Efficacy of ramucirumab combination chemotherapy as second-line treatment in patients with advanced adenocarcinoma of the stomach or gastroesophageal junction after exposure to checkpoint inhibitors and chemotherapy as first-line therapy.

FOLFOX plus nivolumab represents a standard of care for first-line therapy of advanced gastroesophageal cancer (aGEC) with positive PD-L1 expression. The efficacy of second-line VEGFR-2 inhibition with ramucirumab (RAM) plus chemotherapy after progression to immunochemotherapy remains unclear. Medical records of patients with aGEC enrolled in the randomized phase II AIO-STO-0417 trial after treatment failure to first-line FOLFOX plus nivolumab and ipilimumab were retrospectively analyzed. Patients were divided into two groups based on second-line therapy: RAM plus chemotherapy (RAM group) or treatment without RAM (control group). Eighty three patients were included. In the overall population, progression-free survival (PFS) in the RAM group was superior to the control (4.5 vs 2.9 months). Responders (CR/PR) to first-line immunochemotherapy receiving RAM containing second-line therapy had prolonged OS from start of first-line therapy (28.9 vs 16.5 months), as well as second-line OS (9.6 vs 7.5 months), PFS (5.6 vs 2.9 months) and DCR (53% vs 29%) compared to the control. PD-L1 CPS ≥1 was 42% and 44% for the RAM and the control, respectively. Patients with CPS ≥1 in the RAM group showed better tumor control (ORR 25% vs 10%) and improved survival (total OS 11.5 vs 8.0 months; second-line OS 6.5 vs 3.9 months; PFS 4.5 vs 1.6 months) compared to the control. Prior exposure to first-line FOLFOX plus dual checkpoint inhibition followed by RAM plus chemotherapy shows favorable response and survival rates especially in patients with initial response and positive PD-L1 expression and has the potential to advance the treatment paradigm in aGEC.

Adjuvant Gemcitabine Versus Neoadjuvant/Adjuvant FOLFIRINOX in Resectable Pancreatic Cancer: The Randomized Multicenter Phase II NEPAFOX Trial.

BACKGROUND: Although addition of adjuvant chemotherapy is the current standard, the prognosis of pancreatic cancers still remains poor. The NEPAFOX trial evaluated perioperative treatment with FOLFIRINOX in resectable pancreatic cancer. PATIENTS AND METHODS: This multicenter phase II trial randomized patients with resectable or borderline resectable pancreatic cancer without metastases into arm (A,) upfront surgery plus adjuvant gemcitabine, or arm (B,) perioperative FOLFIRINOX. The primary endpoint was overall survival (OS). RESULTS: Owing to poor accrual, recruitment was prematurely stopped after randomization of 40 of the planned 126 patients (A: 21, B: 19). Overall, approximately three-quarters were classified as primarily resectable (A: 16, B: 15), and the remaining patients were classified as borderline resectable (A: 5, B: 4). Of the 12 evaluable patients, 3 achieved partial response under neoadjuvant FOLFIRINOX. Of the 21 patients in arm A and 19 patients in arm B, 17 and 7 underwent curative surgery, and R0-resection was achieved in 77% and 71%, respectively. Perioperative morbidity occurred in 72% in arm A and 46% in arm B, whereas non-surgical toxicity was comparable in both arms. Median RFS/PFS was almost doubled in arm B (14.1 months) compared with arm A (8.4 months) in the population with surgical resection, whereas median OS was comparable between both arms. CONCLUSIONS: Although the analysis was only descriptive owing to small patient numbers, no safety issues regarding surgical complications were observed in the perioperative FOLFIRINOX arm. Thus, considering the small number of patients, perioperative treatment approach appears feasible and potentially effective in well-selected cohorts of patients. In pancreatic cancer, patient selection before initiation of neoadjuvant therapy appears to be critical.

