Resistant Subpopulations Research Articles

Pharmacokinetics and pharmacokinetic/pharmacodynamic-based dosing regimens of long-acting oxytetracycline in Nile tilapia (Oreochromis niloticus) broodstock to minimize selection of drug resistance

This study aimed to determine the pharmacokinetics of long-acting oxytetracycline (OTC-LA) after intraperitoneal (IP) administration in Nile tilapia broodstock. Pharmacokinetic/pharmacodynamic (PK/PD) relationships were evaluated to determine the optimal dosing regimens of OTC-LA against Streptococcus agalactiae. A total of 120 healthy male tilapia (450 ± 37.47 g) were arbitrarily divided into two groups (60 fish/group) that individually received OTC-LA single IP injection at dosages of 50 or 100 mg/kg body weight. Blood samples were collected at various times posttreatment, and plasma oxytetracycline (OTC) was analyzed using high-performance liquid chromatography. For pharmacodynamic study, 56 S. agalactiae isolates were subjected to agar dilution to determine the minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC). Results showed that the Cmax and Tmax of OTC-LA were both 110.70 ± 5.61 μg/mL at 2 h for the dosage of 50 mg/kg and 287.85 ± 8.03 μg/mL at 4 h for the dosage of 100 mg/kg. Plasma OTC levels remained at 3.99 ± 0.48 and 23.00 ± 2.51 μg/mL at 168 h (7 day) after OTC-LA administration at 50 and 100 mg/kg, respectively. The MIC50 and MIC90 of S. agalactiae were 0.5 and 1 μg/mL, respectively, and its MPC50 and MPC90 were 32 and 128 μg/mL, respectively. The mutant selection window of MIC50 and MPC50 was 0.5–32 μg/mL, and that of MIC90 and MPC90 was 1–128 μg/mL. OTC-LA at 50 and 100 mg/kg provided plasma OTC levels above MIC for at least 7 days. However, only OTC-LA at 100 mg/kg can provide plasma OTC to prevent the growth of resistant mutant subpopulation. Our study suggested that a single IP administration of OTC-LA at 100 mg/kg body weight is the optimal dosing regimen to attain therapeutic efficacy against S. agalactiae infection in tilapia and prevent the emergence of resistant mutant subpopulation.

Cefotaxime Exposure Selects Mutations within the CA-Domain of envZ Which Promote Antibiotic Resistance but Repress Biofilm Formation in Salmonella.

ABSTRACTCephalosporins are important beta lactam antibiotics, but resistance can be mediated by various mechanisms including production of beta lactamase enzymes, changes in membrane permeability or active efflux. We used an evolution model to study how Salmonella adapts to subinhibitory concentrations of cefotaxime in planktonic and biofilm conditions and characterized the mechanisms underpinning this adaptation. We found that Salmonella rapidly adapts to subinhibitory concentrations of cefotaxime via selection of multiple mutations within the CA-domain region of EnvZ. We showed that changes in this domain affect the ATPase activity of the enzyme and in turn impact OmpC, OmpF porin expression and hence membrane permeability leading to increased tolerance to cefotaxime and low-level resistance to different classes of antibiotics. Adaptation to cefotaxime through EnvZ also resulted in a significant cost to biofilm formation due to downregulation of curli. We assessed the role of the mutations identified on the activity of EnvZ by genetic characterization, biochemistry and in silico analysis and confirmed that they are responsible for the observed phenotypes. We observed that sublethal cefotaxime exposure selected for heterogeneity in populations with only a subpopulation carrying mutations within EnvZ and being resistant to cefotaxime. Population structure and composition dynamically changed depending on the presence of the selection pressure, once selected, resistant subpopulations were maintained even in extended passage without drug.IMPORTANCE Understanding mechanisms of antibiotic resistance is crucial to guide how best to use antibiotics to minimize emergence of resistance. We used a laboratory evolution system to study how Salmonella responds to cefotaxime in both planktonic and biofilm conditions. In both contexts, we observed rapid selection of mutants within a single hot spot within envZ. The mutations selected altered EnvZ which in turn triggers changes in porin production at the outer membrane. Emergence of mutations within this region was repeatedly observed in parallel lineages in different conditions. We used a combination of genetics, biochemistry, phenotyping and structural analysis to understand the mechanisms. This data show that the changes we observe provide resistance to cefotaxime but come at a cost to biofilm formation and the fitness of mutants changes greatly depending on the presence or absence of a selective drug. Studying how resistance emerges can inform selective outcomes in the real world.

