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Assessment of vancomycin-resistant Enterococcus faecium (VREfm) prevalence upon hospital admission and analysis of risk factors for colonization.From 2014 to 2018, patients were recruited within 72 hours of admission to seven participating German university hospitals, screened for VREfm and questioned for potential risk factors (prior multidrug-resistant organism detection, current/prior antibiotic consumption, prior hospital, rehabilitation or long-term care facility stay, international travel, animal contact and proton pump inhibitor [PPI]/antacid therapy). Genotype analysis was done using cgMLST typing. Multivariable analysis was performed.In 5 years, 265 of 17,349 included patients were colonized with VREfm (a prevalence of 1.5%). Risk factors for VREfm colonization were age (adjusted OR [aOR], 1.02; 95% CI, 1.01-1.03), previous (aOR, 2.71; 95% CI, 1.87-3.92) or current (aOR, 2.91; 95% CI, 2.60-3.24) antibiotic treatment, prior multidrug-resistant organism detection (aOR, 2.83; 95% CI, 2.21-3.63), prior stay in a long-term care facility (aOR, 2.19; 95% CI, 1.62-2.97), prior stay in a hospital (aOR, 2.91; 95% CI, 2.05-4.13) and prior consumption of PPI/antacids (aOR, 1.29; 95% CI, 1.18-1.41). Overall, the VREfm admission prevalence increased by 33% each year and 2% each year of life. 250 of 265 isolates were genotyped and 141 (53.2%) of the VREfm were the emerging ST117. Multivariable analysis showed that ST117 and non-ST117 VREfm colonized patients differed with respect to admission year and prior multidrug-resistant organism detection.Age, healthcare contacts and antibiotic and PPI/antacid consumption increase the individual risk of VREfm colonization. The VREfm admission prevalence increase in Germany is mainly driven by the emergence of ST117.

Efflux by resistance nodulation cell division transporters, such as AcrAB-TolC in

Gram-negative bacteria partly rely on efflux pumps to facilitate growth under stressful conditions and to increase resistance to a wide variety of commonly used drugs. In recent years, Escherichia coli sequence type 131 (ST131) has emerged as a major cause of extraintestinal infection frequently exhibiting a multidrug resistance (MDR) phenotype. The contribution of efflux to MDR in emerging E. coli MDR clones, however, is not well studied. We characterized strains from an international collection of clinical MDR E. coli isolates by MIC testing with and without the addition of the AcrAB-TolC efflux inhibitor 1-(1-naphthylmethyl)-piperazine (NMP). MIC data for 6 antimicrobial agents and their reversion by NMP were analyzed by principal-component analysis (PCA). PCA revealed a group of 17 MDR E. coli isolates (n = 34) exhibiting increased susceptibility to treatment with NMP, suggesting an enhanced contribution of efflux pumps to antimicrobial resistance in these strains (termed enhanced efflux phenotype [EEP] strains). Only 1/17 EEP strains versus 12/17 non-EEP MDR strains belonged to the ST131 clonal group. Whole-genome sequencing revealed marked differences in efflux-related genes between EEP and control strains, with the majority of notable amino acid substitutions occurring in AcrR, MarR, and SoxR. Quantitative reverse transcription-PCR (qRT-PCR) of multiple efflux-related genes showed significant overexpression of the AcrAB-TolC system in EEP strains, whereas in the remaining strains, we found enhanced expression of alternative efflux proteins. We conclude that a proportion of MDR E. coli strains exhibit an EEP, which is linked to an overexpression of the AcrAB-TolC efflux pump and a distinct array of genomic variations. Members of ST131, although highly successful, are less likely to exhibit the EEP.

Inactivating tolC in multidrug-resistant Escherichia coli with differing sequence types and quinolone resistance-determining mutations reveals remarkably potentiated activity of the first-in-class topoisomerase inhibitors gepotidacin and zoliflodacin. Differences between both structurally unrelated compounds in comparison to fluoroquinolones regarding the selectivity of E. coli RND (resistance-nodulation-cell division)-type transporters, efflux inhibitors, and AcrB porter domain mutations were demonstrated.

Inactivating tolC in multidrug-resistant Escherichia coli with differing sequence types and quinolone resistance-determining mutations reveals remarkably potentiated activity of the first-in-class topoisomerase inhibitors gepotidacin and zoliflodacin. Differences between both structurally unrelated compounds in comparison to fluoroquinolones regarding the selectivity of E. coli RND (resistance-nodulation-cell division)-type transporters, efflux inhibitors, and AcrB porter domain mutations were demonstrated. The findings should reinforce efforts to develop efflux-bypassing drugs and provide AcrB targets with critical relevance for this purpose.