Ramucirumab beyond progression plus TAS-102 in patients with advanced or metastatic esophagogastric adenocarcinoma, after treatment failure on a ramucirumab-based therapy.

Based on results of prior trials (TAGS, REGARD, RAINBOW), the combination of ramucirumab beyond progression with TAS-102 (trifluridine/tipiracil) seems to be promising in advanced esophagogastric adenocarcinoma (EGA). In this multicenter, non-randomized, open-label, investigator-initiated pilot trial, ramucirumab-pretreated patients with metastatic EGA received a maximum of 4 cycles of ramucirumab (8 mg/kg i.v. on day 1 and 15, Q2W) plus TAS-102 (35 mg/m2 p.o. bid on day 1-5 and day 8-12; Q2W). Primary endpoint was tolerability and toxicity, defining a positive trial if the SAE rate according to CTCAE 5.0 will increase <30% (up to 55%) compared to historical results from TAGS trial (SAE rate 43%). Secondary endpoints were further evaluation of safety and assessment of efficacy according to tumor response and overall and progression-free survival (OS/PFS). Twenty patients, 20% gastric and 80% GEJ cancers and 55% with ECOG 0 were enrolled. In total, nine SAEs were reported in 25% [95% CI: 8.7-49.1] of the patients, all without relationship to the systemic therapy. The median OS and PFS were 9.1 months [5.4-10.1] and 2.9 months [1.7-4.8], respectively. In addition, a disease control rate of 45% was obtained. The trial showed a favorable safety profile with a numerically lower incidence of SAEs for the combination of ramucirumab with TAS-102 compared to historical TAGS trial. Furthermore, the combination demonstrated efficacy in the beyond progression setting and therefore warrants further evaluation in a randomized trial compared to TAS-102 alone.

Perioperative FLOT plus ramucirumab for resectable esophagogastric adenocarcinoma: A randomized phase II/III trial of the German AIO and Italian GOIM.

This multicenter, randomized phase II/III study evaluated the addition of the vascular endothelial growth factor receptor-2 inhibitor ramucirumab to FLOT as perioperative treatment for resectable esophagogastric adenocarcinoma. Patients received either FLOT alone (Arm A) or combined with ramucirumab followed by ramucirumab monotherapy (Arm B). The primary endpoint for the phase II portion was the pathological complete or subtotal response (pCR/pSR) rate. Baseline characteristics were comparable between both arms with a high rate of tumors signet-ring cell component (A:47% B:43%). No between-arm difference in pCR/pSR rate was seen (A:29% B:26%), therefore the transition to phase III was not pursued. Nevertheless, the combination was associated with a significantly increased R0-resection rate compared with FLOT alone (A:82% B:96%; P = .009). In addition, the median disease-free survival was numerically improved in Arm B (A:21 months B:32 months, HR 0.75, P = 0.218), while the median overall survival was similar in both treatment arms (A:45 months B:46 months, HR 0.94, P = 0.803). Patients with Siewert type I tumors receiving transthoracic esophagectomy with intrathoracic anastomosis showed an increased risk of serious postoperative complications after ramucirumab treatment, therefore recruitment of those patients was stopped after the first-third of the study. Overall, surgical morbidity and mortality was comparable, whereas more non-surgical grade ≥ 3 adverse events were observed with the combination, especially anorexia (A:1% B:11%), hypertension (A:4% B:13%) and infections (A:19% B:33%). The combination of ramucirumab and FLOT as perioperative treatment shows efficacy signals, particularly in terms of R0 resection rates, for a study population with a high proportion of prognostically poor histological subtypes, and further evaluation in this subgroup seems warranted.

Anti-Biofilm Effect of Octenidine and Polyhexanide on Uropathogenic Biofilm-Producing Bacteria.