Heteroresistance Is Associated With in vitro Regrowth During Colistin Treatment in Carbapenem-Resistant Klebsiella pneumoniae.

Polymyxins including polymyxin B and colistin (polymyxin E) are considered the last resort for treating infections caused by carbapenem-resistant gram-negative bacteria. However, in vitro regrowth with the emergence of resistance during treatment is common. Polymyxin heteroresistance, particularly in Acinetobacter baumannii and Klebsiella pneumoniae, has been widely reported. This study was primarily performed to evaluate the prevalence of colistin heteroresistance in carbapenem-resistant K. pneumoniae (CR-KP) and the association between in vitro regrowth and heteroresistance. The mechanisms of colistin resistance and the ability of combination therapies to suppress resistance selection were further investigated. A population analysis profile (PAP) analysis showed that 69 (71.9%) of 96 CR-KP strains had colistin heteroresistance. Time-kill assays revealed that the colistin monotherapy could quickly eliminate the bacterial cells in strains without heteroresistance within the first 6 h. Conversely, it could initially reduce the number of cells in heteroresistant strains, but then regrowth occurred rapidly. Resistance screening at 12 and 24 h in the time-kill assays indicated that susceptible populations were killed, and regrowth was the exact result of the continued growth of resistant subpopulations. Colistin resistance in the regrowth subpopulations was mainly due to the overexpression of phoPQ and pmrD. Colistin combined with tetracyclines (tigecycline or minocycline) or aminoglycosides (amikacin or gentamicin) could effectively suppress the resistance selection and significantly elicit in vitro synergistic effects. These findings suggested that the combination therapy can be used to treat infections caused by CR-KP with colistin heteroresistance. Nevertheless, further in vivo studies considering drugs pharmacokinetics/pharmacodynamics are needed to confirm these findings.

Quantification of long-term doxorubicin response dynamics in breast cancer cell lines to direct treatment schedules.

While acquired chemoresistance is recognized as a key challenge to treating many types of cancer, the dynamics with which drug sensitivity changes after exposure are poorly characterized. Most chemotherapeutic regimens call for repeated dosing at regular intervals, and if drug sensitivity changes on a similar time scale then the treatment interval could be optimized to improve treatment performance. Theoretical work suggests that such optimal schedules exist, but experimental confirmation has been obstructed by the difficulty of deconvolving the simultaneous processes of death, adaptation, and regrowth taking place in cancer cell populations. Here we present a method of optimizing drug schedules in vitro through iterative application of experimentally calibrated models, and demonstrate its ability to characterize dynamic changes in sensitivity to the chemotherapeutic doxorubicin in three breast cancer cell lines subjected to treatment schedules varying in concentration, interval between pulse treatments, and number of sequential pulse treatments. Cell populations are monitored longitudinally through automated imaging for 600–800 hours, and this data is used to calibrate a family of cancer growth models, each consisting of a system of ordinary differential equations, derived from the bi-exponential model which characterizes resistant and sensitive subpopulations. We identify a model incorporating both a period of growth arrest in surviving cells and a delay in the death of chemosensitive cells which outperforms the original bi-exponential growth model in Akaike Information Criterion based model selection, and use the calibrated model to quantify the performance of each drug schedule. We find that the inter-treatment interval is a key variable in determining the performance of sequential dosing schedules and identify an optimal retreatment time for each cell line which extends regrowth time by 40%-239%, demonstrating that the time scale of changes in chemosensitivity following doxorubicin exposure allows optimization of drug scheduling by varying this inter-treatment interval.

In pursuit of biomarkers for predicting susceptibility to activity-based anorexia in adolescent female rats.

ObjectiveIdentifying risk factors that contribute to the development of anorexia nervosa (AN) is critical for the implementation of early intervention strategies. Anxiety, obsessive–compulsive behavior, and immune dysfunction may be involved in the development of AN; however, their direct influence on susceptibility to the condition remains unclear. Here, we used the activity‐based anorexia (ABA) model to examine whether activity, anxiety‐like behavior, compulsive behavior, and circulating immune markers predict the subsequent development of pathological weight loss.MethodFemale Sprague–Dawley rats (n = 44) underwent behavioral testing before exposure to ABA conditions after which they were separated into susceptible and resistant subpopulations. Blood was sampled before behavioral testing and after recovery from ABA to screen for proinflammatory cytokines.ResultsRats that were vulnerable to pathological weight loss differed significantly from resistant rats on all key ABA parameters. While the primary measures of anxiety‐like or compulsive behavior were not shown to predict vulnerability to ABA, increased locomotion and anxiety‐like behavior were both associated with the extent of weight loss in susceptible but not resistant animals. Moreover, the change in expression of proinflammatory markers IL‐4 and IL‐6 evoked by ABA was associated with discrete vulnerability factors. Intriguingly, behavior related to risk assessment was shown to predict vulnerability to ABA.DiscussionWe did not find undisputable behavioral or immune predictors of susceptibility to pathological weight loss in the ABA rat model. Future research should examine the role of cognition in the development of ABA, dysfunction of which may represent an endophenotype linking anorectic, anxiety‐like and compulsive behavior.Public SignificanceAnorexia nervosa (AN) has among the highest mortality rates of all psychiatric disorders and treatment options remain limited in their efficacy. Understanding what types of risk factors contribute to the development of AN is essential for implementing early intervention strategies. This study describes how some of the most common psychological features of AN could be used to predict susceptibility to pathological weight loss in a well‐established animal model.

