Collateral Susceptibility Research Articles

Collateral susceptibility-guided alternation of ceftolozane/tazobactam with imipenem prevents resistance development in XDR Pseudomonas aeruginosa biofilms

ObjectivesNew combinations of β-lactams and β-lactamase inhibitors, such as ceftolozane/tazobactam could be useful to combat biofilm-driven chronic infections by extensively resistant (XDR) Pseudomonas aeruginosa but resistance development by mutations in the Ω-loop of AmpC has been described. However, these mutations confer collateral susceptibility to carbapenems. Thus we aimed to evaluate the therapeutic efficacy and the prevention of resistance development of regimen alternating ceftolozane/tazobactam and imipenem. MethodsA carbapenem-resistant XDR P. aeruginosa clinical strain (ST175, 104-B7) and its isogenic imipenem-susceptible ceftolozane/tazobactam-resistant mutant derivative (AmpC T96I, 104-I9) were used. Experiments of single strains and mixed (104-B7 and 104-I9, 1:0.01 ratio) biofilms were performed. 48h biofilms (flow cell system) were treated for 6 days with either ceftolozane/tazobactam, 4/4 mg/L or the alternation of ceftolozane/tazobactam (2 days)-imipenem 4 mg/L (2 days) - ceftolozane/tazobactam (2 days). After treatment, biofilms were collected and plated on Mueller-Hinton agar± ceftolozane/tazobactam 4/4 mg/L. Structural dynamics were monitored using confocal laser scanning microscopy and images were processed with IMARIS software. At least, three independent triplicate experiments per condition were performed. Emerging resistant mutants were characterized through whole genome sequencing (Illumina). ResultsCeftolozane/tazobactam monotherapy failed to reduce the biofilms of the 104-B7 XDR strain and led to the selection of resistant mutants that showed AmpC Ω-loop mutations (T96I, L244R or aa236Δ7). On the contrary, alternation with imipenem enhanced activity (3 Logs reduction at day 6) and prevented the emergence of ceftolozane/tazobactam-resistant mutants. Likewise, treatment with ceftolozane/tazobactam dramatically amplified the resistant strain 104-I9 in mixed biofilms (>90 % of the population), while the alternation regimen counterselected it. ConclusionsCollateral susceptibility-guided alternation of ceftolozane/tazobactam with imipenem effectively prevented the selection of resistant mutants and thus could be a potential therapeutic strategy for the treatment of P. aeruginosa XDR chronic infections.

Insights into macrolide resistance in Arcobacter butzleri: potential resistance mechanisms and impact on bacterial fitness and virulence.

Macrolides are recommended for treating the emerging enteropathogen Arcobacter butzleri; nonetheless, this bacterium often exhibits highly variable resistance rates, and the mechanisms behind this resistance phenotype remain largely unexplored. To understand the phenotypic and genotypic consequences associated with the acquisition of erythromycin resistance in A. butzleri, as well as the effects on the fitness of this species. Resistant strains resulting from spontaneous mutations and adaptive laboratory evolution under increasing erythromycin concentrations were examined regarding their cross-resistance and collateral susceptibility profiles. Genetic causes of phenotypic antibiotic resistance were analysed by sequencing and bioinformatics, with functional correlation through ethidium bromide accumulation assays. Growth profiles in the presence and absence of erythromycin, motility and biofilm formation abilities were assessed to detect potential changes in fitness and virulence. Clones from spontaneous mutation rate evolution demonstrated decreased susceptibility to erythromycin and other classes of antibiotics, associated with mutations in the transcriptional repressor areR, causing overexpression of the AreABC efflux pump. In turn, WGS analysis of the evolved strain showed additional mutations in the ribosomal proteins L4 and L22 and in the areR gene. Furthermore, the acquisition of macrolide resistance altered A. butzleri virulence and entailed a high biological cost. The findings of this study have proved that efflux activity contributes synergistically with mutations in the ribosomal proteins L4 and L22 to A. butzleri's high-level macrolide resistance. The results further suggest an impact on the bacterial physiology and virulence, with the increased fitness cost justifying the low worldwide prevalence of high-level resistant circulating strains.