BACKGROUND: A catheter allowing a release of antibacterial substances such as antiseptics into the bladder could be a new way of preventing biofilm formation and subsequent catheter-associated urinary tract infections. METHODS: Minimal inhibitory and bactericidal concentration (MIC/MBC) determinations in cation-adjusted Mueller-Hinton broth and artificial urine were performed for 4 antiseptics against 3 uropathogenic biofilm producers, Escherichia coli, Pseudomonas aeruginosa, and Proteus mirabilis. Furthermore, effects of octenidine and polyhexanide against catheter biofilm formation were determined by quantification of biofilm-producing bacteria. RESULTS: Sodium hypochlorite showed MIC/MBC values between 200 and 800 mg/L for all strains tested. Triclosan was efficient against E. coli and P. mirabilis (MIC ≤2.98 mg/L) but ineffective against P. aeruginosa. Octenidine and polyhexanide showed antibacterial activity against all 3 species tested (MIC 1.95-7.8 and 3.9-31.25 mg/L). Both octenidine and polyhexanide were able to prevent biofilm formation on catheter segments in a concentration dependent manner. Furthermore, adding 250 mg/L of each biocide disrupted biofilms formed by E. coli and P. mirabilis, whereas even 500 mg/L was not sufficient to completely destroy P. aeruginosa biofilms. CONCLUSION: Octenidine- and polyhexanide-containing antiseptics showed a broad effect against typical uropathogenic biofilm producers even in high dilutions. This study provides a basis for further investigation of the potential of octenidine and polyhexanide as prophylaxis or treatment of catheter biofilms.

Carbapenem-Containing Combination Antibiotic Therapy against Carbapenem-Resistant Uropathogenic Enterobacteriaceae.

The increasing global prevalence of carbapenem-resistant Enterobacteriaceae (CRE) combined with the decline in effective therapies is a public health care crisis. After respiratory tract infections, urinary tract infections and associated urosepsis are the second most affected by CRE pathogens. By using checkerboard analysis, we tested eight different antibiotics in combination with carbapenems in CAMHB (cation-adjusted Müller-Hinton broth) and artificial urine against seven CRE strains and three susceptible strains. To further determine whether these combinations are also effective in a dynamic model, we have performed growth curves analyses in a dynamic bladder model with three uropathogenic CRE strains. In this model, we simulated the urinary pharmacokinetic after application of 1,000 mg intravenous (i.v.) ertapenem alone or in combination with 500 mg i.v. levofloxacin, 1,000 mg oral rifampin, or 3,000 mg oral fosfomycin. Bacterial growth was measured for 48 h, simulating voiding of the bladder every 3 h. According to the median fractional inhibitory concentration indices (ΣFICIs), the values we found were additive to synergistic results across all tested CRE strains for combinations of carbapenems with colistin sulfate, levofloxacin, fosfomycin, rifampin, and tigecycline in CAMHB and artificial urine. In the dynamic bladder model, all three CRE strains tested showed regrowth after treatment with ertapenem up to 48 h. Regrowth could be prevented by combination with levofloxacin, fosfomycin, or rifampin. Carbapenem-containing combination therapy with fosfomycin or rifampin could be an option for better treatment of urinary tract infections (UTIs) caused by CRE strains. This should be further investigated in clinical studies.

The Lectin-like Domain of TNF Increases ENaC Open Probability through a Novel Site at the Interface between the Second Transmembrane and C-terminal Domains of the α-Subunit.