Polymyxin dose tunes the evolutionary dynamics of resistance in multidrug-resistant Acinetobacter baumannii

ObjectivesEvolutionary principles have informed the design of strategies that slow or prevent antibiotic resistance. However, how antibiotic treatment regimens shape the evolutionary dynamics of resistance mutations remains an open question. Here, we investigate varying concentrations of the last-resort polymyxins on the evolution of resistance in Acinetobacter baumannii. MethodsPolymyxin resistance was measured in 18 multidrug-resistant A. baumannii AB5075 populations treated over 14 days with concentrations of polymyxin B informed by human pharmacokinetics. Time-resolved whole-population sequencing was conducted to track the genetics and population dynamics of susceptible and resistant subpopulations. ResultsA critical threshold concentration of polymyxin B (1 mg/L; i.e. 4 × MIC) was identified. Below this threshold concentration, low levels of resistance repeatedly evolved, but no mutations were fixed, and this resistance was reversed upon removal of the antibiotic. This contrasted with evolution at super-MIC levels (≥4 × MIC) of polymyxin B, which drove the evolution of irreversible resistance, with higher levels of antibiotic correlating with greater rates of molecular evolution. Polymyxin-resistant subpopulations carried mutations in a variety of genes, most commonly pmrB, ompA, glmU/glmS, and wecB/wecC, which contributed to membrane remodelling and virulence in A. baumannii. ConclusionsOur results show that the strength of the selective pressure applied by polymyxin tunes the dynamics of genetic variants within the population, leading to different evolutionary outcomes for the degree, cost and reversibility of resistance. Our study highlights the critical role of integrating evolutionary findings into pharmacokinetics/pharmacodynamics to optimise antibiotic use in patients.

Impaired CXCL12 signaling contributes to resistance of pancreatic cancer subpopulations to T cell-mediated cytotoxicity

ABSTRACT Pancreatic cancer remains largely unresponsive to immune modulatory therapy attributable in part to an immunosuppressive, desmoplastic tumor microenvironment. Here, we analyze mechanisms of cancer cell-autonomous resistance to T cells. We used a 3D co-culture model of cancer cell spheroids from the KPC (LSL-KrasG12D/+ /LSL-Trp53R172H/+ /p48-Cre) pancreatic ductal adenocarcinoma (PDAC) model, to examine interactions with tumor-educated T cells isolated from draining lymph nodes of PDAC-bearing mice. Subpopulations of cancer cells resistant to these tumor-educated T cells were isolated from the in vitro co-culture and their properties compared with sensitive cancer cells. In co-culture with resistant cancer cell subpopulations, tumor-educated T cells showed reduced effector T cell functionality, reduced infiltration into tumor cell spheroids and decreased induction of apoptosis. A combination of comparative transcriptomic analyses, cytometric and immunohistochemistry techniques allowed us to dissect the role of differential gene expression and signaling pathways between sensitive and resistant cells. A decreased expression of the chemokine CXCL12 (SDF-1) was revealed as a common feature in the resistant cell subpopulations. Adding back CXCL12 reversed the resistant phenotype and was inhibited by the CXCR4 inhibitor AMD3100 (plerixafor). We conclude that reduced CXCL12 signaling contributes to PDAC subpopulation resistance to T cell-mediated attack.