We report a case of severe infection with liver abscess and endophthalmitis caused by a hypervirulent Klebsiella pneumoniae strain in an immunocompetent German male patient without travel history to Asia. Phenotypic and molecular characterization showed high similarity to the reference genome NTUH-K2044 isolated in Asia. The isolate was assigned as ST2398 (clonal complex 66). The findings underline global spread of hypervirulent Klebsiella pneumoniae strains to Europe.

Objectives: This study aimed at determining the prevalence of rifaximin resistance in a large collection of Enterobacterales resistant to third-generation cephalosporins. A simple agar screen was d...

A major contribution of the resistance-nodulation-cell division (RND)-transporter AcrB to resistance to oxazolidinones and pleuromutilin derivatives in Escherichia coli was confirmed. However, we discovered significant differences in efflux inhibitor activities, specificities of the homologous pump YhiV (MdtF), and the impact of AcrB pathway mutations. Particularly, entrance channel double-mutation I38F I671T and distal binding pocket mutation F615A revealed class-specific transport routes of oxazolidinones and pleuromutilin derivatives. The findings could contribute to the understanding of the RND-type multidrug transport pathways.

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.

Immunity to mycobacteria involves the formation of granulomas, characterized by a unique macrophage (MΦ) species, so‐called multinucleated giant cells (MGC). It remains unresolved whether MGC are beneficial to the host, that is, by prevention of bacterial spread, or whether they promote mycobacterial persistence. Here, we show that the prototypical antimycobacterial molecule nitric oxide (NO), which is produced by MGC in excessive amounts, is a double‐edged sword. Next to its antibacterial capacity, NO propagates the transformation of MΦ into MGC, which are relatively permissive for mycobacterial persistence. The mechanism underlying MGC formation involves NO‐induced DNA damage and impairment of p53 function. Moreover, MGC have an unsurpassed potential to engulf mycobacteria‐infected apoptotic cells, which adds a further burden to their antimycobacterial capacity. Accordingly, mycobacteria take paradoxical advantage of antimicrobial cellular efforts by driving effector MΦ into a permissive MGC state.

Drug efflux by resistance-nodulation-cell division (RND)-type transporters, such as AcrAB-ToIC of Escherichia can, is an important resistance mechanism in Gram-negative bacteria; however, its contribution to multidrug resistance (MDR) in clinical isolates is poorly defined. We inactivated acrB of a sequence type 131 E. coli human isolate that showed high-level MDR, but had no mutations within the known efflux associated local or global regulators. The resistance profile of the acrB deletion mutant revealed significantly increased susceptibility to drugs from seven antibiotic classes, including agents usually inactive against Gram-negative bacteria, notably the new oxazolidinone, tedizolid (512-fold enhanced susceptibility). AcrB deficiency reduced, but did not abolish, the efflux of dyes, which indicates the activity of at least one more efflux transporter. The findings demonstrate the efficacy of AcrAB-ToIC-mediated broad-spectrum drug efflux, including agents primarily developed for Gram-positive pathogens, in a clinical isolate representative of a globally-emerging lineage. The results illustrate the need to develop molecules modified to impede their transport by AcrAB-ToIC and its homologues and new efflux inhibitors. (C) 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

There is a lack of data on the predictive value of tuberculosis (TB) specific interferon-gamma release assays (TIGRAs) for both immunocompetent and immunocompromised individuals. We retrospectively followed up 460 such patients after QuantiFERON®-TB Gold In-Tube (QFT-GIT) testing (mean follow-up 28 months) by reviewing patient charts. A total of 1/38 QFT-GIT-positive and no QFT-GIT-negative patients developed active TB. The incidence rate of progression to active TB assessed retrospectively in these patients after positive TIGRA was therefore low (1.2 events/100 person-years). The need for careful follow-up and prophylactic therapy after positive TIGRA in this patient group needs to be evaluated prospectively.

