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Human infectious diseases once treatable with antibiotics are now becoming a serious life-threatening problem due to the emergence of multidrug resistant (MDR) micro-organisms. For this, essential oils (EOs) could constitute a viable source of bioactive compounds capable of inhibiting the mechanisms of this antimicrobial resistance. In this context, our goals were to determine for the first time the chemical composition of EO extracted from aerial parts of Rhus albida, its antibacterial and synergistic interactions with fluoroquinolones and finally to evaluate its ability to inhibit the Staphylococcus aureus NorA efflux pump. The chemical profile and antibacterial activity of R. albida EO were determined using Gas Chromatography coupled to Mass Spectrometry and microdilution assays, respectively. While its NorA efflux-pump-inhibiting activity was assessed using a fluorescent real-time efflux method (based on ethidium bromide (EtBr)) against two S. aureus strains: a NorA overproducing strain (SA1199B) and a wild type strain (SA1199). Results showed that the EO, which is characterized by the abundance sesquiterpenes (δ-cadinene (15.6%), α-copaene (6.3%) and caryophyllene (6.2%)), displayed moderate antimicrobial activities against both SA1199 and SA1199B (MIC = 2048 µg/mL). Nevertheless, its combination with fluoroquinolones contributed to the reduction of their intrinsic MICs only against SA1199B by 4- to 8-fold. Furthermore, the EO at 1/32 MIC, contributed to a large accumulation of EtBr and inhibition of its efflux in SA1199B. Our findings demonstrated the ability of R. albida EO to inhibit the NorA efflux pump. Thus, further studies should be performed in order to make the combination of this natural product with approved antimicrobials a viable option for future administrations in infections with S. aureus strains overproducing NorA EPs.

BackgroundAntibiotic-resistantKlebsiella pneumoniaeare a major cause of hospital- and community-acquired infections, including sepsis, liver abscess, and pneumonia, driven mainly by the emergence of successful high-risk clonal lineages. TheK. pneumoniaesequence type (ST) 307 lineage has appeared in several different parts of the world after first being described in Europe in 2008. From June to October 2019, we recorded an outbreak of an extensively drug-resistant ST307 lineage in four medical facilities in north-eastern Germany.MethodsHere, we investigated these isolates and those from subsequent cases in the same facilities. We performed whole-genome sequencing to study phylogenetics, microevolution, and plasmid transmission, as well as phenotypic experiments including growth curves, hypermucoviscosity, siderophore secretion, biofilm formation, desiccation resilience, serum survival, and heavy metal resistance for an in-depth characterization of this outbreak clone.ResultsPhylogenetics suggest a homogenous phylogram with several sub-clades containing either isolates from only one patient or isolates originating from different patients, suggesting inter-patient transmission. We identified three large resistance plasmids, carrying either NDM-1, CTX-M-15, or OXA-48, whichK. pneumoniaeST307 likely donated to otherK. pneumoniaeisolates of different STs and even other bacterial species (e.g.,Enterobacter cloacae) within the clinical settings. Several chromosomally and plasmid-encoded, hypervirulence-associated virulence factors (e.g., yersiniabactin, metabolite transporter, aerobactin, and heavy metal resistance genes) were identified in addition. While growth, biofilm formation, desiccation resilience, serum survival, and heavy metal resistance were comparable to several control strains, results from siderophore secretion and hypermucoviscosity experiments revealed superiority of the ST307 clone, similar to an archetypical, hypervirulentK. pneumoniaestrain (hvKP1).ConclusionsThe combination of extensive drug resistance and virulence, partly conferred through a “mosaic” plasmid carrying both antibiotic resistance and hypervirulence-associated features, demonstrates serious public health implications.

Over the past years, the emergence of multidrug-resistant pathogens has become a pressing public health problem. It is an important cause of morbidity and mortality worldwide and puts a considerably financial burden on the health care system. The development of new antimicrobials is becoming increasingly difficult, time-consuming and expensive. Hence, new approaches are urgently needed to replenish our arsenal of antimicrobial agents. Plant tissues contain a wild variety of antimicrobial constituents discovered and used to reverse drugs resistance and restore their efficiency. For this, essential oil (EO) extracted from aerial parts of Senecio anteuphorbium (L) Haw, a Moroccan endemic plant, was studied for the first time for its: (i) Qualitative and quantitative chemical profile, (ii) antimicrobial activities, (iii) and synergistic interactions with two conventional antifungals and ciprofloxacin. The data of the chemical composition showed that S. anteuphorbium EO was characterized by the abundance of γ-selinene (27.2%), cyperene (21.7%), γ-cadinene (11.4%) and α-cyperone (8.1%). Regarding antimicrobial assays, results demonstrated that S. anteuphorbium EO displayed moderate antimicrobial activities with MIC values in the range of 0.51 mg/mL and 1.02 mg/mL for yeasts and between 2.3 mg/mL and 4.6 mg/mL for bacteria. However, the combination of the studied EO with fluconazole and ciprofloxacin showed a significant decrease in their individual MICs. In fact, at sub-inhibitory concentrations S. anteuophorbium EO contributed to the decrease of fluconazole MICs of Candida strains by 4- to 64- fold and by 2- to 16- fold regarding ciprofloxacin MICs. The overall data suggest that the topical or systemic use of S. anteuphorbium EO in combinational treatments may be a promising strategy to combat infectious diseases and further studies should be performed in order to well determine the main mechanism of these synergistic associations.