Evaluating Paenibacillus odorifer for its potential to reduce shelf life in reworked high-temperature, short-time fluid milk products

Rework is a common practice used in the dairy industry as a strategy to help minimize waste from processing steps or errors that might otherwise render the product unsaleable. Dairy processors may rework their high-temperature, short-time (HTST) fluid milk products up to code date (21 d) at a typical dilution rate of ≤20% rework into ≥80% fresh raw milk. Bacterial spores present in raw milk that can survive pasteurization and grow at refrigeration temperatures are often responsible for milk spoilage. However, the potential impact of growth and thermal resistance of organisms in reworked product has not been investigated. Our objective was to characterize growth, sporulation, and thermal resistance of Paenibacillus odorifer under conditions representative of extreme storage conditions (time and temperature) of reduced fat (2%) and chocolate milk to evaluate whether product containing rework would have a reduced shelf life. Commercial UHT-pasteurized 2% milk and chocolate milk were independently inoculated with 4 strains of P. odorifer at 1 to 2 log cfu/mL and stored at 4°C and 7°C for 30 d. Changes in P. odorifer cell densities were determined by standard serial dilution with spread plating on tryptic soy agar with yeast extract and incubation at 25°C for 48 h. Spore counts were determined following thermal treatment at 80°C for 12 min. Thermal resistance of a cocktail of P. odorifer in milk was determined after treatments at 63°C for 30 min and 72°C for 15 s. Strains of P. odorifer grew rapidly at 7°C and reached a maximum cell density of ~8 log cfu/g in both 2% and chocolate milk within 12 d. All strains grew more slowly at 4°C and had not reached maximum cell density by 21 d. With extreme temperature abuse (25°C, 24 h), P. odorifer will sporulate in milk; however, thermally resistant subpopulations, including spores, did not develop in milk at 4°C until after stationary phase was achieved (>24 d). Vegetative cells of P. odorifer were verified to be sensitive to pasteurization (>7 log reduction); therefore, P. odorifer would not be expected to contribute to reduced shelf life of fluid milk products containing rework, even with extended storage before rework.

Single-cell transcriptomics of neuroblastoma identifies chemoresistance-associated genes and pathways

High-Risk neuroblastoma (NB) survival rate is still <50%, despite treatments being more and more aggressive. The biggest hurdle liable to cancer therapy failure is the drug resistance by tumor cells that is likely due to the intra-tumor heterogeneity (ITH). To investigate the link between ITH and therapy resistance in NB, we performed a single cell RNA sequencing (scRNAseq) of etoposide and cisplatin resistant NB and their parental cells. Our analysis showed a clear separation of resistant and parental cells for both conditions by identifying 8 distinct tumor clusters in etoposide-resistant/parental and 7 in cisplatin-resistant/parental cells. We discovered that drug resistance can affect NB cell identities; highlighting the bi-directional ability of adrenergic-to-mesenchymal transition of NB cells. The biological processes driving the identified resistant cell subpopulations revealed genes such as (BARD1, BRCA1, PARP1, HISTH1 axis, members of RPL family), suggesting a potential drug resistance due to the acquisition of DNA repair mechanisms and to the modification of the drug targets. Deconvolution analysis of bulk RNAseq data from 498 tumors with cell subpopulation signatures showed that the transcriptional heterogeneity of our cellular models reflected the ITH of NB tumors and allowed the identification of clusters associated with worse/better survival.Our study demonstrates the distinct cell populations characterized by genes involved in different biological processes can have a role in NB drug treatment failure. These findings evidence the importance of ITH in NB drug resistance studies and the chance that scRNA-seq analysis offers in the identification of genes and pathways liable for drug resistance.

Bacterial Mixology: Combining Pharmacodynamic Models to Predict In Vitro Competition of MCR-1-Harboring E. coli.

The emergence of mobile colistin resistance (mcr)-mediated polymyxin resistance has resulted in a significant detriment to the utility of the polymyxins in the clinical setting. Though the risk for horizontal transfer of an mcr-containing plasmid is a major component of the transmissibility, selection of polymyxin resistant subpopulations is still a major risk factor for developing polymyxin-resistant infections. Using static time-kills over 24 h (h), we performed competition studies by mixing known inocula of isogenic Escherichia coli strains (wildtype [WT] and mcr-1-harboring) and treating with a concentration array of polymyxin B. These results were then compared to a priori predictions of bacterial-killing effects by polymyxin B on a mixed population of E. coli cells using a previously published mechanism-based model. The data showed that both selective pressure between WT and mcr-1-harboring strains as well as underlying polymyxin B heteroresistance within each of the two strains contributed to bacterial regrowth despite treatment with high concentration polymyxin B. Moreover, the simulations showed that when mcr-1-harboring cells were 1% or 10% of the total population, regrowth by 24 h was still observed in ≥50% of the simulated subjects for both a 106 and 108 inoculum. These results indicate that at lower inoculums with a low proportion of mcr-1-harboring cells, selective pressure from a pharmacokinetic-optimized regimen of polymyxin B still results in regrowth and selection of polymyxin-resistant cells.

