Cryptosporidium parvum causes an opportunistic infection in AIDS patients, and no effective treatments are yet available. This parasite possesses a single fatty acyl-CoA binding protein (CpACBP1) that is localized to the unique parasitophorous vacuole membrane (PVM). The major goal of this study was to identify inhibitors from known drugs against CpACBP1 as potential new anti-Cryptosporidium agents.
A fluorescence assay was developed to detect CpACBP1 activity and to identify inhibitors by screening known drugs. Efficacies of top CpACBP1 inhibitors against Cryptosporidium growth in vitro were evaluated using a quantitative RT–PCR assay.
Nitrobenzoxadiazole-labelled palmitoyl-CoA significantly increased the fluorescent emission upon binding to CpACBP1 (excitation/emission 460/538 nm), which was quantified to determine the CpACBP1 activity and binding kinetics. The fluorescence assay was used to screen a collection of 1040 compounds containing mostly known drugs, and identified the 28 most active compounds that could inhibit CpACBP1 activity with sub-micromolar IC50 values. Among them, four compounds displayed efficacies against parasite growth in vitro with low micromolar IC50 values. The effective compounds were broxyquinoline (IC50 64.9 μM), cloxyquin (IC50 25.1 μM), cloxacillin sodium (IC50 36.2 μM) and sodium dehydrocholate (IC50 53.2 μM).
The fluorescence ACBP assay can be effectively used to screen known drugs or other compound libraries. Novel anti-Cryptosporidium activity was observed in four top CpACBP1 inhibitors, which may be further investigated for their potential to be repurposed to treat cryptosporidiosis and to serve as leads for drug development.
Cryptosporidium parvum; broxyquinoline; cloxyquin; cloxacillin sodium; sodium dehydrocholate
Host iron availability is fundamental to mucormycosis pathogenesis. The combination of liposomal amphotericin B (LAmB) and deferasirox iron chelation therapy synergistically improved survival in diabetic mice with mucormycosis. To determine the safety of combination deferasirox plus LAmB therapy for mucormycosis, a multicentred, placebo-controlled, double-blinded clinical trial was conducted.
Twenty patients with proven or probable mucormycosis were randomized to receive treatment with LAmB plus deferasirox (20 mg/kg/day for 14 days) or LAmB plus placebo (NCT00419770, clinicaltrials.gov). The primary analyses were for safety and exploratory efficacy.
Patients in the deferasirox arm (n = 11) were more likely than those in the placebo arm (n = 9) to have active malignancy, neutropenia and corticosteroid therapy, and were less likely to receive concurrent non-study antifungal therapy. Reported adverse events and serious adverse events were similar between the groups. However, death was more frequent in the deferasirox than in the placebo arm at 30 days (45% versus 11%, P = 0.1) and 90 days (82% versus 22%, P = 0.01). Global success (alive, clinically stable, radiographically improved) for the deferasirox arm versus the placebo arm at 30 and 90 days, respectively, was 18% (2/11) versus 67% (6/9) (P = 0.06) and 18% (2/11) versus 56% (5/9) (P = 0.2).
Patients with mucormycosis treated with deferasirox had a higher mortality rate at 90 days. Population imbalances in this small Phase II study make generalizable conclusions difficult. Nevertheless, these data do not support a role for initial, adjunctive deferasirox therapy for mucormycosis.
antifungal; fungal infections; mould infections; combination therapy
The use of colistin in the treatment of life-threatening Gram-negative infections is associated with a high rate of nephrotoxicity that is dose limiting. This study aimed to examine the nephroprotective effect of ascorbic acid against colistin-induced nephrotoxicity.
Rats were treated intravenously twice daily with saline, colistin (cumulative dose of 36.5 mg/kg), a combination of ascorbic acid (50 or 200 mg/kg) and colistin, or ascorbic acid (200 mg/kg) over 7 days. Colistin-induced apoptosis was examined in rats over 5 days and in vitro using rat renal proximal tubular cells NRK-52E over 24 h with and without ascorbic acid. The effect of co-administered ascorbic acid on colistin pharmacokinetics was investigated.
