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Effective therapeutic options are urgently needed to tackle antibiotic resistance. Furazolidone (FZ), vancomycin (VAN), and sodium deoxycholate (DOC) show promise as their combination can synergistically inhibit the growth of, and kill, multidrug-resistant Gram-negative bacteria that are classified as critical priority by the World Health Organization. Here, we investigated the mechanisms of action and synergy of this drug combination using a transcriptomics approach in the model bacterium Escherichia coli. We show that FZ and DOC elicit highly similar gene perturbations indicative of iron starvation, decreased respiration and metabolism, and translational stress. In contrast, VAN induced envelope stress responses, in agreement with its known role in peptidoglycan synthesis inhibition. FZ induces the SOS response consistent with its DNA-damaging effects, but we demonstrate that using FZ in combination with the other two compounds enables lower dosages and largely mitigates its mutagenic effects. Based on the gene expression changes identified, we propose a synergy mechanism where the combined effects of FZ, VAN, and DOC amplify damage to Gram-negative bacteria while simultaneously suppressing antibiotic resistance mechanisms. IMPORTANCE Synergistic antibiotic combinations are a promising alternative strategy for developing effective therapies for multidrug-resistant bacterial infections. The synergistic combination of the existing antibiotics nitrofurans and vancomycin with sodium deoxycholate shows promise in inhibiting and killing multidrug-resistant Gram-negative bacteria. We examined the mechanism of action and synergy of these three antibacterials and proposed a mechanistic basis for their synergy. Our results highlight much-needed mechanistic information necessary to advance this combination as a potential therapy.

Bacteriophage (phage) therapy is a promising approach to combat the rise of multidrug-resistant bacteria. Currently, the preferred clinical modality is to pair phage with an antibiotic, a practice thought to improve efficacy. However, antagonism between phage and antibiotics has been reported, the choice of phage and antibiotic is not often empirically determined, and the effect of the host factors on the effectiveness is unknown. Here, we interrogate phage-antibiotic interactions across antibiotics with different mechanisms of action. Our results suggest that phage can lower the working MIC for bacterial strains already resistant to the antibiotic, is dependent on the antibiotic class and stoichiometry of the pairing, and is dramatically influenced by the host microenvironment., The continued rise in antibiotic resistance is precipitating a medical crisis. Bacteriophage (phage) has been hailed as one possible therapeutic option to augment the efficacy of antibiotics. However, only a few studies have addressed the synergistic relationship between phage and antibiotics. Here, we report a comprehensive analysis of phage-antibiotic interaction that evaluates synergism, additivism, and antagonism for all classes of antibiotics across clinically achievable stoichiometries. We combined an optically based real-time microtiter plate readout with a matrix-like heat map of treatment potencies to measure phage and antibiotic synergy (PAS), a process we term synography. Phage-antibiotic synography was performed against a pandemic drug-resistant clonal group of extraintestinal pathogenic Escherichia coli (ExPEC) with antibiotic levels blanketing the MIC across seven orders of viral titers. Our results suggest that, under certain conditions, phages provide an adjuvating effect by lowering the MIC for drug-resistant strains. Furthermore, synergistic and antagonistic interactions are highly dependent on the mechanism of bacterial inhibition by the class of antibiotic paired to the phage, and when synergism is observed, it suppresses the emergence of resistant cells. Host conditions that simulate the infection environment, including serum and urine, suppress PAS in a bacterial growth-dependent manner. Lastly, two different related phages that differed in their burst sizes produced drastically different synograms. Collectively, these data suggest lytic phages can resuscitate an ineffective antibiotic for previously resistant bacteria while also synergizing with antibiotics in a class-dependent manner, processes that may be dampened by lower bacterial growth rates found in host environments.

Nontuberculous mycobacteria (NTM) are ubiquitous microbes belonging to the Mycobacterium genus. Among all NTM pathogens, M. avium is one of the most frequent agents causing pulmonary disease, especially in immunocompromised individuals and cystic fibrosis patients. Recently, we reported the first ad hoc designed M. avium efflux pump inhibitor (EPI; 1b) able to strongly boost clarithromycin (CLA) MIC against different M. avium strains. Since the 3-phenylquinolone derivative 1b suffered from toxicity issues toward human macrophages, herein we report a two-pronged medicinal chemistry workflow for identifying new potent and safe NTM EPIs. Initially, we followed a computational approach exploiting our pharmacophore models to screen FDA approved drugs and in-house compounds to identify "ready-to-use" NTM EPIs and/or new scaffolds to be optimized in terms of EPI activity. Although nicardipine 2 was identified as a new NTM EPI, all identified molecules still suffered from toxicity issues. Therefore, based on the promising NTM EPI activity of 1b, we undertook the design, synthesis, and biological evaluation of new 3-phenylquinolones differently functionalized at the C6/C7 as well as N1 positions. Among the 27 synthesized 3-phenylquinolone analogues, compounds 11b, 12b, and 16a exerted excellent NTM EPI activity at concentrations below their CC50 on human cells, with derivative 16a being the most promising compound. Interestingly, 16a also showed good activity in M. avium-infected macrophages both alone as well as in combination with CLA. The antimycobacterial activity observed for 16a only when tested in the ex vivo model suggests a high therapeutic potential of EPIs against M. avium, which seems to need functional efflux pumps to establish intracellular infections.