Antibiotic-resistant Enterobacteriaceae are regularly detected in livestock. As pathogens, they cause difficult-to-treat infections and, as commensals, they may serve as a source of resistance genes for other bacteria. Slaughterhouses produce significant amounts of wastewater containing antimicrobial-resistant bacteria (AMRB), which are released into the environment. We analyzed the wastewater from seven slaughterhouses (pig and poultry) for extended-spectrum β-lactamase (ESBL)-carrying and colistin-resistant Enterobacteriaceae. AMRB were regularly detected in pig and poultry slaughterhouse wastewaters monitored here. All 25 ESBL-producing bacterial strains (19 E. coli and six K. pneumoniae) isolated from poultry slaughterhouses were multidrug-resistant. In pig slaughterhouses 64% (12 of 21 E. coli [57%] and all four detected K. pneumoniae [100%]) were multidrug-resistant. Regarding colistin, resistant Enterobacteriaceae were detected in 54% of poultry and 21% of pig water samples. Carbapenem resistance was not detected. Resistant bacteria were found directly during discharge of wastewaters from abattoirs into water bodies highlighting the role of slaughterhouses for environmental surface water contamination.

The NorA efflux pump is one of the most studied efflux systems in Staphylococcus aureus and confers multidrug resistance to a variety of dyes and antimicrobial compounds. Hence, inhibition of the NorA efflux pump might be a viable option for restoring susceptibility to antibiotics like fluoroquinolones. Fluorescent real-time efflux assays are important tools to identify putative efflux pump inhibitors. Nevertheless, the number of available compounds for usage in Staphylococcus aureus is limited. Previously, a 3-dipropyloxacarbocyanine iodide (DiOC

Summary Background An outbreak of extensively-drug-resistant Klebsiella pneumoniae strain ST307 in a cluster of hospitals in north-east Germany gave rise to the assumption that the epidemiological success of the strain could be based on increased tolerance to biocides. Methods The tolerance of the outbreak strain was compared with epidemiologically unrelated clinical isolates of K. pneumoniae, and reference strains of Pseudomonas aeruginosa (ATCC 15442) and Escherichia coli K12 (NCTC 10538). Tests were performed in a miniaturized assay based on European Standard EN 1040. The widely used biocides benzalkonium chloride (BAC) and didecyl dimethyl ammonium chloride (DDAC), their commercial formulation Descosept spezial (DS), and the antiseptic agent chlorhexidine digluconate (CHG) were selected as test substances. These biocides are used regularly in the hospitals involved in the outbreak. Findings All biocides had a bactericidal effect against all tested strains in the quantitative suspension test within 5 min at typically used concentrations and dilutions. The effectiveness of BAC and DDAC alone and in combination, and CHG antisepsis were not impaired under tested conditions. Conclusion The outbreak strain did not show significantly increased tolerance towards biocides regarding the antiseptic. Thus, the epidemiological success of the strain has to be ascribed to other causes, such as inadequate hand hygiene of visitors.

Escherichia coli KI683 was isolated from blood of a patient who developed septicemia as a complication of a urinary tract infection. Genome sequencing resulted in three contigs with a total genome size of 5,243,173 bp encoding 5,143 genes.

A Raman-based, strain-independent, semi-automated method is presented that allows the rapid (

From June to October 2019, 17 patients (six infected, 11 colonised) with an extensively drug-resistant (XDR) Klebsiella pneumoniae strain were notified from four Western Pomerania medical facilities. The XDR K. pneumoniae produced carbapenemases NDM-1 and OXA-48, and was only susceptible to chloramphenicol, tigecycline and cefiderocol. Synergistic activity was observed for the combination of aztreonam plus ceftazidime-avibactam. Genomic analyses showed all isolates belonged to K. pneumoniae sequence type 307. Control measures and further investigations are ongoing.

The use of molecular assays to rapidly identify pathogens and resistance genes directly from positive blood cultures (BCs) contribute to shortening the time required for the diagnosis of bloodstream infections. In this work, loop-mediated isothermal amplification (LAMP) assays have been examined for their potential use in BC diagnosis. Three different assays were applied. The commercially available eazyplex® MRSA test detects Staphylococcus aureus, S. epidermidis, mecA, and mecC. Two in-house assays [Gram-positive (GP) and Gram-negative (GN)] have been developed for the detection of streptococci, enterococci, vanA, vanB, Pseudomonas spp., Enterobacteriaceae, and the bla CTX-M family. A total of 370 positive BCs were analyzed. LAMP test results were obtained within 30 min, including sample preparation. Amplification was measured by real-time fluorescence detection. The threshold time for fluorescence intensity values ranged from 6.25 to 13.75 min. The specificity and sensitivity of the assays varied depending on the target. Overall, from 87.7% of BCs, true-positive results were obtained, compared to routine standard diagnosis. Twenty-one tests were true-negative because of the lack of an appropriate target (5.7%). The concordance of positive test results for resistance genes with subsequent antibiotic susceptibility testing was 100%. From 15 BC bottles with mixed cultures, eazyplex® assays produced correct results in 73% of the cases. This study shows that LAMP assays are fast and cost-saving tools for rapid BC testing in order to expedite the diagnostic report and improve the antibiotic stewardship for sepsis patients.