The 24 h urinary excretion of N-acetyl-β-d-glucosaminidase, a sensitive marker for tubular damage, was significantly lower (P < 0.0001) in the colistin/ascorbic acid 200 mg/kg group. Significant histological abnormalities (P < 0.01) were detected only in the kidneys of the colistin group, which also had the highest percentage (30.6 ± 7.8%) of apoptotic cells (P < 0.005). In the cell culture studies, the percentage of apoptotic cells was significantly higher in the presence of 0.1 mM colistin alone (51.8 ± 2.0%; P < 0.0001) than in the presence of ascorbic acid, which decreased the apoptotic effect in a concentration-dependent manner. Ascorbic acid (200 mg/kg) altered colistin pharmacokinetics, as the total body clearance decreased from 3.78 ± 0.36 mL/min/kg (colistin group) to 2.46 ± 0.57 mL/min/kg (P = 0.0024).
This is the first study demonstrating the protective effect of ascorbic acid against colistin-induced nephrotoxicity and tubular apoptosis. Co-administration of ascorbic acid has the potential to increase the therapeutic index of colistin.
polymyxin E; vitamin C; rat kidney tubular cells
New classes of drugs are needed to treat tuberculosis (TB) in order to combat the emergence of resistance to existing agents and shorten the duration of therapy. Targeting DNA gyrase is a clinically validated therapeutic approach using fluoroquinolone antibiotics to target the gyrase subunit A (GyrA) of the heterotetramer. Increasing resistance to fluoroquinolones has driven interest in targeting the gyrase subunit B (GyrB), which has not been targeted for TB. The biological activities of two potent small-molecule inhibitors of GyrB have been characterized to validate its targeting as a therapeutic strategy for treating TB.
Materials and methods
Novobiocin and aminobenzimidazole 1 (AB-1) were tested for their activity against Mycobacterium tuberculosis (Mtb) H37Rv and other mycobacteria. AB-1 and novobiocin were also evaluated for their interaction with rifampicin and isoniazid as well as their potential for cytotoxicity. Finally, AB-1 was tested for in vivo efficacy in a murine model of TB.
Novobiocin and AB-1 have both been shown to be active against Mtb with MIC values of 4 and 1 mg/L, respectively. Only AB-1 exhibited time-dependent bactericidal activity against drug-susceptible and drug-resistant mycobacteria, including a fluoroquinolone-resistant strain. AB-1 had potent activity in the low oxygen recovery assay model for non-replicating persistent Mtb. Additionally, AB-1 has no interaction with isoniazid and rifampicin, and has no cross-resistance with fluoroquinolones. In a murine model of TB, AB-1 significantly reduced lung cfu counts in a dose-dependent manner.
Aminobenzimidazole inhibitors of GyrB exhibit many of the characteristics required for their consideration as a potential front-line antimycobacterial therapeutic.
non-replicating bacteria; topoisomerase; benzimidazole; drug resistance; ciprofloxacin; novobiocin; non-tuberculous mycobacteria
Silver carbenes may represent novel, broad-spectrum antimicrobial agents that have low toxicity while providing varying chemistry for targeted applications. Here, the bactericidal activity of four silver carbene complexes (SCCs) with different formulations, including nanoparticles (NPs) and micelles, was tested against a panel of clinical strains of bacteria and fungi that are the causative agents of many skin and soft tissue, respiratory, wound, blood, and nosocomial infections.
MIC, MBC and multidose experiments were conducted against a broad range of bacteria and fungi. Time-release and cytotoxicity studies of the compounds were also carried out. Free SCCs and SCC NPs were tested against a panel of medically important pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Acinetobacter baumannii (MRAB), Pseudomonas aeruginosa, Burkholderia cepacia and Klebsiella pneumoniae.
All four SCCs demonstrated strong efficacy in concentration ranges of 0.5–90 mg/L. Clinical bacterial isolates with high inherent resistance to purified compounds were more effectively treated either with an NP formulation of these compounds or by repeated dosing. Overall, the compounds were active against highly resistant bacterial strains, such as MRSA and MRAB, and were active against the biodefence pathogens Bacillus anthracis and Yersinia pestis. All of the medically important bacterial strains tested play a role in many different infectious diseases.
The four SCCs described here, including their development as NP therapies, show great promise for treating a wide variety of bacterial and fungal pathogens that are not easily killed by routine antimicrobial agents.
antibacterial; antifungal; MIC/MBC; clinical isolates
M. abscessus; metronidazole; PA-824
Candida biofilms, which are often associated with device-related infections, including catheter-related bloodstream infections, are resistant to commonly used antifungal agents. Current microtitre (96-well) plate-based methods to determine the antifungal susceptibility of these biofilms do not involve clinically relevant substrates (e.g. catheters), and are not well suited for evaluating the surface topography and three-dimensional architecture of biofilms. We describe a simple, reproducible catheter-based microtitre plate method to form biofilms and evaluate their antifungal susceptibility.