Regulation of the epithelial sodium channel (ENaC), which regulates fluid homeostasis and blood pressure, is complex and remains incompletely understood. The TIP peptide, a mimic of the lectin-like domain of TNF, activates ENaC by binding to glycosylated residues in the extracellular loop of ENaC-α, as well as to a hitherto uncharacterized internal site. Molecular docking studies suggested three residues, Val567, Glu568, and Glu571, located at the interface between the second transmembrane and C-terminal domains of ENaC-α, as a critical site for binding of the TIP peptide. We generated Ala replacement mutants in this region of ENaC-α and examined its interaction with TIP peptide (3M, V567A/E568A/E571A; 2M, V567A/E568A; and 1M, E571A). 3M and 2M ENaC-α, but not 1M ENaC-α, displayed significantly reduced binding capacity to TIP peptide and to TNF. When overexpressed in H441 cells, 3M mutant ENaC-α formed functional channels with similar gating and density characteristics as the WT subunit and efficiently associated with the ß and γ subunits in the plasma membrane. We subsequently assayed for increased open probability time and membrane expression, both of which define ENaC activity, following addition of TIP peptide. TIP peptide increased open probability time in H441 cells overexpressing wild type and 1M ENaC-α channels, but not 3M or 2M ENaC-α channels. On the other hand, TIP peptide-mediated reduction in ENaC ubiquitination was similar in cells overexpressing either WT or 3M ENaC-α subunits. In summary, this study has identified a novel site in ENaC-α that is crucial for activation of the open probability of the channel, but not membrane expression, by the lectin-like domain of TNF.

Pneumococcal hydrogen peroxide-induced stress signaling regulates inflammatory genes.

Microbial infections can induce aberrant responses in cellular stress pathways, leading to translational attenuation, metabolic restriction, and activation of oxidative stress, with detrimental effects on cell survival. Here we show that infection of human airway epithelial cells with Streptococcus pneumoniae leads to induction of endoplasmic reticulum (ER) and oxidative stress, activation of mitogen-associated protein kinase (MAPK) signaling pathways, and regulation of their respective target genes. We identify pneumococcal H2O2 as the causative agent for these responses, as both catalase-treated and pyruvate oxidase-deficient bacteria lacked these activities. Pneumococcal H2O2 induced nuclear NF-κB translocation and transcription of proinflammatory cytokines. Inhibition of translational arrest and ER stress by salubrinal or of MAPK signaling pathways attenuate cytokine transcription. These results provide strong evidence for the notion that inhibition of translation is an important host pathway in monitoring harmful pathogen-associated activities, thereby enabling differentiation between pathogenic and nonpathogenic bacteria.

Perioperative Atezolizumab Plus Fluorouracil, Leucovorin, Oxaliplatin, and Docetaxel for Resectable Esophagogastric Cancer: Interim Results From the Randomized, Multicenter, Phase II/III DANTE/IKF-s633 Trial.

PURPOSE: This trial evaluates the addition of the PD-L1 antibody atezolizumab (ATZ) to standard-of-care fluorouracil, leucovorin, oxaliplatin, and docetaxel (FLOT) as a perioperative treatment for patients with resectable esophagogastric adenocarcinoma (EGA). METHODS: DANTE started as multicenter, randomized phase II trial, which was subsequently converted to a phase III trial. Here, we present the results of the phase II proportion, focusing on surgical pathology and safety outcomes on an exploratory basis. Patients with resectable EGA (≥cT2 or cN+) were assigned to either four preoperative and postoperative cycles of FLOT combined with ATZ, followed by eight cycles of ATZ maintenance (arm A) or FLOT alone (arm B). RESULTS: Two hundred ninety-five patients were randomly assigned (A, 146; B, 149) with balanced baseline characteristics between arms. Twenty-three patients (8%) had tumors with microsatellite instability (MSI), and 58% patients had tumors with a PD-L1 combined positive score (CPS) of ≥1. Surgical morbidity (A, 45%; B, 42%) and 60-day mortality (A, 3%; B, 2%) were comparable between arms. Downstaging favored arm A versus arm B (ypT0, 23% v 15% [one-sided P = .044]; ypT0-T2, 61% v 48% [one-sided P = .015]; ypN0, 68% v 54% [one-sided P = .012]). Histopathologic complete regression rates (pathologic complete response or TRG1a) were higher after FLOT plus ATZ (A, 24%; B, 15%; one-sided P = .032), and the difference was more pronounced in the PD-L1 CPS ≥10 (A, 33%; B, 12%) and MSI (A, 63%; B, 27%) subpopulations. Complete margin-free (R0) resection rates were relatively high in both arms (A, 96%; B, 95%). The incidence and severity of adverse events were similar in both groups. CONCLUSION: Within the limitations of the exploratory nature of the data, the addition of ATZ to perioperative FLOT is safe and improved postoperative stage and histopathologic regression.