Phe-Arg-β-naphthylamide (PAβN) has been characterized as an efflux pump inhibitor (EPI) acting on the major multidrug resistance efflux transporters of Gram-negative bacteria, such as AcrB in Eschericha coli. In the present study, in vitro random mutagenesis was used to evolve resistance to the sensitizing activity of PAβN with the aim of elucidating its mechanism of action. A strain was obtained that was phenotypically similar to a previously reported mutant from a serial selection approach that had no efflux-associated mutations. We could confirm that acrB mutations in the new mutant were unrelated to PAβN resistance. The next-generation sequencing of the two mutants revealed loss-of-function mutations in lpxM. An engineered lpxM knockout strain showed up to 16-fold decreased PAβN activity with large lipophilic drugs, while its efflux capacity, as well as the efficacy of other EPIs, remained unchanged. LpxM is responsible for the last acylation step in lipopolysaccharide (LPS) synthesis, and lpxM deficiency has been shown to result in penta-acylated instead of hexa-acylated lipid A. Modeling the two lipid A types revealed steric conformational changes due to underacylation. The findings provide evidence of a target site of PAβN in the LPS layer, and prove membrane activity contributing to its drug-sensitizing potency.

Rapid diagnosis of bloodstream infections remains a challenge for the early targeting of an antibiotic therapy in sepsis patients. In recent studies, the reliability of the Nanosphere Verigene Gram-positive and Gram-negative blood culture (BC-GP and BC-GN) assays for the rapid identification of bacteria and resistance genes directly from positive BCs has been demonstrated. In this work, we have developed a model to define treatment recommendations by combining Verigene test results with knowledge on local antibiotic resistance patterns of bacterial pathogens. The data of 275 positive BCs were analyzed. Two hundred sixty-three isolates (95.6%) were included in the Verigene assay panels, and 257 isolates (93.5%) were correctly identified. The agreement of the detection of resistance genes with subsequent phenotypic susceptibility testing was 100%. The hospital antibiogram was used to develop a treatment algorithm on the basis of Verigene results that may contribute to a faster patient management.

Sepsis guidelines recommend obtaining blood cultures before starting anti-infective therapy in patients with sepsis. However, little is known of how antibiotic treatment before sampling affects bacterial growth. The aim of this study was to compare the results of blood cultures drawn before and during antibiotic therapy.Prospective clinical cohort study of septic patients. Adult intensive care unit patients with two or three blood culture sets at the beginning of sepsis between 2010 and 2017 were included. Patients with blood culture samples obtained before antibiotic therapy were compared with patients with samples taken during antibiotic therapy. Blood culture positivity, defined as presence of a microbiological pathogen, was compared between the groups. Logistic regression was performed to adjust the impact of different factors with respect to blood culture positivity.In total, 559 patients with 1364 blood culture sets at the beginning of sepsis were analysed. Blood culture positivity was 50.6% (78/154) among patients with sepsis who did not receive antibiotics and only 27.7% (112/405) in those who were already receiving antibiotics (p0.001). Logistic regression revealed antibiotic therapy as an independent factor for less pathogen identification (odds ratio 0.4; 95% CI 0.3-0.6). Gram-positive pathogens (28.3% (111/392) versus 11.9% (116/972); p0.001) and also Gram-negative pathogens (16.3% (64/392) versus 9.3% (90/972); p0.001) were more frequent in blood culture sets drawn before antibiotic therapy compared with sets obtained during antibiotic therapy.Obtaining blood cultures during antibiotic therapy is associated with a significant loss of pathogen detection. This strongly emphasizes the current recommendation to obtain blood cultures before antibiotic administration in patients with sepsis.

Aim To describe the indication and technique of microbiological wound investigation in treating infected or colonised wounds with or without multi-drug resistant microorganisms (MDROs). The clinical symptoms and criteria of locally or systemically infected wounds are summarized and described. Method An interdisciplinary consensus of microbiological wound diagnosis was elaborated between representatives of different medical specialities. Moreover, some perspectives on future wound diagnostics were presented. Results The panel agreed on ten clinical signs, situations and symptoms, which give the indication for further microbiological wound investigation: formation of pus, clinical signs or suspicion of an infection, suspicion or presence of a surgical site infection, localised progredient infection with or without systemic involvement (e.g. joint empyema, phlegmone), gangrene formation and necrotising fasciitis, ulcerative neoplasia, burn wounds with > 15% thermally injured surface area, exacerbation of dermatological disorders, localised wound infection without sign of systemic involvement after 3 days treatment with antiseptics without obvious clinical improvement, and chronic skin lesion with indication to screen for MDROs. The panel agreed that no indication for microbiological investigation is given in acute wounds without signs of infection and uncomplicated superficial wounds. Today, the gold standard for microbiological investigation is processing tissue biopsies in the laboratory. However, in many instances it may be easier and more rapid to perform a wound swab. If swabs are obtained, the Essener circle or the Levine technique shall be applied. Wound swabs, however, are the method of choice in screening for wound colonisation with MDROs. Additionally, information is summarised pertaining to transport and processing of microbiological wound specimens Also, potential applicability of future wound diagnostic methods based on genomic, metabolomic and proteomic approach are discussed. Conclusion Beside a full understanding of all clinical symptoms and clinical experience of wound experts the correct microbiological wound investigation will be the basis for a succesfull management of infected wounds.