Biofilms were formed by Candida species on 5 mm catheter discs placed in microtitre plates and quantified using metabolic conversion of a formazan dye (XTT). The morphology, surface topography and three-dimensional architecture of these biofilms were evaluated by fluorescence, confocal scanning laser and scanning electron microscopy, respectively. The optimized XTT method was used to evaluate the antifungal susceptibility of formed Candida biofilms to fluconazole, voriconazole, itraconazole and anidulafungin.
Maximum XTT activity was achieved within 90 min. All tested Candida strains formed robust biofilms on catheter discs at both 24 and 48 h (P = 0.66). Biofilms exhibited typical gross morphology, surface topography and architecture, and no difference in biofilm thickness (P = 0.37). The three tested azoles were not active against the biofilms (MIC ≥64 mg/L), but anidulafungin possessed potent activity against them (MIC = 0.063–0.125 mg/L).
The developed method is simple, rapid and reproducible, and requires relatively small amounts of drug. It can be used to perform both high-resolution microscopic analysis of the topography and architecture of biofilms, and evaluation of their antifungal susceptibility.
biofilm architecture; surface topography; resistance
Smartphone usage amongst clinicians is widespread. Yet smartphones are not widely used for the dissemination of policy or as clinical decision support systems. We report here on the development, adoption and implementation process of the Imperial Antimicrobial Prescribing Application across five teaching hospitals in London.
Doctors and clinical pharmacists were recruited to this study, which employed a mixed methods in-depth case-study design with focus groups, structured pre- and post-intervention survey questionnaires and live data on application uptake. The primary outcome measure was uptake of the application by doctors and its acceptability. The development and implementation processes were also mapped.
The application was downloaded by 40% (376) of junior doctors with smartphones (primary target user group) within the first month and by 100% within 12 months. There was an average of 1900 individual access sessions per month, compared with 221 hits on the Intranet version of the policy. Clinicians (71%) reported that using the application improved their antibiotic knowledge.
Clinicians rapidly adopted the mobile application for antimicrobial prescribing at the point of care, enabling the policy to reach a much wider audience in comparison with paper- and desktop-based versions of the policy. Organizations seeking to optimize antimicrobial prescribing should consider utilizing mobile technology to deliver point-of-care decision support. The process revealed a series of barriers, which will need to be addressed at individual and organizational levels to ensure safe and high-quality delivery of local policy at the point of care.
eHealth; decision support; antimicrobial management; technology adoption
Although candidaemia is a well-known complication of hospital stay and has a crude mortality of ∼40%, few data are available for episodes diagnosed within 10 days after hospital admission. In this paper, we compared the risk factors for mortality according to the onset of candidaemia.
This was a retrospective study of hospitalized patients with early-onset candidaemia (EOC; ≤10 days) or late-onset candidaemia (LOC; >10 days) to identify any distinct clinical characteristics and risk factors for 30 day mortality in two Italian academic centres.
A total of 779 patients were included in the study: 183 EOC and 596 LOC. Mortality was significantly lower in EOC (71/183, 38.8% versus 283/596, 47.5%, P = 0.03). In EOC, multivariate analysis showed that inadequate initial antifungal therapy (IIAT) (P = 0.005, OR 3.02, 95% CI 1.40–6.51), Candida albicans aetiology (P = 0.02, OR 2.17, 95% CI 1.11–4.26) and older age (P < 0.001, OR 1.05, 95% CI 1.02–1.07) were independent risk factors for mortality. In LOC, liver disease (P = 0.003, OR 2.46, 95% CI 1.36–4.43), IIAT (P = 0.002, OR 2.01, 95% CI 1.28–3.15) and older age (P < 0.001, OR 1.03, 95% CI 1.02–1.04) were independently associated with a fatal outcome, while treatment with caspofungin was associated with survival (P < 0.001, OR 0.42, 95% CI 0.26–0.67).
EOC has different clinical characteristics and risk factors for mortality compared with LOC. Although EOC mortality is significantly lower, the rate of inappropriate antifungal treatment is higher. Treatment with caspofungin is significantly associated with survival in patients with LOC. Efforts are needed to improve the diagnosis and treatment of EOC.
caspofungin; risk factors; mortality; healthcare-associated infections; early-onset candidaemia
Despite a decreasing mortality and morbidity in treated HIV-1 patients, highly active antiretroviral treatment (HAART) can still fail due to the development of drug resistance. Especially, multidrug-resistant viruses pose a threat to efficient therapy. We studied the changing prevalence of multidrug resistance (MDR) over time in a cohort of HIV-1-infected patients in Portugal.