Pharmacodynamics of Linezolid Plus Fosfomycin Against Vancomycin–Resistant Enterococcus faecium in a Hollow Fiber Infection Model

The optimal therapy for severe infections caused by vancomycin-resistant Enterococcus faecium (VREfm) remains unclear, but the combination of linezolid and fosfomycin may be a good choice. The 24-h static-concentration time-kill study (SCTK) was used to preliminarily explore the pharmacodynamics of linezolid combined with fosfomycin against three clinical isolates. Subsequently, a hollow-fibre infection model (HFIM) was used for the first time to further investigate the pharmacodynamic activity of the co-administration regimen against selected isolates over 72 h. To further quantify the relationship between fosfomycin resistance and bacterial virulence in VREfm, the Galleria mellonella infection model and virulence genes expression experiments were also performed. The results of SCTK showed that the combination of linezolid and fosfomycin had additive effect on all strains. In the HFIM, the dosage regimen of linezolid (12 mg/L, steady-state concentration) combined with fosfomycin (8 g administered intravenously every 8 h as a 1 h infusion) not only produced a sustained bactericidal effect of 3∼4 log10 CFU/mL over 72 h, but also completely eradicated the resistant subpopulations. The expression of virulence genes was down-regulated to at least 0.222-fold in fosfomycin-resistant strains compared with baseline isolate, while survival rates of G. mellonella was increased (G. mellonella survival ≥45% at 72 h). For severe infections caused by VREfm, neither linezolid nor fosfomycin monotherapy regimens inhibited amplification of the resistant subpopulations, and the development of fosfomycin resistance was at the expense of the virulence of VREfm. The combination of linezolid with fosfomycin produced a sustained bactericidal effect and completely eradicated the resistant subpopulations. Linezolid plus Fosfomycin is a promising combination for therapy of severe infections caused by VREfm.

LB16. Surveillance Rates of Cefiderocol Heteroresistance Correlate With All-cause Mortality in the APEKS-NP and CREDIBLE-CR Trials

BackgroundCefiderocol is a recently FDA approved, novel siderophore beta-lactam antibiotic. Conventional antimicrobial susceptibility testing (AST) suggests that a variety of Gram-negative pathogens, including carbapenem-resistant (CR) isolates, are overwhelmingly susceptible to cefiderocol.. This antibiotic performed well in the APEKS-NP trial for the treatment of nosocomial pneumonia caused largely by carbapenem susceptible isolates. However, in the CREDIBLE-CR trial involving exclusively CR Gram-negative bacteria, cefiderocol was associated with a higher rate of all-cause mortality. We hypothesized one explanation for these discrepant data might be undetected cefiderocol heteroresistance (HR).HR is a form of antibiotic resistance in which an isolate harbors a minority resistant subpopulation of cells co-existing with a majority susceptible population, and is often undetected by standard AST. Isolates exhibiting undetected HR, with as low as 1 in 1 million resistant cells, can cause treatment failure in in vivo models.MethodsWe quantified HR to cefiderocol by population analysis profile (PAP) of 161 Acinetobacter, 180 Klebsiella, and 108 Pseudomonas isolates collected in Georgia, USA.ResultsWe observed CR isolates exhibited a high frequency of HR, which was largely undetected by standard AST, and correlated with all-cause mortality in the CREDIBLE-CR study (Table). Carbapenem-susceptible isolates exhibited no or low rates of cefiderocol HR (Table). Cephalosporin-resistant bacteria mostly exhibited increased rates of cefiderocol HR, but below those of CR strains. These differences in rates of cefiderocol HR correlated with the mortality data from the APEKS-NP and CREDIBLE-CR trials, across the bacterial species tested (Table).Table: Surveillance rates of cefiderocol heteroresistance correlate with all-cause mortality in the APEKS-NP and CREDIBLE-CR trials ConclusionThese data suggest that the lower rates of cefiderocol HR in carbapenem-susceptible isolates that predominated the APEKS-NP trial may explain the enhanced efficacy of the drug in that study as compared to the CREDIBLE-CR trial. Importantly, the widespread, undetected cefiderocol HR observed among CR pathogens may explain the discordance between this drug’s excellent in vitro susceptibility profile and increased patient mortality in the CREDIBLE-CR trial.Disclosures All Authors: No reported disclosures

Heteroresistance to Amikacin in Carbapenem-Resistant Klebsiella pneumoniae Strains.