Resistant bacteria are spreading worldwide, which makes fast antibiotic susceptibility testing and determination of the minimal inhibitory concentration (MIC) urgently necessary to select appropriate antibiotic therapy in time and, by this, improve patient's outcome and, at the same time, avoid inappropriate treatment as well as the unnecessary use of broad spectrum antibiotics that would foster further spread of resistant bacteria. Here, a simple and fast Raman spectroscopy-based procedure is introduced to identify antimicrobial susceptibilities and determine the MIC within only 2 h total analysis, marking a huge time savings compared to established phenotypic methods nowadays used in diagnostics. Sample preparation is fast and easy as well as comparable to currently established tests. The use of a dielectrophoresis chip allows automated collection of the bacteria in a micron-sized region for high-quality Raman measurement directly from bacterial suspensions. The new Raman spectroscopic MIC test was validated with 13 clinical E. coli isolates that show a broad range of ciprofloxacin resistance levels and were collected from patients with blood-stream infection. Micro-Raman spectroscopy was able to detect ciprofloxacin-induced changes in E. coli after only 90 min interaction time. Principal component analysis as well as a simple computed ratio of the Raman marker bands at 1458 and 1485 cm-1 show a clear concentration-dependent effect. The MIC values determined with the new Raman method are in good agreement with MICs obtained by reference methods (broth microdilution, Vitek-2, E-test) and can be used to provide a classification as sensitive, intermediate, or resistant using the clinical breakpoints provided by EUCAST.

Efflux is an important mechanism of bacterial multidrug resistance (MDR), and the inhibition of MDR pumps by efflux pump inhibitors (EPIs) could be a promising strategy to overcome MDR. 1-(1-Naphthylmethyl)-piperazine (NMP) and phenylalanine-arginine-β-naphthylamide (PAβN) are model EPIs with activity in various Gram-negative bacteria expressing AcrB, the major efflux pump of Escherichia coli , or similar homologous pumps of the resistance-nodulation-cell division class. The aim of the present study was to generate E. coli AcrB mutants resistant to the inhibitory action of the two model EPIs and to identify putative EPI target residues in order to better understand mechanisms of pump inhibition. Using an in vitro random mutagenesis approach focusing on the periplasmic domain of AcrB, we identified the double mutation G141D N282Y, which substantially compromised the synergistic activity of NMP with linezolid, was associated with similar intracellular linezolid concentrations in the presence and absence of NMP, and did not impair the intrinsic MICs of various pump substrates and dye accumulation. We propose that these mutations near the outer face of the distal substrate binding pocket reduce NMP trapping. Other residues found to be relevant for efflux inhibition by NMP were G288 and A279, but mutations at these sites also changed the susceptibility to several pump substrates. Unlike with NMP, we were unable to generate AcrB periplasmic domain mutants with resistance or partial resistance to the EPI activity of PAβN, which is consistent with the modes of action of PAβN differing from those of NMP.

Introduction To determine whether on-site incubation of blood cultures at the intensive care unit (ICU) improves not only the time to incubation but also time to positivity, time to knowledge of positivity and time to results (identification and antibiotic susceptibility testing). Methods This observational single-centre study in ICU patients with severe sepsis and septic shock investigated the impact of blood culture incubation immediately on-site at the ICU (ICU group) by comparison with traditional processing in a remote laboratory (LAB group) on different time intervals of blood culture diagnostics from obtaining blood to clinician notification of final result. The effect of on-site incubation was evaluated in Kaplan-Meier estimates for the time to positivity, time to knowledge of positivity and time to microbiological results and a linear mixed model was built. Results A total of 3,549 blood culture sets from 657 ICU patients were analysed: 2,381 in the LAB group and 1,168 in the ICU group. Overall, 660 (18.6%) blood culture sets were positive and 2,889 (81.4%) sets remained negative. On-site incubation was associated with reduced time to knowledge of positivity (46.9 h [CI 43.4–50.8 h] vs. 28.0 h [CI 23.6–32.2 h], p < 0.001) and reduced time to result (61.4 h [CI 58.4–64.8 h] vs. 42.1 h [CI 39.1–47.5 h], p < 0.001). In blood cultures processed instantaneously at the ICU compared to incubation in the remote laboratory within 4 h, the time to microbiological result was significantly reduced by 8.5 h (p < 0.001). Pre-existing anti-infective therapy had no significant impact on diagnostic time intervals. Conclusions Instantaneous incubation of blood cultures in the ICU compared to incubation in a remote laboratory significantly improves time to knowledge to positivity and time to result. These effects are even more pronounced during off-hours of the microbiological laboratory. The results underline the importance of 24/7 diagnostics to provide round-the-clock processing of blood culture samples in patients with sepsis and septic shock and an immediate to communication of the results to the clinicians.