Patients and methods
We used data of 8065 HIV-1-infected patients followed from July 2001 up to April 2012 in 22 hospitals located in Portugal. MDR at a specific date of sampling was defined as no more than one fully active drug (excluding integrase and entry inhibitors) at that time authorized by the Portuguese National Authority of Medicines and Health Products (INFARMED), as interpreted with the Rega algorithm version 8.0.2. A generalized linear mixed model was used to study the time trend of the prevalence of MDR.
We observed a statistically significant decrease in the prevalence of MDR over the last decade, from 6.9% (95% CI: 5.7–8.4) in 2001–03, 6.0% (95% CI: 4.9–7.2) in 2003–05, 3.7% (95% CI: 2.8–4.8) in 2005–07 and 1.6% (95% CI: 1.1–2.2) in 2007–09 down to 0.6% (95% CI: 0.3–0.9) in 2009–12 [OR = 0.80 (95% CI: 0.75–0.86); P < 0.001]. In July 2011 the last new case of MDR was seen.
The prevalence of multidrug-resistant HIV-1 is decreasing over time in Portugal, reflecting the increasing efficiency of HAART and the availability of new drugs. Therefore, in designing a new drug, safety and practical aspects, e.g. less toxicity and ease of use, may need more attention than focusing mainly on efficacy against resistant strains.
resistance; drug susceptibility; therapy failure; antiretroviral therapy; AIDS
Long-term chemoprophylaxis using neuraminidase inhibitors may be needed during influenza epidemics but safety data are limited to several weeks. We sought to assess the tolerability of oseltamivir and zanamivir as primary prophylaxis over 16 weeks.
We conducted a parallel group, double blind, 2 (active drug) :1 (placebo) randomized trial of oral oseltamivir/placebo or inhaled zanamivir/placebo over 16 weeks in healthy, Thai hospital professionals at two Bangkok hospitals. The primary endpoint was study withdrawal due to drug-related (possibly, probably, definitely) serious or adverse events (AEs) graded ≥2.
Recruited subjects numbered 129 oseltamivir/65 placebo and 131 zanamivir/65 placebo. A total of 102 grade ≥2 AEs were reported or detected in 69 subjects: 23/129 (17.8%) versus 15/65 (23.1%) (P = 0.26), and 23/131 (17.6%) versus 8/65 (12.3%) (P = 0.28). Intercurrent infections/fevers [26/102 (25.5%)], abnormal biochemistry [25/102 (24.5%)] and gastrointestinal symptoms [18/102 (17.6%)] were the most frequently reported AEs. There were no drug-related study withdrawals. Eight serious AEs were all due to intercurrent illnesses. Laboratory, lung function and ECG parameters were similar between drugs and placebos.
Oseltamivir and zanamivir were well tolerated in healthy hospital professionals. Both drugs can be recommended for primary influenza prophylaxis for up to 16 weeks.
neuraminidase inhibitors; pandemic; adverse event; tolerability
Concern has been raised over the practice of unnecessary double anaerobic coverage therapy (DACT) in the hospital setting. However, the incidence of and risk factors for unnecessary DACT are not well studied. On 8 September 2008, the antimicrobial stewardship programme (ASP) at our institution was modified such that several antibiotics, including ampicillin/sulbactam and metronidazole, no longer required pre-approval. We anticipated that this change would increase both unnecessary DACT and target antibiotic consumption.
A nested case–control study was conducted to determine the cumulative incidence of and risk factors for unnecessary DACT. Cases were subjects who received unnecessary DACT while controls were subjects who did not receive DACT or who received necessary DACT. Segmented regression analysis was subsequently performed to evaluate the impact of ASP changes on unnecessary DACT and consumption of target antibiotics.
From October 2007 to September 2009, the cumulative incidence of unnecessary DACT was 2.3% [95% confidence interval (CI) 1.7–3.1]. Independent risk factors for unnecessary DACT [adjusted odds ratio (95% CI); P value] included hospitalization on a surgical ward [3.51 (1.03–12.02); P = 0.002], hospitalization on an obstetrics and gynaecology ward [9.07 (2.54–32.40); P = 0.002] and underlying metastatic malignancy [3.18 (1.38–7.09); P = 0.006]. The ASP change was associated with an increase in ampicillin/sulbactam and metronidazole consumption. However, there was no significant impact on unnecessary DACT prescribing.
