Carbapenem-associated multidrug-resistant Acinetobacter baumannii (MDR-Ab) are common among clinical isolates worldwide and are a major therapeutic challenge. Previously it was shown that exogenous polyamines (spermine and spermidine) enhanced susceptibility to β-lactams but induced resistance to polymyxins in Pseudomonas aeruginosa. This study aimed to explore the possible availability of exogenous polyamines in treating carbapenem-associated MDR-Ab. The effects of polyamines on the growth rate of A. baumannii, minimal inhibitory concentrations (MICs) of antibiotics, and time–kill and checkerboard assays were determined. Roles of lipopolysaccharide (LPS) and β-lactamase activity of A. baumannii were also assessed for the polyamine effects. Growth of A. baumannii was unaffected at 4 mM spermine and 16 mM spermidine alone, but was significantly inhibited by a subinhibitory concentration of aztreonam (AZT) (8 μg/mL) and those concentrations of the polyamines. MICs to AZT alone (≥128 μg/mL) were reduced to the range 0.25–8 μg/mL in combination with polyamines in all carbapenem-associated MDR-Ab. MICs to penicillins, but not to ceftazidime and meropenem, were also significantly reduced, whilst MICs to other antibiotics, including polymyxin B, were unaffected in combination with polyamines for all tested A. baumannii. Polyamine effects on AZT were strongly synergistic with bactericidal activity and were retained at concentrations of 5 mM MgCl2 (or CaCl2) or 200 mM NaCl. Roles of LPS and β-lactamase in the polyamine effects were excluded. Overall results suggest that AZT in combination with polyamines may be useful for the treatment of carbapenem-associated MDR-Ab.
Polyamines; β-Lactam susceptibility; Acinetobacter baumannii
Invasive fungal infections (IFIs) have emerged as a major cause of morbidity and mortality among critically ill patients. Cancer patients admitted in the intensive care unit (ICU) have multiple risk factors for IFIs. The vast majority of IFIs in the ICU are due to Candida spp. The incidence of invasive candidiasis (IC) has increased over the last decades, especially in the ICU. A shift in the distribution of Candida spp. from Candida albicans to non-albicans Candida spp. has been observed both in ICUs and oncology units in the last two decades. Timely diagnosis of IC remains a challenge despite the introduction of new microbiology techniques. Delayed initiation of antifungal therapy is associated with increased mortality. Therefore, prediction rules have been developed and validated prospectively in order to identify those ICU patients at high risk for IC and likely to benefit from early treatment. These rules, however, have not been validated in cancer patients. Similarly, major clinical studies on the efficacy of newer antifungals typically do not include cancer patients. Despite the introduction of more potent and less toxic antifungals, mortality of IFIs among cancer patients remains high. In recent years, aspergillosis and mucormycosis have also emerged as significant causes of morbidity and mortality among ICU patients with haematological cancer.
Fungal infections; Cancer patients; Intensive care unit
Diarrhoeagenic Escherichia coli (DEC) are an important cause of diarrhoea in children and are associated with high antibiotic resistance. However, there are few studies on the molecular mechanisms of resistance in this group of bacteria. The aim of this study was to determine the mechanisms associated with antibiotic resistance in the most common phenotypes of DEC. A total of 369 E. coli strains [commensal strains and DEC from children with (‘DEC-diarrhoea’) or without (‘DEC-control’) diarrhoea] isolated from children aged <1 year in periurban districts of Lima, Peru, were analysed. In total, 154 ampicillin-resistant strains (36 commensals, 33 DEC-control and 85 DEC-diarrhoea) were studied by PCR for the most prevalent resistance mechanisms to ampicillin, trimethoprim/sulfamethoxazole (SXT), tetracycline and chloramphenicol as well as for integrase types 1 and 2. In additional, restriction fragment length polymorphism was performed for SXT-resistant strains. Commensal strains were more frequently resistant to nalidixic acid and ciprofloxacin (68% and 28%, respectively) than DEC strains (23% and 2%, respectively) (P < 0.05). DEC-diarrhoea strains were more frequently SXT-resistant (78%) compared with DEC-control strains (65%) and commensal strains (60%) (P < 0.05). The most frequent mechanisms of antibiotic resistance in DEC strains were: for β-lactams, blaTEM (31%; 37/118); for SXT, sul2 (48%; 49/103); for tetracycline, tetA (27%; 23/84); and for chloramphenicol, cat (80%; 28/35). The genes sul1 and dfrA1, related to SXT resistance, were more frequent in the DEC-diarrhoea group (41% and28%, respectively) than in the other two groups (P < 0.05). There was a high diversity of resistance genes in DEC, including symptomatic strains.