Efflux pump inhibitors (EPIs) are attractive compounds to reverse multidrug-resistance in clinically relevant bacterial pathogens. In this study we tested the ability of the neuroleptic drug pimozide to inhibit the Escherichia coli AcrAB-TolC efflux pump, whose overproduction confers resistance to various antimicrobial agents. A real-time Nile red efflux assay in the AcrAB – overproducing strain 3-AG100 revealed that pimozide was capable of full inhibition of this pump at a concentration of 100 µM, which is far below its intrinsic MIC (>1mM). However, MIC assay demonstrated very little effect of pimozide with regard to reduction in MICs of various antimicrobial compounds. Only oxacillin MICs were reduced twofold in the presence of pimozide at 100 and 200 µM. Since pimozide did considerably enhance accumulation of ethidium bromide in a fluorescence assay, ethidium bromide MIC assays in the presence and absence of this putative EPI were performed. They revealed that pimozide was able to reduce the MICs of ethidium bromide by 4-fold. In line with previous reports we suggest that the capability of EPIs to restore the susceptibility to antimicrobial agents can be highly substrate-specific due to different substrate binding sites.

AcrAB-TolC is the major efflux protein complex in Escherichia coli extruding a vast variety of antimicrobial agents from the cell. The inner membrane component AcrB is a homotrimer, and it has been postulated that the monomers cycle consecutively through three conformational stages designated loose (L), tight (T), and open (O) in a concerted fashion. Binding of drugs has been shown at a periplasmic deep binding pocket in the T conformation. The initial drug-binding step and transport toward this drug-binding site has been elusive thus far. Here we report high resolution structures (1.9–2.25 Å) of AcrB/designed ankyrin repeat protein (DARPin) complexes with bound minocycline or doxorubicin. In the AcrB/doxorubicin cocrystal structure, binding of three doxorubicin molecules is apparent, with one doxorubicin molecule bound in the deep binding pocket of the T monomer and two doxorubicin molecules in a stacked sandwich arrangement in an access pocket at the lateral periplasmic cleft of the L monomer. This access pocket is separated from the deep binding pocket apparent in the T monomer by a switch-loop. The localization and conformational flexibility of this loop seems to be important for large substrates, because a G616N AcrB variant deficient in macrolide transport exhibits an altered conformation within this loop region. Transport seems to be a stepwise process of initial drug uptake in the access pocket of the L monomer and subsequent accommodation of the drug in the deep binding pocket during the L to T transition to the internal deep binding pocket of the T monomer.

Real-time fluorescent efflux assays are commonly used for measuring the efflux of bacterial pumps. Here we describe an optimized protocol for the NorA efflux pump in S. aureus using DiOC3 instead of ethidium bromide. Glucose and sodium formate were tested as energy carriers. This novel method is fast and reproducible.

article i nfo The AcrA/AcrB/TolC complex is responsible for intrinsic multidrug resistance (MDR) in Escherichia coli. Together with the periplasmic adaptor protein AcrA and the outer membrane channel TolC, the inner membrane component AcrB forms an efflux complex that spans both the inner and outer membrane and bridges the periplasm of the Gram-negative cell. Within the entire tripartite complex, homotrimeric AcrB plays a central role in energy transduction and substrate selection. In vitro selected designed ankyrin repeat proteins (DARPin) that specifically bind to the periplasmic domain of AcrB were shown to ameliorate diffraction resolution of AcrB/DARPin protein co-crystals (G. Sennhauser, P. Amstutz, C. Briand, O. Storchenegger, M.G. Grutter, Drug export pathway of multidrug exporter AcrB revealed by DARPin inhibitors, PLoS Biol 5 (2007) e7). Structural analysis by X-ray crystallography revealed that 2 DARPin molecules were bound to the trimeric AcrB wildtype protein in the crystal, whereas the V612F and G616N AcrB variant crystal structures show 3 DARPin molecules bound to the trimer. These specific stoichiometric differences were analyzed in solution via densitometry after microchannel electrophoresis, analytical ultracentrifugation and via laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS). Using the latter technology, we investigated the gradual disassembly of the AcrB trimer and bound DARPin ligands in dependence on laser intensity in solution. At low laser intensity, the release of the detergent molecule micelle from the AcrB/ DARPin complex was observed. By increasing laser intensity, dimeric and monomeric AcrB species with bound DARPin molecules were detected showing the high affinity binding of DARPin to monomeric AcrB species. High laser intensity LILBID MS experiments indicated a spectral shift of the monomeric AcrB peak of 3.1 kDa, representing a low molecular weight ligand in all detergent-solubilized AcrB samples and in the AcrB crystal. The identity of this ligand was further investigated using phospholipid analysis of purified AcrB and AcrB variant samples, and indicated the presence of phosphatidylethanolamine and possibly cardiolipin, both constituents of the Escherichia coli membrane.