Heteroresistance can lead to treatment failure and is difficult to detect by the methods currently employed by clinical laboratories. The aim of this study was to investigate the prevalence of the amikacin-heteroresistant Klebsiella pneumoniae strains and explore potential amikacin heteroresistance mechanism through whole-genome sequencing (WGS) and quantitative reverse-transcription PCR (qRT-PCR). In this study, 13 isolates (8.39%) were considered as amikacin-heteroresistant K. pneumoniae strains among a total of 155 K. pneumoniae strains. The majority of the heterogeneous phenotypes (11/13, 84.61%) was unstable and the minimal inhibitory concentrations (MICs) fully or partially reverted back to the level of susceptibility of the parental isolate. The frequency of heteroresistant subpopulation ranged from 2.94×10−7 to 5.59×10−6. Whole-genome sequencing and single-nucleotide variants (SNVs) analysis showed that there were different nucleotide and resultant amino acid alterations among an amikacin-heteroresistant strain S38 and the resistant subpopulation S38L in several genes. Quantitative reverse-transcription PCR analysis revealed that the increased expression of aminoglycoside resistance genes detected in amikacin-heteroresistant K. pneumoniae strains might be associated with amikacin heteroresistance. The findings raise concerns for the emergence of amikacin-heteroresistant K. pneumoniae strains and the use of amikacin as therapy for the treatment of multidrug-resistant K. pneumoniae strains.

Impact of Carbapenem Heteroresistance Among Multidrug-Resistant ESBL/AmpC-Producing Klebsiella pneumoniae Clinical Isolates on Antibiotic Treatment in Experimentally Infected Mice.

PurposeAntibiotic resistance is a growing health crisis that is further complicated by treatment failures caused by bacteria that exhibit heterogeneous susceptibility to antibiotics. The aim of this study was to describe imipenem (IPM)-heteroresistant strains among multidrug-resistant (MDR) ESBL/AmpC-producing Klebsiella pneumoniae clinical isolates, investigate their molecular phenotypic characteristics, and elucidate the outcome of antibiotic treatment in mice infected with the heteroresistant isolates.Materials and MethodsAntimicrobial susceptibility of K. pneumoniae isolates was determined by the disk diffusion and E-test methods. Heteroresistance to IPM was confirmed by population analysis profile (PAP) assays. PCR and sequencing were employed to detect MDR determinants. Molecular differences between the susceptible and resistant subpopulations were evaluated by sequencing and quantitative real-time reverse transcription PCR (qRT-PCR) analysis. The effect of the carbapenem-heteroresistant strains on antibiotic treatment was assessed using a mouse model of peritonitis with heteroresistant K. pneumoniae and subsequent treatment with IPM.ResultsIn total, 37 MDR ESBL/AmpC-producing clinical isolates of K. pneumoniae were identified between September 2018 and December 2019. These strains were notably resistant to conventional antimicrobials other than carbapenems. Among the isolates, three strains exhibited heteroresistance to IPM and carried several ESBL and/or AmpC genes. Mice infected with a lethal dose of any of the three heteroresistant isolates were unable to survive in the presence of IPM treatment, as the percentage of the IPM-resistant subpopulation of each strain was increased in the peritoneum of these mice at 24 h after infection. The resistant subpopulation of the strains presented pulsed-field gel electrophoresis (PFGE) profiles that were identical to those of the susceptible subpopulation, but ompK36 porin showed a reduction in gene expression (0.09- to 0.50-fold) in the resistant subpopulation.ConclusionCarbapenem-heteroresistant strains were present among the MDR K. pneumoniae isolates producing ESBL/AmpC β-lactamases, and these heteroresistant strains failed IPM therapy in experimentally infected mice.

Azithromycin and cefixime combination versus azithromycin alone for the out-patient treatment of clinically suspected or confirmed uncomplicated typhoid fever in South Asia: a randomised controlled trial protocol.

