AcrB drug-binding pocket substitution confers clinically relevant resistance and altered substrate specificity

The incidence of multidrug-resistant bacterial infections is increasing globally and the need to understand the underlying mechanisms is paramount to discover new therapeutics. The efflux pumps of Gram-negative bacteria have a broad substrate range and transport antibiotics out of the bacterium, conferring intrinsic multidrug resistance (MDR). The genomes of pre- and posttherapy MDR clinical isolates of Salmonella Typhimurium from a patient that failed antibacterial therapy and died were sequenced. In the posttherapy isolate we identified a novel G288D substitution in AcrB, the resistance-nodulation division transporter in the AcrAB-TolC tripartite MDR efflux pump system. Computational structural analysis suggested that G288D in AcrB heavily affects the structure, dynamics, and hydration properties of the distal binding pocket altering specificity for antibacterial drugs. Consistent with this hypothesis, recreation of the mutation in standard Escherichia coli and Salmonella strains showed that G288D AcrB altered substrate specificity, conferring decreased susceptibility to the fluoroquinolone antibiotic ciprofloxacin by increased efflux. At the same time, the substitution increased susceptibility to other drugs by decreased efflux. Information about drug transport is vital for the discovery of new antibacterials; the finding that one amino acid change can cause resistance to some drugs, while conferring increased susceptibility to others, could provide a basis for new drug development and treatment strategies.The incidence of multidrug-resistant (MDR, also used herein for “multidrug resistance”) bacterial infections is increasing, and the 2013 World Economic Forum Global Risks report listed antibiotic-resistant bacteria as one of the greatest threats to human health (1). Resistance-nodulation division (RND) efflux pumps of Gram-negative bacteria confer intrinsic and acquired MDR in clinically relevant infections by exporting antibiotics out of the bacterial cell, allowing bacteria to survive at increased drug concentrations.To date the best-characterized efflux pump is AcrAB-TolC of Escherichia coli, composed of the inner membrane RND antiporter AcrB that functions in a tripartite assembly with a periplasmic adaptor protein, AcrA, and the outer membrane channel, TolC. The AcrB pump is an asymmetric homotrimer whose monomers undergo a functional rotation through three states: access, binding, and extrusion (labeled A, B, and E, respectively) to pump substrates from the periplasm, or outer leaflet of the inner membrane, to outside of the cell (2, 3). Drug binding within AcrB is complex. The distal binding pocket has a phenylalanine-rich region that binds low-molecular-weight drugs and a proximal binding pocket and vestibule bind larger compounds such as erythromycin (4, 5).To date, MDR in clinically relevant infections mediated by RND efflux pumps, including AcrB, has been documented to be due to overexpression of the efflux pump and concomitant increased efflux of antibiotics from the bacterial cell (6). Overproduction of efflux pumps can occur via four mechanisms: (i) mutation of the local repressor gene (7, 8), (ii) mutation in a global regulatory gene (9, 10), (iii) mutation of the promoter region of the efflux pump gene (11), or (iv) insertion elements upstream of the transporter gene (12, 13).This study focused on elucidating the mechanism of resistance in a unique set of clinical isolates collected over the course of a complex Salmonella infection (Fig. S1) (14, 15). The isolates were obtained from a 52-y-old male patient admitted for repair of a leaking abdominal aortic aneurysm graft. Salmonella Typhimurium (L3) was isolated before ciprofloxacin treatment and was susceptible to ampicillin, sulphonamide, trimethoprim, cefuroxime, chloramphenicol, gentamicin, and ciprofloxacin. Over the course of the infection the patient received i.v. ciprofloxacin, oral ciprofloxacin, i.v. ceftazidime, and i.v. aztreonam. Isolates were taken throughout infection, and during treatment MDR strains were isolated. L18, the last MDR strain to be isolated, was from wound drainage fluid when the patient had received no antibiotics for 2 wks. The patient died soon afterward with the infection unresolved. Compared with the pretherapy isolate L3, the MDR posttherapy isolate, L18, was less susceptible to numerous agents, including ciprofloxacin and β-lactams, and accumulated less ciprofloxacin and Hoechst dye (16). This set of isolates has provided a unique opportunity to investigate in vivo evolution of MDR in response to clinically validated courses of antimicrobial treatment.Here, we report that whole genome sequencing revealed a new mechanism of clinically significant MDR selected during therapy: substitution in a transporter protein, which altered the specificity of the efflux pump for antibacterial drugs. The structural impact of the substitution was investigated by molecular dynamics (MD) simulations.     

Efficacy of cardiac resynchronization therapy in very old patients: the Insync/Insync ICD Italian Registry.

