Non-antibiotic pharmaceuticals are toxic against <i>Escherichia coli</i> with no evolution of cross-resistance to antibiotics

Antimicrobial resistance can arise in the natural environment via prolonged exposure to the effluent released by manufacturing facilities. In addition to antibiotics, pharmaceutical plants also produce non-antibiotic pharmaceuticals, both the active ingredients and other components of the formulations. The effect of these on the surrounding microbial communities is less clear. We aimed to assess whether non-antibiotic pharmaceuticals and other compounds produced by pharmaceutical plants have inherent toxicity, and whether long-term exposure might result in significant genetic changes or select for cross-resistance to antibiotics. To this end, we screened four non-antibiotic pharmaceuticals (acetaminophen, ibuprofen, propranolol, metformin) and titanium dioxide for toxicity against Escherichia coli K-12 MG1655 and conducted a 30 day selection experiment to assess the effect of long-term exposure. All compounds reduced the maximum optical density reached by E. coli at a range of concentrations including one of environmental relevance, with transcriptome analysis identifying upregulated genes related to stress response and multidrug efflux in response ibuprofen treatment. The compounds did not select for significant genetic changes following a 30 day exposure, and no evidence of selection for cross-resistance to antibiotics was observed for population evolved in the presence of ibuprofen in spite of the differential gene expression after exposure to this compound. This work suggests that these compounds, at environmental concentrations, do not select for cross-resistance to antibiotics in E. coli

New multidrug efflux inhibitors for Gram-negative bacteria

Multidrug-resistant Gram-negative bacteria pose a serious threat to human and animal health. Molecules that inhibit multidrug efflux offer an alternative approach to resolving the challenges caused by antibiotic resistance, by potentiating the activity of old, licensed, and new antibiotics. We have developed, validated, and implemented a high-throughput screen and used it to identify efflux inhibitors from two compound libraries selected for their high chemical and pharmacological diversity. We found that the new high-throughput screen is a valuable tool to identify efflux inhibitors, as evidenced by the 43 new efflux inhibitors described in this study.Active efflux of antibiotics preventing their accumulation to toxic intracellular concentrations contributes to clinically relevant multidrug resistance. Inhibition of active efflux potentiates antibiotic activity, indicating that efflux inhibitors could be used in combination with antibiotics to reverse drug resistance. Expression of ramA by Salmonella enterica serovar Typhimurium increases in response to efflux inhibition, irrespective of the mode of inhibition. We hypothesized that measuring ramA promoter activity could act as a reporter of efflux inhibition. A rapid, inexpensive, and high-throughput green fluorescent protein (GFP) screen to identify efflux inhibitors was developed, validated, and implemented. Two chemical compound libraries were screened for compounds that increased GFP production. Fifty of the compounds in the 1,200-compound Prestwick chemical library were identified as potential efflux inhibitors, including the previously characterized efflux inhibitors mefloquine and thioridazine. There were 107 hits from a library of 47,168 proprietary compounds from L. Hoffmann La Roche; 45 were confirmed hits, and a dose response was determined. Dye efflux and accumulation assays showed that 40 Roche and three Prestwick chemical library compounds were efflux inhibitors. Most compounds had specific efflux-inhibitor-antibiotic combinations and/or species-specific synergy in antibiotic disc diffusion and checkerboard assays performed with Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Salmonella Typhimurium. These data indicate that both narrow-spectrum and broad-spectrum combinations of efflux inhibitors with antibiotics can be found. Eleven novel efflux inhibitor compounds potentiated antibiotic activities against at least one species of Gram-negative bacteria, and data revealing an E. coli mutant with loss of AcrB function suggested that these are AcrB inhibitors

Antimicrobial resistance and gene regulation in Enteroaggregative Escherichia coli from Egyptian children with diarrhoea:Similarities and differences

Enteroaggregative Escherichia coli (EAEC) is a common diarrhoeagenic human pathogen, isolated from patients in both developing and industrialized countries, that is becoming increasingly resistant to many frontline antibiotics. In this study, we screened 50 E. coli strains from children presenting with diarrhea at the outpatients clinic of Assiut University Children's Hospital, Egypt. We show that all of these isolates were resistant to multiple classes of antibiotics and identified two as being typical EAEC strains. Using whole genome sequencing, we determined that both isolates carried, amongst others, bla CTX-M and bla TEM antibiotic resistance genes, as well as many classical EAEC virulence determinants, including the transcriptional regulator, AggR. We demonstrate that the expression of these virulence determinants is dependent on AggR, including aar, which encodes for a repressor of AggR, Aar. Since biofilm formation is the hallmark of EAEC infection, we examined the effect of Aar overexpression on both biofilm formation and AggR-dependent gene expression. We show that whilst Aar has a minimal effect on AggR-dependent transcription it is able to completely disrupt biofilm formation, suggesting that Aar affects these two processes differently. Taken together, our results suggest a model for the induction of virulence gene expression in EAEC that may explain the ubiquity of EAEC in both sick and healthy individuals