Antibiotics; Antibiotic resistance mechanism; Children; Diarrhoeagenic E. coli; Commensal E. coli
Plasma concentrations of antimicrobial drugs have long been used to correlate exposure with effect, yet one cannot always assume that unbound plasma and tissue concentrations are similar. Knowledge about unbound tissue concentrations is important in the development of antimicrobial drugs, since most infections are localised in tissues. Therefore, a clinical microdialysis study was conducted to evaluate the distribution of tedizolid (TR-700), the active moiety of the antimicrobial prodrug tedizolid phosphate (TR-701), into interstitial fluid (ISF) of subcutaneous adipose and skeletal muscle tissues following a single oral 600 mg dose of tedizolid phosphate in fasting conditions. Twelve healthy adult subjects were enrolled. Two microdialysis probes were implanted into the thigh of each subject, one into the vastus medialis muscle and one into subcutaneous adipose tissue. Probes were calibrated using retrodialysis. Dialysate samples were collected every 20 min for 12 h following a single oral dose of 600 mg tedizolid phosphate, and blood samples were drawn over 24 h. Unbound tedizolid levels in plasma were similar to those in muscle and adipose tissue. The ratios of unbound (free) AUC in tissues over unbound AUC in plasma (fAUCtissue/fAUCplasma) were 1.1 ± 0.2 and 1.2 ± 0.2 for adipose and muscle tissue, respectively. The median half-life was 8.1, 9.2 and 9.6 h for plasma, adipose tissue and muscle tissue, respectively. Mean protein binding was 87.2 ± 1.8%. The study drug was very well tolerated. The results of this study show that tedizolid distributes well into ISF of adipose and muscle tissues. Unbound levels of tedizolid in plasma, adipose tissue and muscle tissue were well correlated. Free plasma levels are indicative of unbound levels in the ISF of muscle and adipose tissues.
Microdialysis; Tissue distribution; Tedizolid; Pharmacokinetics
Human β-defensin-3 (HBD3) is a small, cationic, host defence peptide with broad antimicrobial activities and diverse innate immune functions. HBD3 binds to many microbial antigens and, in this study, we hypothesised that the known binding of HBD3 to Porphyromonas gingivalis recombinant haemagglutinin B (rHagB) alters, but does not inhibit, the binding of rHagB to human dendritic cells. To test this, human myeloid dendritic cells were incubated for 5 min with rHagB, HBD3 + rHagB (10:1 molar ratio), HBD3 or 0.1 M phosphate-buffered saline (PBS) (pH 7.2) and were then rapidly fixed and processed for confocal microscopy and ultramicrotomy. rHagB and HBD3 could be detected with primary monoclonal mouse antibody to rHagB (MoAb 1858) or polyclonal rabbit antibody to HBD3 (P241) and secondary fluorescent-labelled anti-mouse or anti-rabbit antibodies (confocal microscopy) or protein A–colloidal gold (immunoelectron microscopy). In cells incubated with rHagB only, fluorescence and protein A–colloidal gold were seen at the cell surface and throughout the cytoplasm. In cells incubated with HBD3 + rHagB, fluorescence was observed only at the cell surface in a ‘string of pearls’ configuration. Overall, these results suggest that HBD3 binding to rHagB alters, but does not inhibit, the binding of rHagB to human myeloid dendritic cells.