Climate warming promotes collateral antibiotic resistance development in cyanobacteria

Both cyanobacterial blooms and antibiotic resistance have aggravated worldwide and posed a great threat to public health in recent years. As a significant source and reservoir of water environmental resistome, cyanobacteria exhibit confusing discrepancy between their reduced susceptibility and their chronic exposure to antibiotic mixtures at sub-inhibitory concentrations. How the increasing temperature affects the adaptive evolution of cyanobacteria-associated antibiotic resistance in response to low-level antibiotic combinations under climate change remains unclear. Here we profiled the antibiotic interaction and collateral susceptibility networks among 33 commonly detected antibiotics in 600 cyanobacterial strains isolated from 50 sites across four eutrophicated lakes in China. Cyanobacteria-associated antibiotic resistance level was found positively correlated to antibiotic heterogeneity across all sites. Among 528 antibiotic combinations, antagonism was observed for 62 % interactions and highly conserved within cyanobacterial species. Collateral resistance was detected in 78.5 % of pairwise antibiotic interaction, leading to a widened or shifted upwards mutant selection window for increased opportunity of acquiring second-step mutations. We quantified the interactive promoting effect of collateral resistance and increasing temperature on the evolution of both phenotypic and genotypic cyanobacteria-associated resistance under chronic exposure to environmental level of antibiotic combinations. With temperature increasing from 16 °C to 36 °C, the evolvability index and genotypic resistance level increased by 1.25 – 2.5 folds and 3 – 295 folds in the collateral-resistance-informed lineages, respectively. Emergence of resistance mutation pioneered by tolerance, which was jointly driven by mutation rate and persister fraction, was found to be accelerated by increased temperature and antibiotic switching rate. Our findings provided mechanic insights into the boosting effect of climate warming on the emergence and development of cyanobacteria-associated resistance against collateral antibiotic phenotypes.

O03 The effect of antibiotic selection on collateral susceptibility and evolvability of uropathogenic Escherichia coli

Abstract Urinary tract infections (UTIs) are a common infection in healthy adult women and as such account for 1%–3% of all medical consultations and the second most common reason for prescribing antimicrobials in the UK. Escherichia coli is a frequent aetiological agent of uncomplicated UTIs, causing 75% of cases, associated with recurrence of infection (40%) and a major source of bacteraemia (∼40%). Trimethoprim and nitrofurantoin are currently recommended as first-line antibiotics for the treatment of uncomplicated UTIs. However, 48700 deaths in Europe in 2019 have been associated with antimicrobial-resistant UTIs and resistance to trimethoprim in community-acquired UTIs caused by E. coli is high (33.4%). With little to no antimicrobial development in recent years, new strategies are therefore required to maintain efficacy in currently available antibiotics. One such strategy is to use evolutionary principles to understand how antimicrobial resistance (AMR) to one antibiotic can affect the activity and probability of resistance development to a second antibiotic. Effect on antimicrobial activity can be assessed through collateral susceptibility, where prior resistance to one antibiotic either increases or decreases susceptibility to a second, unrelated class of antibiotic. While the probability of resistance development can be assessed by the mutant selection window (MSW), an antibiotic concentration range where AMR mutations can be selected for. This ranges from the MIC (the lowest concentration to select for resistance) to the mutant prevention concentration (MPC, the highest concentration to select for resistance). In this study, we aimed to determine the effect trimethoprim resistant derivatives of three clinical strains of uropathogenic E. coli on collateral susceptibility and MSWs to nitrofurantoin. We found that trimethoprim-resistant mutants conferred the expected decrease in susceptibility to trimethoprim and an increase in the MSW. This suggests prior selection with trimethoprim increases the probability of treatment failure and the selection of further mutations conferring high-level resistance to trimethoprim. We also observed collateral resistance to nitrofurantoin in the trimethoprim-resistant mutants. Importantly, however, there was a reduction in the MPC, and therefore a narrowing of the MSW, reducing the probability of selection of higher levels of nitrofurantoin resistance. This suggests that using nitrofurantoin after trimethoprim may reduce the probability of developing nitrofurantoin resistance compared with using nitrofurantoin first. These data provide a potential evidence base to use evolutionary principles when designing antimicrobial regimens to limit AMR development and improve treatment success.

Evolution of β-lactamase-mediated cefiderocol resistance.