Activation of the unfolded protein response by Listeria monocytogenes.

The endoplasmic reticulum (ER) responds to perturbation of homeostasis with stress. To maintain ER function, a signalling-circuitry has evolved which, when engaged, attempts to reduce a surplus of unfolded proteins by triggering the unfolded protein response (UPR). Several studies have implicated UPR in viral infections, neurodegenerative disorders and metabolic diseases but UPR has not yet been widely linked to bacterial infections. Here we demonstrate that the facultative intracellular pathogen Listeria monocytogenes (Lm) induces ER expansion and UPR prior to host cell entry. Lm activated protein kinase RNA-like ER kinase (PERK) evidenced by the phosphorylation of the α-subunit of eukaryotic translation initiation factor-2 (eIF2α), inositol-requiring protein-1 (IRE1) as shown by detection of spliced X-box binding protein-1 (XBP1) and activating transcription factor-6 (ATF6) as demonstrated by depletion of its inactive form. A mutant LmΔhly strain that did not produce listeriolysin (LLO) lacked the UPR response. Conversely purified LLO activated UPR. Sustained infection with Lm resulted in apoptosis. Induction of ER stress by thapsigargin or tunicamycin reduced intracellular bacterial number. Our findings suggest that UPR plays an important role in the cell autonomous defence responses to bacterial infection.

Comparative transcriptomic and proteomic signature of lung alveolar macrophages reveals the integrin CD11b as a regulatory hub during pneumococcal pneumonia infection.

Introduction: Streptococcus pneumoniae is one of the main causes of community-acquired infections in the lung alveoli in children and the elderly. Alveolar macrophages (AM) patrol alveoli in homeostasis and under infectious conditions. However, the molecular adaptations of AM upon infections with Streptococcus pneumoniae are incompletely resolved. Methods: We used a comparative transcriptomic and proteomic approach to provide novel insights into the cellular mechanism that changes the molecular signature of AM during lung infections. Using a tandem mass spectrometry approach to murine cell-sorted AM, we revealed significant proteomic changes upon lung infection with Streptococcus pneumoniae. Results: AM showed a strong neutrophil-associated proteomic signature, such as expression of CD11b, MPO, neutrophil gelatinases, and elastases, which was associated with phagocytosis of recruited neutrophils. Transcriptomic analysis indicated intrinsic expression of CD11b by AM. Moreover, comparative transcriptomic and proteomic profiling identified CD11b as the central molecular hub in AM, which influenced neutrophil recruitment, activation, and migration. Discussion: In conclusion, our study provides novel insights into the intrinsic molecular adaptations of AM upon lung infection with Streptococcus pneumoniae and reveals profound alterations critical for effective antimicrobial immunity.

Interleukin-1 receptor associated kinase 1 (IRAK1) is epigenetically activated in luminal epithelial cells in prostate cancer.