Defensins; Human β-defensin-3; HBD3; Porphyromonas gingivalis; Haemagglutinin B; Dendritic cells; Confocal microscopy
Natural antimicrobial peptides (AMPs) are promising candidates for developing a generation of new antimicrobials to meet the challenge of antibiotic-resistant pathogens such as meticillin-resistant Staphylococcus aureus (MRSA). To facilitate the search for new candidates, we have utilised the Antimicrobial Peptide Database (APD), which contains natural AMPs from bacteria, fungi, plants and animals. This study demonstrates the identification of novel templates against MRSA by screening 30 peptides selected from the APD. These peptides are short (<25 residues), cysteine-free, cationic and represent candidates from different biological sources such as bacteria, insects, arachnids, tunicates, amphibians, fish and mammals. Six peptides, including ascaphin-8, database-screened antimicrobial peptide 1 (DASamP1), DASamP2, lycotoxin I, maculatin 1.3 and piscidin 1, were found to exert potent antimicrobial activity against an MRSA USA300 isolate. Although five of the six peptides showed broad-spectrum antibacterial activity, DASamP1 displayed killing of MRSA in vitro but not of Escherichia coli, Bacillus subtilis or Pseudomonas aeruginosa. In addition, DASamP1 suppressed early biofilm formation in a mouse model of catheter-associated MRSA infection. DASamP1 is a novel, short and potent peptide that will be a useful starting template for further developing novel anti-MRSA peptides.
Antimicrobial peptides; Biofilms; Meticillin-resistant Staphylococcus aureus
Salicylidene acylhydrazide compounds have been shown to inhibit bacterial pathogens, including Chlamydia and Neisseria gonorrhoeae. If such compounds could also target HIV-1, their potential use as topical microbicides to prevent sexually transmitted infections would be considerable. We determined the in vitro anti-HIV-1 activity, cytotoxicity and mechanism of action of several salicylidene acylhydrazides.
Inhibitory activity was assessed using TZMbl cells and primary peripheral blood mononuclear cells (PBMCs) as targets for HIV-1 infection. Anti-viral activity was measured against cell-free and cell-associated virus and in vaginal fluid and semen simulants. Since the anti-bacterial activity of salicylidene acylhydrazides is reversible by Fe2+, we determined whether Fe2+ and other cations could reverse the anti-HIV-1 activity of the compounds. We also employed real-time PCR to determine the stage affected in the HIV-1 replication cycle.
We identified four compounds with 50% HIV-1 inhibitory concentrations of 1 to 7 μM. In vitro toxicity varied but was generally limited. Activity was similar against three R5 clade B primary isolates and whether targets for virus replication were TZMbl cells or PBMCs. Compounds inhibited cell-free and cell-associated virus and were active in vaginal fluid and semen simulants. Fe2+, but not other cations, reversed the anti-HIV-1 effect. Finally, inhibitory effect of the compounds occurred at a post-integration step.
We identified salicylidene acylhydrazides with in vitro anti-HIV-1 activity in the μM range. The activity of these compounds against other sexually transmitted pathogens makes them potential candidates to formulate for use as a broad-spectrum topical genital microbicide.
Salicylidene acylhydrazides; HIV; microbicide; iron chelation
In a project to characterise new antibacterial chemotypes from plants, hyperenone A and hypercalin B were isolated from the hexane and chloroform extracts of the aerial parts of Hypericum acmosepalum. The structures of both compounds were characterised by extensive one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy and were confirmed by mass spectrometry. Hyperenone A and hypercalin B exhibited antibacterial activity against multidrug-resistant strains of Staphylococcus aureus, with minimum inhibition concentration ranges of 2–128 mg/L and 0.5–128 mg/L, respectively. Hyperenone A also showed growth-inhibitory activity against Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG at 75 mg/L and 100 mg/L. Neither hyperenone A nor hypercalin B inhibited the growth of Escherichia coli and both were non-toxic to cultured mammalian macrophage cells. Both compounds were tested for their ability to inhibit the ATP-dependent MurE ligase of M. tuberculosis, a crucial enzyme in the cytoplasmic steps of peptidoglycan biosynthesis. Hyperenone A inhibited MurE selectively, whereas hypercalin B did not have any effect on enzyme activity.
Hypericum acmosepalum; Hyperenone A; Hypercalin B; Staphylococcus aureus; Tuberculosis; Peptidoglycan; MurE ligase