To evaluate the antimicrobial and synergistic (hypothetically due to the inhibition of efflux pumps) effects of selective serotonin reuptake inhibitors (SSRIs) in Escherichia coli strains overproducing various resistance-nodulation-division (RND) efflux pumps.MICs of various SSRIs and of clinically relevant antibiotics in the presence and absence of sertraline were determined for E. coli strains overproducing the RND efflux pumps AcrAB, AcrEF, MdtEF and MexAB. The effect of sertraline on Nile red efflux was evaluated in a real-time efflux assay. Expression of marA and acrB was monitored using quantitative RT-PCR.In MIC assays there was limited synergy of sertraline with tetracycline, oxacillin, linezolid and clarithromycin, depending on the individual pump overexpressed and on whether rich or minimal medium was used. Sertraline, as the most potent SSRI with regard to bacterial growth inhibition, led to rapid dose-dependent Nile red efflux inhibition, and was also found to increase the expression of marA and acrB.A possible explanation for the discrepancy between the MIC and real-time efflux assays was that sertraline is a weak inducer of marA and acrB, thereby reducing its initial antibacterial and sensitizing effects over time. The results indicate that sertraline may be useful as a model efflux pump inhibitor for in vitro short-term experiments in E. coli, but is unlikely to be clinically useful as a co-drug against Gram-negative bacteria.

In this study, we tested five compounds belonging to a novel series of piperazine arylideneimidazolones for the ability to inhibit the AcrAB-TolC efflux pump. The biphenylmethylene derivative (BM-19) and the fluorenylmethylene derivative (BM-38) were found to possess the strongest efflux pump inhibitor (EPI) activities in the AcrAB-TolC-overproducing Escherichia coli strain 3-AG100, whereas BM-9, BM-27, and BM-36 had no activity at concentrations of up to 50 μM in a Nile red efflux assay. MIC microdilution assays demonstrated that BM-19 at 1/4 MIC (intrinsic MIC, 200 μM) was able to reduce the MICs of levofloxacin, oxacillin, linezolid, and clarithromycin 8-fold. BM-38 at 1/4 MIC (intrinsic MIC, 100 μM) was able to reduce only the MICs of oxacillin and linezolid (2-fold). Both compounds markedly reduced the MIC of rifampin (BM-19, 32-fold; and BM-38, 4-fold), which is suggestive of permeabilization of the outer membrane as an additional mechanism of action. Nitrocefin hydrolysis assays demonstrated that in addition to their EPI activity, both compounds were in fact weak permeabilizers of the outer membrane. Moreover, it was found that BM-19, BM-27, BM-36, and BM-38 acted as near-infrared-emitting fluorescent membrane probes, which allowed for their use in a combined influx and efflux assay and thus for tracking of the transport of an EPI across the outer membrane by an efflux pump in real time. The EPIs BM-38 and BM-19 displayed the most rapid influx of all compounds, whereas BM-27, which did not act as an EPI, showed the slowest influx.

Background Multidrug-resistant Escherichia coli and other enteric bacteria producing extended-spectrum β-lactamases (ESBL) have emerged as an important cause of invasive infection. Targeting the primary (intestinal) niche by decolonization may be a valuable approach to decrease the risk of relapsing infections and to reduce transmission of ESBL-producing enteric pathogens. Methods In a retrospective observational study we evaluated the efficacy of intestinal decolonization treatment using orally administered colistin or other non-absorbable agents given for 2 to 4 weeks in adult patients with previous relapsing infection and persistent intestinal colonization with ESBL-positive Enterobacteriaceae (ESBL-E). Eradication success was defined as negative rectal swab or stool culture at the end of treatment and at follow up-2 weeks after treatment discontinuation. Results First-line decolonization treatment led to eradication of ESBL-E in 19/45 patients (42 %, 7/18 low-dose [4 × 1 million units] colistin, 3/12 high-dose [4 × 2 million units] colistin, 9/15 rifaximin [2 × 400 mg]), and secondary/salvage treatment was successful in 8/13 patients (62 %, 20 treatment episodes). Late follow-up showed that 7/13 patients (54 %) with successful initial or salvage decolonization became recolonized within 3 months after post-treatment assessment while all eight of the patients failing initial or salvage decolonization treatment with late follow-up remained colonized. A narrative review of the literature confirms the limited efficacy of non-absorbable antibiotics including conventional selective digestive tract decolonization (SDD)-like combination regimens for eradicating multidrug-resistant enteric bacteria from the intestinal tract. Conclusions At present, there is no clear evidence of a significant decolonization efficacy using single-drug treatment with oral non-absorbable antibiotics. More effective regimens are needed and a better definition of at risk patients is required for planning meaningful randomized controlled studies in this field.

The spread of carbapenem-non-susceptible Klebsiella pneumoniae strains bearing different resistance determinants is a rising problem worldwide. Especially infections with KPC (Klebsiella pneumoniae carbapenemase) - producers are associated with high mortality rates due to limited treatment options. Recent clinical studies of KPC-blood stream infections revealed that colistin-based combination therapy with a carbapenem and/or tigecycline was associated with significantly decreased mortality rates when compared to colistin monotherapy. However, it remains unclear if these observations can be transferred to K. pneumoniae harboring other mechanisms of carbapenem resistance. A three-dimensional synergy analysis was performed to evaluate the benefits of a triple combination with meropenem, tigecycline and colistin against 20 K. pneumoniae isolates harboring different β-lactamases. To examine the mechanism behind the clinically observed synergistic effect, efflux properties and outer membrane porin (Omp) genes (ompK35 and ompK36) were also analyzed. Synergism was found for colistin-based double combinations for strains exhibiting high minimal inhibition concentrations against all of the three antibiotics. Adding a third antibiotic did not result in further increased synergistic effect in these strains. Antagonism did not occur. These results support the idea that colistin-based double combinations might be sufficient and the most effective combination partner for colistin should be chosen according to its MIC.