The use of immune adjuvants such as toll-like receptor (TLR) agonists reflects a novel strategy in prostate cancer (PCa) therapy. However, interleukin-1 receptor associated kinase 1 (IRAK1), a central effector of TLR signaling, has been shown to be responsible for resistance to radiation-induced tumor cell death. In order to better understand the function and epigenetic regulation of IRAK1 in PCa, we performed in vitro cell culture experiments together with integrative bioinformatic studies using the latest single-cell RNA-sequencing data of human PCa and normal prostate (NOR), and data from The Cancer Genome Atlas. We focused on key effectors of TLR signaling, the Myddosome-complex components IRAK1, IRAK4 and MYD88 (myeloid differentiation primary response 88), and TRAF6 (tumor-necrosis-factor receptor associated factor 6). In PCa, IRAK1-mRNA was specifically enriched in luminal epithelial cells, representing 57% of all cells, whereas IRAK4 and MYD88 were predominantly expressed in leukocytes, and TRAF6, in endothelial cells. Compared to NOR, only IRAK1 was significantly overexpressed in PCa (Benjamini-Hochberg adjusted p<2x10-8), whereas the expression of IRAK4, MYD88, and TRAF6 was unchanged in PCa, and IRAK1-expression was inversely correlated with a specific differentially methylated region (IRAK1-DMR) within a predicted promoter region enriched for H3K27ac (Spearman correlation r<-0.36; Fisher's test, p<10-10). Transcription factors with high binding affinities in IRAK1-DMR were significantly enriched for canonical pathways associated with viral infection and carcinogenic transformation in the Kyoto Encyclopedia of Gene and Genomes analysis. DU145 cells, exhibiting hypermethylated IRAK1-DMR and low IRAK1-expression, reacted with 4-fold increased IRAK1-expression upon combined treatment with 5-aza-2-deoxycytidine and trichostatin A, and were unresponsive to infection with the uropathogenic Escherichia coli strain UTI89. In contrast, PC3 and LNCaP cells, exhibiting hypomethylated IRAK1-DMR and high endogenous IRAK1-mRNA levels, responded with strong activation of IRAK1-expression to UTI89 infection. In summary, exclusive overexpression of IRAK1 was observed in luminal epithelial cells in PCa, suggesting it has a role in addition to Myddosome-dependent TLR signaling. Our data show that the endogenous epigenetic status of PCa cells within IRAK1-DMR is decisive for IRAK1 expression and should be considered as a predictive marker when selective IRAK1-targeting therapies are considered.

FLOT Versus FLOT/Trastuzumab/Pertuzumab Perioperative Therapy of Human Epidermal Growth Factor Receptor 2-Positive Resectable Esophagogastric Adenocarcinoma: A Randomized Phase II Trial of the AIO EGA Study Group.

PURPOSE: High pathologic complete response (pCR) rates and comparably good survival data were seen in a phase II trial combining perioperative fluorouracil, leucovorin, oxaliplatin, and docetaxel (FLOT) chemotherapy with trastuzumab for resectable, esophagogastric adenocarcinoma (EGA). The current trial evaluates the addition of trastuzumab and pertuzumab to FLOT as perioperative treatment for human epidermal growth factor receptor 2-positive resectable EGA. METHODS: In this multicenter, randomized phase II/III trial, patients with human epidermal growth factor receptor 2-positive, resectable EGA (≥ clinical tumor 2 or clinical nodal-positive) were assigned to four pre- and postoperative cycles of either FLOT alone (arm A) or combined with trastuzumab and pertuzumab, followed by nine cycles of trastuzumab/pertuzumab (arm B). The primary end point for the phase II part was the rate of pCR. RESULTS: The trial was closed prematurely, without transition into phase III, after results of the JACOB trial were reported. Eighty-one patients were randomly assigned (A: 41/B: 40) during the phase II part. The pCR rate was significantly improved with the trastuzumab/pertuzumab treatment (A: 12%/B: 35%; P = .02). Similarly, the rate of pathologic lymph node negativity was higher with trastuzumab/pertuzumab (A: 39%/B: 68%), whereas the R0 resection rate (A: 90%/B: 93%) and surgical morbidity (A: 43%/B: 44%) were comparable. Moreover, the inhouse mortality was equal in both arms (overall 2.5%). The median disease-free survival was 26 months in arm A and not yet reached in arm B (hazard ratio, 0.58; P = .14). After a median follow-up of 22 months, the median overall survival was not yet reached (hazard ratio, 0.56; P = .24). Disease-free survival and overall survival rates at 24 months were 54% (95% CI, 38 to 71) and 77% (95% CI, 63 to 90) in arm A and 70% (95% CI, 55 to 85) and 84% (95% CI, 72 to 96) in arm B, respectively. More ≥ grade 3 adverse events were reported with trastuzumab/pertuzumab, especially diarrhea (A: 5%/B: 41%) and leukopenia (A: 13%/B: 23%). CONCLUSION: The addition of trastuzumab/pertuzumab to perioperative FLOT significantly improved pCR and nodal negativity rates at the price of higher rates of diarrhea and leukopenia.