To the Editors: Immunocompromised patients with various causes and degrees of immunodeficiencies, such as stem cell and solid organ transplant recipients, patients with autoimmune diseases, patients with chronic renal failure or HIV-positive patients, are at increased risk of progression from latent Mycobacterium tuberculosis infection to active disease. Therefore, screening for latent tuberculosis and preventive treatment is recommended in this patient population. Tuberculosis-specific interferon (IFN)-γ release assays (TIGRAs) lacking cross-reactivity with Mycobacterium bovis bacille Calmette-Guerin have been introduced into the routine diagnosis of latent tuberculosis infection (LTBI) in the last few years as a more specific alternative to tuberculin skin test (TST). More recent results implicate that QuantiFERON®-TB Gold in tube (QFT-G-IT; Cellestis, Carnegie, Australia) may better predict progression from latent to active disease compared with TST 1, 2. TIGRAs using stimulation of T-cells with phytohemagglutinin (PHA) as a positive control for identification of false negatives and the classification of indeterminate results may be a better alternative for the prediction of LTBI in immunocompromised patients where IFN-γ release may be affected by immunosuppression. The growing list of data existing on reliability of TIGRAs in immunocompromised patients show that the prevalence of indeterminate results may vary depending on the degree of immunosuppression and the TIGRA test used 3. In addition, PHA and recall antigens use different IFN-γ secretion pathways 4, which may be differentially affected by immunosuppressive conditions. Thus, an in-depth analysis of factors influencing PHA-associated IFN-γ secretion is important for the assessment of TIGRAs in immunocompromised patients. Since disease group dependent and independent risk factors of indeterminate results in immunocompromised patients have not been evaluated prospectively using the third generation QFT-G-IT test, we tested QFT-G-IT in patients with diverse conditions of immunosuppression to determine the rate of and identify risk factors for indeterminate results. After approval of …

Efflux pumps of the resistance nodulation cell division (RND) transporter family, such as AcrB of Escherichia coli , play an important role in the development of multidrug resistance, but the molecular basis for their substrate promiscuity is not yet completely understood. From a collection of highly clarithromycin-resistant AcrB periplasmic domain mutants derived from in vitro random mutagenesis, we identified variants with an unusually altered drug resistance pattern characterized by increased susceptibility to many drugs of lower molecular weight, including fluoroquinolones, tetracyclines, and oxazolidinones, but unchanged or increased resistance to drugs of higher molecular weight, including macrolides. Sequencing of 14 such “divergent resistance” phenotype mutants and 15 control mutants showed that this unusual phenotype was associated with mutations at residues I38 and I671 predominantly to phenylalanine and threonine, respectively, both conferring a similar susceptibility pattern. Reconstructed I38F and I671T single mutants as well as an engineered I38F I671T double mutant with proved efflux competence revealed an equivalent phenotype with enhanced or unchanged resistance to many large AcrB substrates but increased susceptibility to several lower-molecular-weight drugs known to bind within the distal binding pocket. The two isoleucines located in close vicinity to each other in the lower porter domain of AcrB beneath the bottom of the proximal binding pocket may be part of a preferential small-drug entrance pathway that is compromised by the mutations. This finding supports recent indications of distinct entrance channels used by compounds with different physicochemical properties, of which molecular size appears to play a prominent role.

Therapy of AIDS patients infected with Mycobacterium avium is problematic due to its intrinsic resistance to antibiotics. We have characterized the efflux pump activity of M. avium wild-type strain through an automated fluorometric method and correlated it with intrinsic resistance to antibiotics.M. avium ATCC 25291(T) and Mycobacterium smegmatis mc(2)155 were evaluated for accumulation and efflux of ethidium bromide in the presence or absence of the efflux pump inhibitors (EPIs) thioridazine, chlorpromazine, verapamil and the proton uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). For this purpose, a new automated fluorometric method was used that separately assesses accumulation and extrusion of ethidium bromide.The automated fluorometric method described in this paper allowed the detection and quantification of ethidium bromide transport across M. avium and M. smegmatis cell walls. Accumulation of ethidium bromide was found to be temperature-dependent and significantly increased by EPIs thioridazine, chlorpromazine, verapamil and CCCP in a concentration-dependent manner. Efflux of ethidium bromide under optimum conditions of temperature and glucose is inhibited by the above agents. At half their intrinsic MICs, both thioridazine and chlorpromazine, similarly to verapamil and CCCP, significantly increased the susceptibility of M. avium to erythromycin, suggesting an effect upon an efflux pump with ethidium bromide and erythromycin as substrates. A similar effect was observed for M. smegmatis with verapamil only.M. avium and M. smegmatis intrinsic resistance is affected by EPIs such as thioridazine or chlorpromazine, an effect that might be important in research and development of new, more effective antimycobacterial therapies.