Objectives 1-(1-Naphthylmethyl)-piperazine (NMP) has been shown to reverse multidrug resistance (MDR) in Escherichia coli overexpressing resistance-nodulation-cell division type efflux pumps, but there is no data on its activity in clinical isolates of E. coli. Methods The antimicrobial susceptibility of 60 clinical isolates of E. coli to a variety of antimicrobial agents was determined in the absence and presence of NMP and, for comparison, of Phe-Arg-beta-naphthylamide (PAbetaN), another putative efflux pump inhibitor (EPI). The intracellular accumulation of ethidium bromide was measured to confirm efflux pump inhibition as the likely mechanism of action of NMP. Results Based on a 4-fold or greater reduction of the MIC after the addition of NMP in >50% of the isolates, significant effects of NMP at a concentration of 100 mg/L were seen for levofloxacin, linezolid and ethidium bromide. The ethidium bromide MIC changes after NMP addition correlated with differences in the ethidium bromide intracellular accumulation as measured by fluorometry in whole cell accumulation experiments. The activity of PAbetaN was different from that of NMP, in particular regarding macrolide resistance reversal, suggesting different modes of action of the two putative EPIs. Conclusions NMP is moderately active in reversing MDR in clinical isolates of E. coli and can partially restore fluoroquinolone susceptibility through inhibition of efflux pumps.

We describe the first isolation of Mycobacterium hassiacum, a rapid-growing, partial acid-resistant mycobacterium, in a respiratory specimen from a patient with exacerbated chronic obstructive pulmonary disease. To provide therapeutic recommendation for future cases, antibiotic susceptibility testing of 3 clinical isolates was performed by broth microdilution. All strains tested showed susceptibility to clarithromycin, imipenem, ciprofloxacin, and doxycycline. The role of M hassiacum as a respiratory pathogen remains unclear and needs to be evaluated by future reports.

To evaluate the importance of phenylalanine residues for substrate transport in the Escherichia coli efflux pump protein AcrB, we subjected Phe-to-Ala binding pocket mutants to a real-time efflux assay with the novel near-infrared lipophilic membrane probe 1,2'-dinaphthylamine (1,2'-DNA). All mutations, with the exception of F617A, led to considerable retardation of efflux. F610A was the point mutation with the most pronounced impact, followed by F628A, F615A, F136A, and F178A. This is the first study to demonstrate the importance of single phenylalanine residues within the AcrB binding pocket for real-time substrate transport.

AcrAB-TolC is the major constitutively expressed efflux pump system that provides resistance to a variety of antimicrobial agents and dyes in Escherichia coli . However, no systematically optimized real-time dye efflux assay has been published for the measurement of its activity and for detection of possible competition between substrates. Here, we report on the development of such an assay using a lipophilic dye, Nile Red. Energy-depleted cells were loaded with the dye in the presence of low (10 μM or less) concentrations of the proton conductor carbonyl cyanide m -chlorophenylhydrazone (CCCP). The CCCP was then removed, and efflux was triggered by energization with glucose. Various known efflux pump inhibitors and antimicrobials were checked for the ability to slow down Nile Red efflux, presumably through competition. Besides the known inhibitors Phe-Arg-β-naphthylamide and 1-naphthyl-methylpiperazine, several tetracyclic compounds (doxorubicin, minocycline, chlortetracycline, doxycycline, and tetracycline) and tetraphenylphosphonium chloride were found to interfere with dye efflux. This inhibition could not be explained by the depletion of proton motive force. None of the other tested antimicrobials, including macrolides, fluoroquinolones, and β-lactams, had any impact on Nile Red efflux, even at concentrations of up to 1 mM.

Objectives: YhiV (MdtF) is an resistance nodulation division (RND) type efflux pump in Escherichia coli with significant homology to AcrB but usually expressed at a low level in clinical isolates. When overexpressed the pump confers decreased susceptibility to a variety of substances including erythromycin and ethidium bromide (EtBr). We characterized two mutants of E. coli E12 (DacrB DacrF) overexpressing yhiV that showed surprising differences in their spectrum of multidrug resistance (MDR). Methods: The two mutants obtained after repeated exposure of E. coli E12 to levofloxacin were tested for antimicrobial susceptibility to a variety of agents and for intracellular accumulation of selected pump substrates. Gene expression was studied by quantitative RT–PCR, and yhiV was sequenced. Gene inactivation and replacement were done by phage l-based homologous recombination. Results: Mutant DKO20/1 overexpressed yhiV, showed a wild-type yhiV sequence and had >2-fold increased MICs of fluoroquinolones, novobiocin, macrolides/ketolides, EtBr, oxacillin and Phe-Arg-bnaphthylamide (PAbN, a putative efflux pump inhibitor) compared with the E12 parent. A second mutant, strain DKO1/17 that had the Val-610!Phe point mutation in YhiV differed from DKO20/1 by faster growth, >2-fold increased MICs of linezolid and tetracycline, but >2-fold decreased MICs of PAbN, azithromycin and telithromycin. Inactivation of yhiV in DKO1/17 and reintroduction of the wild-type and mutant yhiV sequence confirmed that the differing MICs of most of the drugs were associated with the observed single point mutation. Intracellular drug accumulation studies with linezolid and PAbN were consistent with the MIC results. Conclusions: The region around amino acid Val-610 in YhiV appears to be involved in determining recognition and efficiency of export of a number of MDR efflux pump substrates. This single point mutation in the periplasmic loop of the pump can increase resistance to a given drug such as a fluoroquinolone while decreasing resistance to another one.