Although uncommon, unnecessary DACT is more prevalent in specific services. Future qualitative studies focusing on these specific subgroups would be useful in elucidating this problem more clearly. The ASP changes were not associated with increases in unnecessary DACT.
ampicillin/sulbactam; metronidazole; antibiotic stewardship programme
The presence of herpes simplex virus-2 (HSV-2) shedding episodes correlates with transmission to sexual partners and neonates, and some episodes correlate with disease manifestations. HSV-2-targeted guanosine analogues are effective when given on a prophylactic basis, but do not completely eliminate recurrences, asymptomatic shedding or transmission. We sought to describe the impact of twice-daily aciclovir and famciclovir on shedding episodes.
We used pooled results from crossover clinical trials to construct frequency histograms for viral shedding episode duration, peak copy number, expansion kinetics and decay kinetics.
Suppressive aciclovir and famciclovir decreased the frequency of episodes of >24 h duration by 50%, lowered the mean early episode expansion rate (from 8.2 to 7.2 HSV DNA logs/day, P = 0.004), decreased the mean peak values for shedding episodes (from 4.9 to 3.9 log10 HSV DNA copies/mL, P < 0.001) and lowered the mean episode duration (from 4.8 to 2.1 days, P < 0.001) primarily by decreasing the probability of viral re-expansion during episodes. The mean rate of late viral decay was similar for persons on and off antiviral medications (−6.0 versus −5.8 HSV DNA logs/day, P = 0.61).
HSV-2-targeted antiviral therapy limits episode severity by decreasing the rate of early viral expansion and the likelihood of episode re-expansion. Late clearance of episodes in the immunocompetent host is not affected by antiviral therapy, suggesting that local immune response is critical for clearance of episodes both on and off treatment.
viral dynamics; aciclovir; famciclovir
There is a general lack of effective and non-toxic chemotherapeutic agents for leishmaniasis and there is as yet no study about the effect of HIV peptidase inhibitors (HIV PIs) on Leishmania/HIV-coinfected patients. In the present work, we performed a comparative analysis of the spectrum of action of HIV PIs on different Leishmania spp., including strains obtained from HIV-positive patients receiving or not receiving antiretroviral treatment.
The effects of nelfinavir and saquinavir on Leishmania proliferation were assessed by means of a colorimetric assay (MTT). Subsequently, the effect of nelfinavir on aspartic peptidase activity from Leishmania spp. was assessed by following the degradation of the fluorogenic substrate MCA-G-K-P-I-L-F-F-R-L-K-DNP-Arg-NH2.
Nelfinavir was capable of significantly reducing the multiplication of many Leishmania reference strains and isolates obtained from HIV-positive patients receiving or not receiving antiretroviral treatment. Leishmania major growth was inhibited by ∼50%, while all other flagellates were strongly inhibited (at least 94%), except for a Leishmania chagasi strain obtained from an HIV-positive patient under treatment with highly active antiretroviral therapy (HAART). Culture of this isolate in the presence of nelfinavir induced a considerable reduction in the aspartic peptidase activity. In addition, nelfinavir was also capable of inhibiting the aspartic peptidase activity of all Leishmania strains tested.
The present data contribute to the study of the effect of HIV PIs on Leishmania infection and add new insights into the possibility of exploiting aspartic peptidases as promising targets in order to generate novel medications to treat leishmaniasis.
aspartyl peptidases; Leishmania/HIV coinfection; HIV peptidase inhibitors; chemotherapy; leishmaniasis; proteases
To determine whether pan-protease inhibitor (PI)-resistant virus populations are composed predominantly of viruses with resistance to all PIs or of diverse virus populations with resistance to different subsets of PIs.
We performed deep sequencing of plasma virus samples from nine patients with high-level genotypic and/or phenotypic resistance to all licensed PIs. The nine virus samples had a median of 12 PI resistance mutations by direct PCR Sanger sequencing.
For each of the nine virus samples, deep sequencing showed that each of the individual viruses within a sample contained nearly all of the mutations detected by Sanger sequencing. Indeed, a median of 94.9% of deep sequence reads had each of the PI resistance mutations present as a single chromatographic peak in the Sanger sequence. A median of 5.0% of reads had all but one of the Sanger mutations that were not part of an electrophoretic mixture.