BackgroundCefiderocol is a novel siderophore β-lactam with improved hydrolytic stability toward β-lactamases, including carbapenemases, achieved by combining structural moieties of two clinically efficient cephalosporins, ceftazidime and cefepime. Consequently, cefiderocol represents a treatment alternative for infections caused by MDR Gram-negatives.ObjectivesTo study the role of cefiderocol on resistance development and on the evolution of β-lactamases from all Ambler classes, including KPC-2, CTX-M-15, NDM-1, CMY-2 and OXA-48.MethodsDirected evolution, using error-prone PCR followed by selective plating, was utilized to investigate how the production and the evolution of different β-lactamases cause changes in cefiderocol susceptibility determined using microbroth dilution assays (MIC and IC50).ResultsWe found that the expression of blaOXA-48 did not affect cefiderocol susceptibility. On the contrary, the expression of blaKPC-2, blaCMY-2, blaCTX-M-15 and blaNDM-1 substantially reduced cefiderocol susceptibility by 4-, 16-, 8- and 32-fold, respectively. Further, directed evolution on these enzymes showed that, with the acquisition of only 1–2 non-synonymous mutations, all β-lactamases were evolvable to further cefiderocol resistance by 2- (NDM-1, CTX-M-15), 4- (CMY-2), 8- (OXA-48) and 16-fold (KPC-2). Cefiderocol resistance development was often associated with collateral susceptibility changes including increased resistance to ceftazidime and ceftazidime/avibactam as well as functional trade-offs against different β-lactam drugs.ConclusionsThe expression of contemporary β-lactamase genes can potentially contribute to cefiderocol resistance development and the acquisition of mutations in these genes results in enzymes adapting to increasing cefiderocol concentrations. Resistance development caused clinically important cross-resistance, especially against ceftazidime and ceftazidime/avibactam.

Evolutionary Instability of Collateral Susceptibility Networks in Ciprofloxacin-Resistant Clinical Escherichia coli Strains.

ABSTRACTCollateral sensitivity and resistance occur when resistance development toward one antimicrobial either potentiates or deteriorates the effect of others. Previous reports on collateral effects on susceptibility focus on newly acquired resistance determinants and propose that novel treatment guidelines informed by collateral networks may reduce the evolution, selection, and spread of antimicrobial resistance. In this study, we investigate the evolutionary stability of collateral networks in five ciprofloxacin-resistant, clinical Escherichia coli strains. After 300 generations of experimental evolution without antimicrobials, we show complete fitness restoration in four of five genetic backgrounds and demonstrate evolutionary instability in collateral networks of newly acquired resistance determinants. We show that compensatory mutations reducing efflux expression are the main drivers destabilizing initial collateral networks and identify rpoS as a putative target for compensatory evolution. Our results add another layer of complexity to future predictions and clinical application of collateral networks.

Inactivation of a New Potassium Channel Increases Rifampicin Resistance and Induces Collateral Sensitivity to Hydrophilic Antibiotics in Mycobacterium smegmatis

Rifampicin is a critical first-line antibiotic for treating mycobacterial infections such as tuberculosis, one of the most serious infectious diseases worldwide. Rifampicin resistance in mycobacteria is mainly caused by mutations in the rpoB gene; however, some rifampicin-resistant strains showed no rpoB mutations. Therefore, alternative mechanisms must explain this resistance in mycobacteria. In this work, a library of 11,000 Mycobacterium smegmatis mc2 155 insertion mutants was explored to search and characterize new rifampicin-resistance determinants. A transposon insertion in the MSMEG_1945 gene modified the growth rate, pH homeostasis and membrane potential in M. smegmatis, producing rifampicin resistance and collateral susceptibility to other antitubercular drugs such as isoniazid, ethionamide and aminoglycosides. Our data suggest that the M. smegmatis MSMEG_1945 protein is an ion channel, dubbed MchK, essential for maintaining the cellular ionic balance and membrane potential, modulating susceptibility to antimycobacterial agents. The functions of this new gene point once again to potassium homeostasis impairment as a proxy to resistance to rifampicin. This study increases the known repertoire of mycobacterial ion channels involved in drug susceptibility/resistance to antimycobacterial drugs and suggests novel intervention opportunities, highlighting ion channels as druggable pathways.

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 128μgml-1 against PMB-adapted ABKACC , while MICs of CIP, MER, TET and TOB were decreased from 2 to 1μgml-1 , 16 to 1μgml-1 , 16 to 2μgml-1 and 64 to 16μgml-1 , respectively. The PMB-adapted ABCCARM was resistant to CIP (32μgml-1 ) and PMB (64μgml-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 β-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.

613. Suppression of Daptomycin Resistance Development in Staphylococcus aureus Is a Class Effect of β-Lactams and Is Independent of Daptomycin–β Lactam Synergy