Endocarditis is a rare but serious manifestation of Listeria monocytogenes (LM). However, the optimal treatment strategy for LM endocarditis has yet to be established. Current antibiotic strategies for listeriosis include penicillin G or ampicillin (AMP) monotherapy, or AMP + gentamicin combination therapy which is often favored for endocarditis. The primary objective of our investigation was to assess the utility of AMP + ceftriaxone (CRO) and AMP + daptomycin (DAP) against LM, modeling less nephrotoxic antibiotic combinations traditionally used to manage resistant enterococcal endocarditis. Here we report a case of LM endocarditis, review the world literature, and evaluate alternative treatment strategies for listeriosis utilizing in vitro and ex vivo studies. The combination of AMP + CRO and AMP + DAP were each noted to have synergistic activity against a LM endocarditis isolate. Additionally, co-incubation of the isolate with sub-lethal concentrations of antibiotics (AMP, CRO, DAP, AMP + CRO or AMP + DAP) sensitized the bacterium to whole blood killing while pretreatment with CRO and DAP (at 1/4 MIC) sensitized the bacterium to neutrophil killing. However, these effects did not reflect potentiation of antibiotic activity to human cathelicidin peptide LL-37, which is abundant in neutrophils and highly active against LM. Interestingly, AMP pretreatment of the LM endocarditis isolate resulted in increased DAP binding to the bacterium when assessed by fluorescence microscopy. These in vitro and ex vivo studies suggest further investigation of combination therapy using AMP + CRO or AMP + DAP as an alternative treatment for LM infection is warranted.

Patients with cystic fibrosis (CF) suffer from chronic bacterial infection of the airways, and many patients are infected with multiresistant bacteria. Combination antibiotic susceptibility tests, or antibiotic synergy tests, are in vitro tests that have been developed to allow clinicians to choose combinations of antibiotics that should be more effective at killing, or inhibiting, multiresistant bacterial pathogens. Only one randomised controlled clinical trial has been performed to determine whether combination antibiotic susceptibility testing leads to an improved clinical outcome for patients with acute pulmonary exacerbations of CF. The results of this clinical trial were disappointing - treatment based on combination antibiotic susceptibility testing was no more effective than treatment based on conventional culture and sensitivity testing. The adoption of antibiotic synergy testing as routine practice for patients with CF would be costly and would not be justified as there is insufficient evidence to suggest that the routine use of these tests improves clinical outcomes.

Combination of antibiotics in order to achieve antimicrobial synergy is often necessary in the treatment of infections due to resistant bacterial strains. Therefore, several in-vitro test systems have been developed with the purpose of predicting in-vivo action of antibiotics, and the fractional inhibitory concentration (FIC) index has been used to interpret results obtained in different test systems. Using these systems it seems that only antibiotic synergy in vitro is predictive of the results of treatment. It is therefore of interest to have an in-vivo test system that makes it possible to describe antibiotic interaction in detail. Animal infection models such as the foreign body model system enable the measurement of many parameters at the site of infection, such as bactericidal effect (BE) and antibiotic concentrations. A new calculation of drug interaction is suggested in which the measurements used are the BE and the time during which the MIC is exceeded, for the individual drugs and the combination. This calculation enables us to penetrate into observed antibiotic efficacy in vivo to find out whether an observed high BE is due to real synergy or simply to optimal pharmacokinetics of antibiotics at the site of infection.

We assessed the reproducibility of the microdilution checkerboard method for measuring antibiotic synergy. Five strains of Pseudomonas aeruginosa were tested with four antibiotic combinations by using 10 replicates each. Twenty-five percent of replicate sets gave discordant classification results (i.e., a 7:3 or worse split in categorization). Determination of the individual MICs of each antibiotic alone was excellent; all 10 replicates were within 1 twofold dilution for 95% of the 80 sets of 10 replicates. The microdilution checkerboard method either should not be used or should be used with at least five replicates per determination, with > or = 80% agreement among the replicates required for classification.

Current methods of measuring bactericidal antibiotic synergy are particularly susceptible to error because of their use of arbitrary endpoints and criteria; their results are not easily tested for statistical significance and the procedures require considerable time and effort. A single-stage method is described in which bacteria are exposed to antibiotics for a short period and then transferred twice to drug-free medium by means of a filter membrane. The results are available the next day in the form of survival counts which can be compared statistically. In an examination of 21 strains of faecal streptococcus exposed to penicillin and gentamicin the bactericidal synergy observed was smoothly variable, a finding with a bearing on the management of infections with this organism, in that the recognition of strains showing an intermediate degree of synergy is possible.

Thirty-two human isolates of enterococci were tested for antibiotic synergy by using penicillin and one of six aminoglycosides. Three methods were used: synergy screen, microdilution checkerboard, and time-kill curves. The synergy screen accurately predicted synergy for gentamicin-penicillin combinations, and this synergy was later confirmed by time-kill curves. The microdilution checkerboard method suffered from inherent variation, and agreement with time-kill curves ranged from 92% (twofold reduction in minimum inhibitory concentration) to 4.2% (fourfold reduction in minimum inhibitory concentration). We suggest that enterococci be screened for synergy (i.e., presence or absence of high-level resistance) by using the criterion of growth or no growth in the presence of 2,000 microgram of an aminoglycoside per ml. The microdilution checkerboard test for synergy is not recommended.