed. Recently, a phenomenon called collateral sensitivity (CS) emerged as a new strategy to hamper MDR acting as a synthetic lethality, where the genetic changes developed upon the acquisition of resistance towards a specific agent are followed by the development of hypersensitivity towards a second agent. Among our library of sigma ligands acting as MDR modulators, we identified three compounds, F397, F400, and F421, acting as CS-promoting agents. We deepened their CS mechanisms in the "pure" model of MRP1-expressing cells (MDCK-MRP1) and in MRP1-expressing/drug resistant non-small cell lung cancer cells (A549/DX). The in vitro results demonstrated that (i) the three ligands are highly cytotoxic for MRP1-expressing cells; (ii) their effect is MRP1-mediated; (iii) they increase the cytotoxicity induced by cis-Pt, the therapeutic agent commonly used in the treatment of lung tumors; and (iv) their effect is ROS-mediated. Moreover, a preclinical in vivo study performed in lung tumor xenografts confirms the in vitro findings, making the three CS-promoting agents candidates for a novel therapeutic approach in lung resistant tumors.

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.

ß-lactamase expression induces collateral sensitivity in Escherichia coli.

Major antibiotic groups are losing effectiveness due to the uncontrollable spread of antimicrobial resistance (AMR) genes. Among these, ß-lactam resistance genes -encoding ß-lactamases- stand as the most common resistance mechanism in Enterobacterales due to their frequent association with mobile genetic elements. In this context, novel approaches that counter mobile AMR are urgently needed. Collateral sensitivity (CS) occurs when the acquisition of resistance to one antibiotic increases susceptibility to another antibiotic and can be exploited to eliminate AMR selectively. However, most CS networks described so far emerge as a consequence of chromosomal mutations and cannot be leveraged to tackle mobile AMR. Here, we dissect the CS response elicited by the acquisition of a prevalent antibiotic resistance plasmid to reveal that the expression of the ß-lactamase gene blaOXA-48 induces CS to colistin and azithromycin. We next show that other clinically relevant mobile ß-lactamases produce similar CS responses in multiple, phylogenetically unrelated E. coli strains. Finally, by combining experiments with surveillance data comprising thousands of antibiotic susceptibility tests, we show that ß-lactamase-induced CS is pervasive within Enterobacterales. These results highlight that the physiological side-effects of ß-lactamases can be leveraged therapeutically, paving the way for the rational design of specific therapies to block mobile AMR or at least counteract their effects.

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.