BackgroundPrevious studies demonstrate that adding oxacillin during daptomycin (DAP) exposure can prevent DAP resistance development in community-acquired (ST8/USA300) MRSA, presumably by preventing mprF mutation. Hospital-acquired strains, such as MRSA sequence types 5 and 239, typically have higher β-lactam (BL) minimum inhibitory concentrations (MICs) than their community-acquired counterparts and are often less toxigenic, more multidrug-resistant and more refractory to primary antistaphylococcal therapies. It is unknown whether DAP resistance prevention occurs in hospital-acquired MRSA lineages or if augmenting DAP therapy with BL antibiotics other than oxacillin would prevent DAP resistance development.MethodsMRSA ST5/USA100 (D592) and ST239 (JKD6004) differ in the degree to which BL enhances DAP activity. D592 and JKD6004 were passaged in escalating concentrations of DAP in a stepwise fashion in vitro as described previously. Following 28 days of serial passage all replicates were passaged twice on mannitol-salt agar and tested for DAP MIC by Etest. Parallel passages were performed in media supplemented with BL antibiotics. Between-group differences in DAP MIC suppression effectiveness among individual BLs compared with nafcillin was evaluated using Kruskal–Wallis rank-sum testing with Holm-adjusted post-hoc Dunn testing.ResultsPassage of D592 or JKD6004 in DAP resulted in highly DAP-resistant isolates (median ≥256 mg/L, IQR [96,256]). In contrast, when passages were performed in the presence of DAP+BL, DAP resistance development was suppressed. No between-group differences in DAP MIC suppression effectiveness were observed among individual BLs compared with nafcillin. Highly DAP-resistant isolates demonstrated variable collateral susceptibility to BL monotherapy but were frequently susceptible to combination antibiotic exposure.ConclusionAddition of β-lactams to DAP can prevent DAP resistance development in vitro in ST5/USA100 and ST239 MRSA, consistent with findings in ST8/USA300 lineages. Furthermore, this ability appears to be a class effect of β-lactam antibiotics and is independent of the extent of DAP-BL synergy. This provides evidence to support the use of BL combination therapy without regard to staphylococcal lineage or specific BL used.Disclosures All authors: No reported disclosures.

Mechanisms and phenotypic consequences of acquisition of tigecycline resistance by Stenotrophomonas maltophilia

To elucidate the potential mutation-driven mechanisms involved in the acquisition of tigecycline resistance by the opportunistic pathogen Stenotrophomonas maltophilia. The mutational trajectories and their effects on bacterial fitness, as well as cross-resistance and/or collateral susceptibility to other antibiotics, were also addressed. S. maltophilia populations were submitted to experimental evolution in the presence of increasing concentrations of tigecycline for 30 days. The genetic mechanisms involved in the acquisition of tigecycline resistance were determined by WGS. Resistance was evaluated by performing MIC assays. Fitness of the evolved populations and individual clones was assessed by measurement of the maximum growth rates. All the tigecycline-evolved populations attained high-level resistance to tigecycline following different mutational trajectories, yet with some common elements. Among the mechanisms involved in low susceptibility to tigecycline, mutations in the SmeDEF efflux pump negative regulator smeT, changes in proteins involved in the biogenesis of the ribosome and modifications in the LPS biosynthesis pathway seem to play a major role. Besides tigecycline resistance, the evolved populations presented cross-resistance to other antibiotics, such as aztreonam and quinolones, and they were hypersusceptible to fosfomycin, suggesting a possible combination treatment. Further, we found that the selected resistance mechanisms impose a relevant fitness cost when bacteria grow in the absence of antibiotic. Mutational resistance to tigecycline was easily selected during exposure to this antibiotic. However, the fitness cost may compromise the maintenance of S. maltophilia tigecycline-resistant populations in the absence of antibiotic.

Molecular mechanisms of action of herbal antifungal alkaloid berberine, in Candida albicans.

Candida albicans causes superficial to systemic infections in immuno-compromised individuals. The concomitant use of fungistatic drugs and the lack of cidal drugs frequently result in strains that could withstand commonly used antifungals, and display multidrug resistance (MDR). In search of novel fungicidals, in this study, we have explored a plant alkaloid berberine (BER) for its antifungal potential. For this, we screened an in-house transcription factor (TF) mutant library of C. albicans strains towards their susceptibility to BER. Our screen of TF mutant strains identified a heat shock factor (HSF1), which has a central role in thermal adaptation, to be most responsive to BER treatment. Interestingly, HSF1 mutant was not only highly susceptible to BER but also displayed collateral susceptibility towards drugs targeting cell wall (CW) and ergosterol biosynthesis. Notably, BER treatment alone could affect the CW integrity as was evident from the growth retardation of MAP kinase and calcineurin pathway null mutant strains and transmission electron microscopy. However, unlike BER, HSF1 effect on CW appeared to be independent of MAP kinase and Calcineurin pathway genes. Additionally, unlike hsf1 null strain, BER treatment of Candida cells resulted in dysfunctional mitochondria, which was evident from its slow growth in non-fermentative carbon source and poor labeling with mitochondrial membrane potential sensitive probe. This phenotype was reinforced with an enhanced ROS levels coinciding with the up-regulated oxidative stress genes in BER-treated cells. Together, our study not only describes the molecular mechanism of BER fungicidal activity but also unravels a new role of evolutionary conserved HSF1, in MDR of Candida.