AIMS: To assess the effects of cardiac resynchronization therapy (CRT) in > or =80-year-old patients vs. patients <80 years, in terms of clinical, functional, and echocardiographic parameters after 12 month of CRT, survival, and incidence of arrhythmic events. METHODS AND RESULTS: The study population consisted of 1181 CRT patients (85 were > or =80 years old). They were enrolled in a national observational registry and underwent baseline evaluation and periodical follow-up visits. In the overall population, New York Heart Association class and ejection fraction (EF) improved and ventricular diameters decreased. Similar changes were observed in the two groups. In the study population, 157 patients died, 144 (13%) in the <80 years group and 13 (15%) in the > or =80 years group. There was a higher all-cause mortality (log-rank test, P = 0.015) among > or =80 years patients, with a trend towards higher sudden cardiac death (SCD) (P = 0.057), but similar non-SCD (P = 0.293). Using the combined endpoint of SCD or appropriate shock from a defibrillator for ventricular fibrillation, no significant differences resulted between groups (P = 0.455). In both groups, lower EF was associated with higher mortality. CONCLUSION: Cardiac resynchronization therapy demonstrated similar efficacy in patients aged > or =80 years and in those under 80, in terms of clinical and functional parameters and reverse remodelling. Similarly, CRT resulted in comparable effects on death for heart failure and on SCD.     

Unexpected embolization of Teflon pledget in the left main stem during: a Bentall operation

We report the case of an 83-year-old man in whom acute left ventricular failure with ventricular arrhythmic storm developed during a Bentall operation. During re-exploration of the annular and coronary ostial anastomoses, no abnormality was seen, and none of the common sequelae of aortic root replacement was evident. The application of retrograde cardioplegia yielded a Teflon pledget that had migrated into the distal part of the left main stem. The pledget was removed, the anastomoses were reestablished, and the patient recovered uneventfully. This case suggests that left ostial anastomosis re-exploration should be carefully considered when no other cause of coronary insufficiency is obvious, and that retrograde cardioplegia may be useful to detect embolization in the left coronary system.     

Overexpression of the cholesterol-binding protein MLN64 induces liver damage in the mouse

AIM: To examine the in vivo phenotype associated with hepatic metastatic lymph node 64 (MLN64) over-expression. METHODS: Recombinant-adenovirus-mediated MLN64 gene transfer was used to overexpress MLN64 in the livers of C57BL/6 mice. We measured the effects of MLN64 overexpression on hepatic cholesterol content, bile flow, biliary lipid secretion and apoptosis markers. For in vitro studies cultured CHO cells with transient MLN64 overexpression were utilized and apoptosis by TUNEL assay was measured. RESULTS: Livers from Ad.MLN64-infected mice exhibited early onset of liver damage and apoptosis. This response correlated with increases in liver cholesterol content and biliary bile acid concentration, and impaired bile flow. We investigated whether liver MLN64 expression could be modulated in a murine model of hepatic injury. We found increased hepatic MLN64 mRNA and protein levels in mice with chenodeoxycholic acid-induced liver damage. In addition, cultured CHO cells with transient MLN64 overexpression showed increased apoptosis. CONCLUSION: In summary, hepatic MLN64 over- expression induced damage and apoptosis in murinelivers and altered cholesterol metabolism. Further studies are required to elucidate the relevance of these fi ndings under physiologic and disease conditions.     

Variable susceptibility to dynamic coronary obstruction: An elusive link between coronary atherosclerosis and angina pectoris

Growing evidence suggests that dynamic coronary obstructions play an important but elusive role in the genesis of ischemic events. Dynamic coronary obstructions can develop during certain phases of coronary disease as a result of a variable combination of vasoconstriction, arterial wall lesions, and increased thrombotic tendency. In a certain phase of their disease some patients develop dynamic coronary obstruction, while others with a similar degree of fixed atherosclerotic obstruction do not.     

The Osteoprint: A bioinspired two-photon polymerized 3-D structure for the enhancement of bone-like cell differentiation

The need for a better understanding of cell behavior and for exploiting cell functions in various healthcare applications has driven biomedical research to develop increasingly complex fabrication strategies to reproduce the natural biological microenvironment in vitro. Different approaches have led to the development of refined examples of 2- and 3-D structures able to sustain cellular proliferation, differentiation and functionality very similar to those normally occurring in living organisms. One such approach is two-photon polymerization. In this paper, we present a trabecula-like structure (which we have named “Osteoprint”) that resembles to the typical microenvironment of trabecular bone cells. Starting from microtomography images of the trabecular bone, we prepared several Osteoprints through two-photon polymerization and tested the behavior of SaOS-2 bone-like cells cultured on our structures. Interestingly, we found that Osteoprints deeply affect cellular behavior, determining an exit from the cell cycle and an enhancement of osteogenic differentiation. Indeed, we found an up-regulation of the genes involved in SaOS-2 cell maturation and an increase in hydroxyapatite production and accumulation upon SaOS-2 culture on the Osteoprints. The findings we obtained are extremely interesting, and open up new perspectives in “bioinspired” approaches for tissue engineering and regenerative medicine.