Background: Typhoid and paratyphoid fever (enteric fever) is a common cause of non-specific febrile infection in adults and children presenting to health care facilities in low resource settings such as the South Asia. A 7-day course of a single oral antimicrobial such as ciprofloxacin, cefixime, or azithromycin is commonly used for its treatment. Increasing antimicrobial resistance threatens the effectiveness of these treatment choices. We hypothesize that combined treatment with azithromycin (active mainly intracellularly) and cefixime (active mainly extracellularly) will be a better option for the treatment of clinically suspected and culture-confirmed typhoid fever in South Asia. Methods: This is a phase IV, international multi-center, multi-country, comparative participant-and observer-blind, 1:1 randomised clinical trial. Patients with suspected uncomplicated typhoid fever will be randomized to one of the two interventions: Arm A: azithromycin 20mg/kg/day oral dose once daily (maximum 1gm/day) and cefixime 20mg/kg/day oral dose in two divided doses (maximum 400mg bd) for 7 days, Arm B: azithromycin 20mg/kg/day oral dose once daily (max 1gm/day) for 7 days AND cefixime-matched placebo for 7 days. We will recruit 1500 patients across sites in Bangladesh, India, Nepal, and Pakistan. We will assess whether treatment outcomes are better with the combination after one week of treatment and at one- and three-months follow-up. Discussion: Combined treatment may limit the emergence of resistance if one of the components is active against resistant sub-populations not covered by the other antimicrobial activity. If the combined treatment is better than the single antimicrobial treatment, this will be an important result for patients across South Asia and other typhoid endemic areas. Clinicaltrials.gov registration: NCT04349826 (16/04/2020).

Azithromycin and cefixime combination versus azithromycin alone for the out-patient treatment of clinically suspected or confirmed uncomplicated typhoid fever in South Asia: a randomised controlled trial protocol

Background: Typhoid and paratyphoid fever (enteric fever) is a common cause of non-specific febrile infection in adults and children presenting to health care facilities in low resource settings such as the South Asia. A 7-day course of a single oral antimicrobial such as ciprofloxacin, cefixime, or azithromycin is commonly used for its treatment. Increasing antimicrobial resistance threatens the effectiveness of these treatment choices. We hypothesize that combined treatment with azithromycin (active mainly intracellularly) and cefixime (active mainly extracellularly) will be a better option for the treatment of clinically suspected and culture-confirmed typhoid fever in South Asia. Methods: This is a phase IV, international multi-center, multi-country, comparative participant-and observer-blind, 1:1 randomised clinical trial. Patients with suspected uncomplicated typhoid fever will be randomized to one of the two interventions: Arm A: azithromycin 20mg/kg/day oral dose once daily (maximum 1gm/day) and cefixime 20mg/kg/day oral dose in two divided doses (maximum 400mg bd) for 7 days, Arm B: azithromycin 20mg/kg/day oral dose once daily (max 1gm/day) for 7 days AND cefixime-matched placebo for 7 days. We will recruit 1500 patients across sites in Bangladesh, India, Nepal, and Pakistan. We will assess whether treatment outcomes are better with the combination after one week of treatment and at one- and three-months follow-up. Discussion: Combined treatment may limit the emergence of resistance if one of the components is active against resistant sub-populations not covered by the other antimicrobial activity. If the combined treatment is better than the single antimicrobial treatment, this will be an important result for patients across South Asia and other typhoid endemic areas. Clinicaltrials.gov registration: NCT04349826 (16/04/2020)

Is the Fixed Periodic Treatment Effective for the Tumor System without Complete Information?

ObjectiveThe treatment plans designed with the guidance of the mathematical model and adaptive strategy can trap tumor subpopulations in a periodic and controllable loop. But this process requires detailed information about the tumor system, which is difficult to obtain. Therefore, we wondered whether the fixed periodic treatment plans designed with the typical values of population parameters could be applied to a similar tumor system without complete information.MethodsA binary tumor system constructed by an EGFR-mutant and a KRAS-mutant cell line was used to explore the applicability of the fixed periodic treatment plans. The dynamics of this system were described by combining the Lotka-Volterra model with the framework of the nonlinear mixed-effects model. The typical values of population parameters were used to design the plans, and the robust plans were screened through parameter variation. These screened plans were examined their applicability in animal experiments and simulations.ResultsIn animal experiments where system parameters vary from −30% to 30%, the “osimertinib administration, withdrawal, FK866 administration and withdrawal” plan can trap subpopulations of the system in periodic cycles. In simulation, when there was an unknown resistant subpopulation, the screened fixed periodic treatment plans can still delay the evolution of resistance. The median outcomes of screened plans were better than conventional sequential treatment in most cases. There was no significant difference between the outcomes of the screened plan with median stability and the optimal therapy. The evolutionary trajectories of these two plans were similar.ConclusionAccording to the results, these fixed periodic plans should be tried in treatment even the information of the tumor system was incomplete.

Does cefiderocol heteroresistance explain the discrepancy between the APEKS-NP and CREDIBLE-CR clinical trial results?