Ethics and neuropsychiatric genetics: a review of major issues.

Advances in neuropsychiatric genetics hold great hopes for improved prevention, diagnosis and treatment. However, the power of genetic testing to identify individuals at increased risk for disorders and to convey information about relatives creates a set of complex ethical issues. Public attitudes are inevitably affected by the shadow of eugenics, with its history of distorting scientific findings to serve socio-political ends. Nonetheless, the growing availability of genetic tests means that more patients will seek genetic information and physicians must manage the process of informed consent to allow meaningful decisions. Patients should be helped to understand the often-limited predictive power of current knowledge, potential psychological impact, risks of stigma and discrimination and possible implications for family members. Decisions for predictive testing of children raise additional concerns, including distortions of family dynamics and negative effects on children's self-image; testing is best deferred until adulthood unless preventive interventions exist. Pharmacogenomic testing, part of personalized medicine, may bring collateral susceptibility information for which patients should be prepared. The implications of genetic findings for families raise the question of whether physicians have duties to inform family members of implications for their health. Finally, participation in research in neuropsychiatric genetics evokes a broad range of ethical concerns, including the contentious issue of the extent to which results should be returned to individual subjects. As genetic science becomes more widely applied, the public will become more sophisticated and will be likely to demand a greater role in determining social policy on these issues.

Cross-resistance patterns related to glutathione metabolism in primary human renal cell carcinoma

In 59 cases of primary human renal cell carcinoma (RCC), cross-resistance and collateral susceptibility patterns were determined in an MTT microculture assay. Concomitantly, the glutathione (GSH) content and the enzymatic activity of gamma-glutamyl transpeptidase (GGT) were measured as distinct resistance characteristics. Resistance or chemoresponse towards Vinca alkaloids and anthracyclines were found to be highly coincident, suggesting that the classical multidrug resistance mechanism is active in human RCC. Strong resistance to platinum complexes combined with relative sensitivity to bleomycin was significantly associated with elevated glutathione levels, providing evidence for another pathway instigating chemoresistance. In contrast, despite substantial enzymatic activity, GGT effects revealed no correlation to the chemoresistance pattern. This result implies that it is the GSH-linked binding and reduction potential rather than the GGT-associated transportation capacity that has an impact on the expression of chemoresistance in human RCC.

Drug-resistant Leptomonas: cross-resistance in trypanocide-resistant clones.

A Leptomonas of insect origin was highly susceptible to several standard trypanocides and leishmanicides in vitro. Resistance was induced to some of these drugs; clones were isolated from each strain. Cross-resistance patterns of the clones were derived for diamidines, quinapyramine (Antrycide), acriflavin, phenanthridines, and other drugs active against trypanosomes and leishmanias. Clones tested included two each that were resistant to acriflavin, Antrycide, diminazene aceturate (Berenil), and pentamidine and one that was resistant to stilbamidine. Appreciable cross-resistance was evident for all clones. Differences were observed between clones from the same parent strain. Collateral susceptibility towards isometamidium and oxophenarsine was detected in most clone-derived populations. In clones passaged without drug to test for drug fastness, acriflavin and pentamidine clones lost resistance within 10 transfers, whereas Berenil and Antrycide clones retained considerable resistance after 20 to 30 subcultures without drug. Considerations of differences in life cycles suggest that the clone collection may be useful in screening for agents effective against leishmanias and stercorarian trypanosomes rather than against salivary trypanosomes.

Cross-resistance and collateral susceptibility to antifolic antimalarial compounds.

Series of strains of Streptococcus faecium ATCC 8043, Lactobacillus casei ATCC 7469, and Pediococcus cerevisiae ATCC 8081 with increasing resistance to the active antifolate antimalarial drugs chlorguanide triazine (CGT), pyrimethamine (PM), and trimethoprim (TMP) were isolated. These mutant strains, stable for at least 3 to 5 years, were examined for cross-resistance and collateral susceptibility to the above compounds and to methotrexate (MTX). Generally, they exhibited cross-resistance to all four compounds, but resistance of a strain to one compound did not predict accurately its resistance to another drug. Unexpectedly, L. casei resistant to CGT exhibited collateral susceptibility to MTX, TMP, and PM varying from 5- to 20-fold. P. cerevisiae developed resistance to CGT readily but maintained its susceptibility to PM and TMP after prolonged exposure to these compounds. Resistance to these antimalarial antifolates was accompanied by only low-grade cross-resistance to MTX, a representative antileukemic antifolate agent.