Natural Bred ε2-Phages Have an Improved Host Range and Virulence against Uropathogenic Escherichia coli over Their Ancestor Phages.

Alternative treatments for Escherichia coli infections are urgently needed, and phage therapy is a promising option where antibiotics fail, especially for urinary tract infections (UTI). We used wastewater-isolated phages to test their lytic activity against a panel of 47 E. coli strains reflecting the diversity of strains found in UTI, including sequence type 131, 73 and 69. The plaquing host range (PHR) was between 13 and 63%. In contrast, the kinetic host range (KHR), describing the percentage of strains for which growth in suspension was suppressed for 24 h, was between 0% and 19%, substantially lower than the PHR. To improve the phage host range and their efficacy, we bred the phages by mixing and propagating cocktails on a subset of E. coli strains. The bred phages, which we termed evolution-squared ε2-phages, of a mixture of Myoviridae have KHRs up to 23% broader compared to their ancestors. Furthermore, using constant phage concentrations, Myoviridae ε2-phages suppressed the growth of higher bacterial inocula than their ancestors did. Thus, the ε2-phages were more virulent compared to their ancestors. Analysis of the genetic sequences of the ε2-phages with the broadest host range reveals that they are mosaic intercrossings of 2-3 ancestor phages. The recombination sites are distributed over the whole length of the genome. All ε2-phages are devoid of genes conferring lysogeny, antibiotic resistance, or virulence. Overall, this study shows that ε2-phages are remarkably more suitable than the wild-type phages for phage therapy.

Anti-Bacterial Effects of Essential Oils against Uropathogenic Bacteria.

Given the increasing antimicrobial resistance in urinary tract infections (UTI), alternative strategies need to be investigated. Determination of minimal inhibitory and bactericidal concentrations of essential oils from cajeput, lemongrass, tea tree, and thyme in artificial urine, revealed bactericidal activity of all four tested essential oils against seven uropathogenic species with values ranging between 0.78-50 mg/mL. Tea tree and thyme essential oils were more efficient than lemongrass and cajeput. In addition, antibiotic-resistant strains showed similar susceptibility as antibiotic-sensitive strains, suggesting no cross-resistance between antibiotics and these essential oils. Checkerboard assays revealed a synergistic activity of the combination of thyme and tea tree. Furthermore, the combination with thyme and tea tree essential oils increased the activity of fosfomycin and pivmecillinam, but not nitrofurantoin, against Escherichia coli. This study provides a basis for further investigation of the potential of thyme and tea tree oil as an alternative or additional treatment of UTI.

Effect of Different Media on the Bactericidal Activity of Colistin and on the Synergistic Combination With Azidothymidine Against mcr-1-Positive Colistin-Resistant Escherichia coli.