Objectives: 1-(1-Naphthylmethyl)-piperazine (NMP) has been shown to reverse multidrug resistance (MDR) in Escherichia coli overexpressing RND type efflux pumps, but there is no data on its activity in Enterobacteriaceae other than E. coli. Methods: The antimicrobial susceptibilities of laboratory strains and 167 clinical isolates of Enterobacteriaceae to a variety of antimicrobial agents were determined in the absence and presence of NMP and, for comparison, of Phe-Arg-beta-naphthylamide (PA beta N), another putative efflux pump inhibitor (EPI). A 4-fold or greater reduction of the MIC after EPI addition was considered significant. Results: NMP consistently reduced the MIC of linezolid in Citrobacter freundii, Enterobacter aerogenes and Klebsiella pneumoniae clinical isolates. Significant effects of NMP addition in > 50% of tested isolates were also seen for levofloxacin, tetracycline and chloramphenicol in E. aerogenes, and for levofloxacin and tetracycline in K. pneumoniae, whereas no or minor effects were observed in Serratia marcescens. MDR reversal by NMP was more likely in isolates with decreased susceptibility to fluoroquinolones. In most fluoroquinolone-resistant strains the activity was sufficient to render isolates drug-susceptible at clinically achievable concentrations. The activity of PA beta N was different from that of NMP, suggesting different modes of action of the two putative EPIs. Conclusion: NMP has moderate activity in reversing MDR in many but not all members of the Enterobacteriaceae family including clinical isolates. Its effects on resistance reversal depend on bacterial species and drug, and are different from those seen with PA beta N.

OBJECTIVES 1-(1-Naphthylmethyl)-piperazine (NMP) has been shown to reverse multidrug resistance (MDR) in Escherichia coli overexpressing RND-type efflux pumps but there are no data on its activity in non-fermenters like Acinetobacter. METHODS Antimicrobial susceptibility in the absence and presence of NMP and, for comparison, phenyl-arginine-beta-naphthylamide (PAbetaN), another putative efflux pump inhibitor (EPI), was tested in laboratory and mutant strains with differing intracellular dye accumulation and expression of adeB, and in clinical isolates of Acinetobacter baumannii. RESULTS Based on a 4-fold or greater MIC reduction, the effects of both EPIs at low concentrations (25 mg/L) were limited. PAbetaN had a highly selective action on the reduction in the MIC of rifampicin and clarithromycin. At a higher concentration of the putative EPIs (100 mg/L), NMP was more active than PAbetaN. This effect was not limited to strains with adeB overexpression, but affected the susceptibility to linezolid, chloramphenicol and tetracycline most, and was enhanced in clinical isolates with reduced fluoroquinolone susceptibility. CONCLUSION NMP can partially reverse MDR in A. baumannii and differs substantially in its activity from that of PAbetaN.

The aim of the present study was to investigate the epidemiologic evolution of fluoroquinolone resistance of E. coli clinical isolates from patients admitted to a hematology-oncology service where fluoroquinolone prophylaxis during neutropenia was recommended as the standard of care for many years but was then discontinued in a trial conducted in patients with acute leukemia. Fluoroquinolones had been shown to decrease the incidence of gram-negative bacteremia in cancer patients with neutropenia, yet it was thought that the emergence of resistance in Escherichia coli and other gram-negative bacteria may have caused a progressive lack of efficacy of fluoroquinolone prophylaxis. Epidemiologic surveillance of fluoroquinolone resistance of E. coli clinical isolates at our cancer center since 1992 showed a continuing influx of new clones not previously observed in the population of cancer patients, an increase in the number of cancer patients per year colonized and/or infected by fluoroquinolone-resistant E. coli (1992-1994, 10-16 patients; 1995-1997, 24-27 patients), and a resistance rate of50% among E. coli bloodstream isolates of hematology-oncology patients. A 6-month fluoroquinolone prophylaxis discontinuation intervention trial in 1998 suggested that despite increasing resistance among E. coli isolates, fluoroquinolone prophylaxis in acute leukemia patients was still effective in the prevention of gram-negative bacteremia (incidence rates, 8% during the pre-intervention period vs. 20% after discontinuation; p0.01). The resumption of fluoroquinolone prophylaxis in acute leukemia patients thereafter decreased the incidence of gram-negative bacteremia to the pre-intervention level (9%; p=0.03), while the proportion of in vitro fluoroquinolone resistance in E. coli bacteremia isolates again increased (from 15% during the intervention period to50% in the post-intervention period). Relative rates of resistance thus were a poor indicator of the potential clinical benefits associated with fluoroquinolone prophylaxis in cancer patients.