Does cefiderocol heteroresistance explain the discrepancy between the APEKS-NP and CREDIBLE-CR clinical trial results?

Phenotypic Detection of Hemin-Inducible Trimethoprim-Sulfamethoxazole Heteroresistance in Staphylococcus aureus.

ABSTRACTTrimethoprim-sulfamethoxazole (SXT) is a valuable second-line antimicrobial agent to treat methicillin-resistant Staphylococcus aureus infections. Discrepancies between various antibiotic susceptibility testing (AST) methods for SXT susceptibility in S. aureus have been described. Here, we describe a hemin-inducible heteroresistance phenotype in S. aureus. We compared the results of the Vitek 2 AST on a set of 95 S. aureus clinical isolates with broth microdilution, disk diffusion using standard Mueller-Hinton agar, and disk diffusion using Mueller-Hinton agar supplemented with 5% horse blood (MHF). To investigate the potential clinical relevance of SXT heteroresistance, an in vivo Galleria mellonella infection assay was performed. All Vitek 2 SXT-susceptible (n = 17) isolates were concordant with AST results by other methods applied in this study. In 32/78 (41%) of Vitek 2 SXT-resistant isolates, we observed a heteroresistant growth phenotype on MHF. The heteroresistance phenotype was associated with the presence of dfr genes, encoding trimethoprim resistance. The addition of a hemin-impregnated disk in a double disk diffusion method on standard Mueller-Hinton agar was able to induce growth in the SXT zone of inhibition. An in vivo infection assay with G. mellonella suggested that the SXT heteroresistance phenotype resulted in lethality similar to that of the SXT-resistant phenotype. In this study, we describe a novel hemin-inducible heteroresistance phenotype in S. aureus. This heteroresistance phenotype may be missed by standard AST methods but can be detected by performing disk diffusion using Mueller-Hinton agar supplemented with 5% horse blood, commonly used for AST of fastidious organisms. This phenomenon may partly explain the discrepancies of AST methods in determining SXT resistance in S. aureus.IMPORTANCE Staphylococcus aureus is one of most important pathogens in clinical medicine. Besides its virulence, the acquisition or emergence of resistance toward antibiotic agents, in particular to beta-lactam antibiotics (methicillin-resistant S. aureus [MRSA]), poses a major therapeutic challenge. Trimethoprim-sulfamethoxazole (SXT) is one of the effective antimicrobial agents of last resort to treat MRSA infections. Here, we report the detection of a SXT-heteroresistant phenotype which is inducible by hemin and can be detected using Mueller-Hinton agar supplemented with horse blood. Heteroresistance describes the presence or emergence of resistant subpopulations, which may potentially lead to inaccurate antibiotic susceptibility testing results and influence the success of antibiotic therapy.

Optimizing Adaptive Therapy Based on the Reachability to Tumor Resistant Subpopulation.

Simple SummaryThe intra-competition among tumor subpopulations is a promising target to modify and control the outgrowth of the resistant subpopulation. Adaptive therapy lives up to this principle well, but the gain of tumors with an aggressive resistant subpopulation is not superior to maximum tolerated dose therapy (MTD). How to integrate these two therapies to maximize the outcome? According to the model and system reachability, the ‘restore index’ is proposed to evaluate the timing of the transition from the treatment cycle of adaptive therapy to high-frequency administration, and to juggle the benefits of intra-competition and killing of the sensitive subpopulation. Based on the simulation and animal experiment, the effectiveness of this method in treating tumors with an aggressive resistant subpopulation has been confirmed.Adaptive therapy exploits the self-organization of tumor cells to delay the outgrowth of resistant subpopulations successfully. When the tumor has aggressive resistant subpopulations, the outcome of adaptive therapy was not superior to maximum tolerated dose therapy (MTD). To explore methods to improve the adaptive therapy’s performance of this case, the tumor system was constructed by osimertinib-sensitive and resistant cell lines and illustrated by the Lotka-Volterra model in this study. Restore index proposed to assess the system reachability can predict the duration of each treatment cycle. Then the threshold of the restore index was estimated to evaluate the timing of interrupting the treatment cycle and switching to high-frequency administration. The introduced reachability-based adaptive therapy and classic adaptive therapy were compared through simulation and animal experiments. The results suggested that reachability-based adaptive therapy showed advantages when the tumor has an aggressive resistant subpopulation. This study provides a feasible method for evaluating whether to continue the adaptive therapy treatment cycle or switch to high-frequency administration. This method improves the gain of adaptive therapy by taking into account the benefits of tumor intra-competition and the tumor control of killing sensitive subpopulation.