Antimicrobial susceptibility testing (AST) performed according to defined guidelines is important to identify resistance and to predict the clinical success or failure of specific antibiotic therapy. However, these guidelines do not cover all physiological conditions that can have a tremendous impact on in vivo resistance. In this study, we tested the susceptibility of thirteen mcr-1-positive Escherichia coli strains against colistin, one of the last resort antibiotics for treating multi-drug resistant pathogens, in media recommended for ASTs as well as - physiologically more relevant - in human serum and artificial urine (AU). Minimal inhibitory concentration (MIC) values in heat-inactivated human serum were similar to those in cation-adjusted Mueller-Hinton broth (CAMHB), but reduced in native serum for almost all strains that could grow in this media. In AU MIC values for mcr-1 positive E. coli were increased significantly up to 16-fold compared to that in CAMBH, which did not apply to the colistin-susceptible E. coli strains tested. Although different growth media could affect the MIC of colistin alone, their impact on the synergistic effect of the combination with the antiviral drug azidothymidine was minimal. The higher divalent cation concentration combined with acidic pH values is most likely responsible for the increased MIC values of the mcr-1 harboring E. coli strains tested against colistin in AU compared to that in CAMHB. Antimicrobial susceptibility screening procedures for colistin using CAMHB only could lead to an underestimation of resistance under different physiological conditions. Therefore, not only pharmacokinetic but also pharmacodynamic studies in urine are as important as in serum or plasma.

Amidochelocardin Overcomes Resistance Mechanisms Exerted on Tetracyclines and Natural Chelocardin.

The reassessment of known but neglected natural compounds is a vital strategy for providing novel lead structures urgently needed to overcome antimicrobial resistance. Scaffolds with resistance-breaking properties represent the most promising candidates for a successful translation into future therapeutics. Our study focuses on chelocardin, a member of the atypical tetracyclines, and its bioengineered derivative amidochelocardin, both showing broad-spectrum antibacterial activity within the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) panel. Further lead development of chelocardins requires extensive biological and chemical profiling to achieve favorable pharmaceutical properties and efficacy. This study shows that both molecules possess resistance-breaking properties enabling the escape from most common tetracycline resistance mechanisms. Further, we show that these compounds are potent candidates for treatment of urinary tract infections due to their in vitro activity against a large panel of multidrug-resistant uropathogenic clinical isolates. In addition, the mechanism of resistance to natural chelocardin was identified as relying on efflux processes, both in the chelocardin producer Amycolatopsis sulphurea and in the pathogen Klebsiella pneumoniae. Resistance development in Klebsiella led primarily to mutations in ramR, causing increased expression of the acrAB-tolC efflux pump. Most importantly, amidochelocardin overcomes this resistance mechanism, revealing not only the improved activity profile but also superior resistance-breaking properties of this novel antibacterial compound.

Urinary bactericidal activity of colistin and azidothymidine combinations against mcr-1-positive colistin-resistant Escherichia coli.

A phase 1 clinical study was performed to assess the pharmacokinetics and safety of intravenous (i.v.) administration of colistin methanesulfonate (CMS) and azidothymidine (AZT) alone and in combination. Seven healthy subjects received three (every 12 h) 1-h i.v. infusions of 4, 2 and 2 million international units (MIU) of CMS co-administered with 200, 100 and 100 mg of AZT, respectively. In an ex vivo study, urinary bactericidal titres (UBTs) and time-kill curve determinations were performed in artificial urine spiked with colistin sulfate and AZT according to median and minimum peak concentrations in urine measured after the first and third dose using four mcr-1-positive colistin-resistant and five colistin-susceptible Gram-negative isolates. Reciprocal UBTs for the different colistin concentrations obtained in urine ranged from 1-128 and 0-2 for colistin-susceptible and colistin-resistant isolates, respectively. Combination with AZT could increase UBTs up to two dilution steps each for the Enterobacteriaceae and Acinetobacter strains tested. In contrast, the combination had no activity against Pseudomonas strains. In time-kill curves, the combination showed bactericidal activity against colistin-resistant strains even when the substances alone were not bactericidal. Thus, combination of CMS with AZT shows promising synergistic activity against Gram-negative uropathogens, including colistin-resistant Enterobacteriaceae. According to the urinary bactericidal activity, a maintenance dosage of 2 MIU of CMS combined with 100 mg of AZT twice daily may be sufficient for the treatment of urinary tract infections (UTIs) caused by colistin-susceptible strains. However, the dosage requires optimisation for efficient treatment of UTIs caused by colistin-resistant strains.