The collinearity of PI resistance mutations in the nine virus samples demonstrated that pan-PI-resistant viruses are able to replicate in vivo despite their highly mutated protease enzymes. We hypothesize that the marked collinearity of PI resistance mutations in pan-PI-resistant virus populations results from the unique requirements for multi-PI resistance and the extensive cross-resistance conferred by many of the accessory PI resistance mutations.
drug resistance; deep sequencing; minority variants; Sanger sequencing
Despite extensive research on the emergence of and treatments for methicillin-resistant Staphylococcus aureus (MRSA), prior studies have not rigorously evaluated the impact of methicillin resistance on the overall incidence of S. aureus infections. Yet, there are direct clinical and research implications of determining whether methicillin-susceptible S. aureus (MSSA) infection rates remain stable in the face of increasing MRSA prevalence or whether MSSA will be replaced over time. A synthesis of prior studies indicates that the emergence of healthcare-associated MRSA (HA-MRSA) and community-associated MRSA (CA-MRSA) has led to an increase in the overall incidence of S. aureus infections, with MRSA principally adding to, rather than replacing, MSSA. However, colonization with CA-MRSA may at least partially replace colonization with MSSA. So far, evidence indicates that MSSA still accounts for many infections. Therefore, eradication of MRSA alone is not sufficient to address the public health burden of S. aureus.
bacterial resistance; staphylococcal infections; healthcare-associated infections; community-associated infections
We investigated whether Acinetobacter baumannii isolates of veterinary origin shared common molecular characteristics with those described in humans.
Nineteen A. baumannii isolates collected in pets and horses were analysed. Clonality was studied using repetitive extragenic palindromic PCR (rep-PCR) and multilocus sequence typing (MLST). PCR and DNA sequencing for various β-lactamase, aminoglycoside-modifying enzyme, gyrA and parC, ISAba1 and IS1133, adeR and adeS of the AdeABC efflux pump, carO porin and class 1/2/3 integron genes were performed.
Two main clones [A (n = 8) and B (n = 9)] were observed by rep-PCR. MLST indicated that clone A contained isolates of sequence type (ST) ST12 (international clone II) and clone B contained isolates of ST15 (international clone I). Two isolates of ST10 and ST20 were also noted. Seventeen isolates were resistant to gentamicin, 12 to ciprofloxacin and 3 to carbapenems. Isolates of ST12 carried blaOXA-66, blaADC-25, blaTEM-1, aacC2 and IS1133. Strains of ST15 possessed blaOXA-69, blaADC-11, blaTEM-1 and a class 1 integron carrying aacC1 and aadA1. ISAba1 was found upstream of blaADC (one ST10 and one ST12) and/or blaOXA-66 (seven ST12). Twelve isolates of different STs contained the substitutions Ser83Leu in GyrA and Ser80Leu or Glu84Lys in ParC. Significant disruptions of CarO porin and overexpressed efflux pumps were not observed. The majority of infections were hospital acquired and in animals with predisposing conditions for infection.
STs and the molecular background of resistance observed in our collection have been frequently described in A. baumannii detected in human patients. Animals should be considered as a potential reservoir of multidrug-resistant A. baumannii.
MLST; MDR; PCR/ESI-MS; rep-PCR; T5000
Mycoplasma pneumoniae respiratory infection is a common cause of acute respiratory infection in children and adults. We evaluated the efficacy of increasing dosages of clarithromycin for the optimized therapy of M. pneumoniae respiratory infection in a mouse model.
BALB/c mice were intranasally inoculated once with M. pneumoniae or SP4 broth (control). Groups of mice were treated with increasing dosages of clarithromycin (10, 25 or 75 mg/kg/day) or placebo subcutaneously daily. Groups of mice were evaluated after 1, 2, 3, 6 and 12 days of therapy. Outcome variables included quantitative M. pneumoniae culture, histopathological score of the lungs, bronchoalveolar lavage (BAL) cytokine/chemokine/growth factor concentrations and plethysmography after aerosolized methacholine to assess airway hyperresponsiveness.
Elevated dosages of clarithromycin resulted in greater antimicrobial efficacy with significantly reduced M. pneumoniae quantitative cultures (P < 0.05), as well as greater improvement in markers of disease severity with significantly reduced lung histopathology scores, BAL cytokine concentrations and airway hyperresponsiveness (P < 0.05).
Escalated dosing of clarithromycin resulted in significantly greater therapeutic efficacy in the treatment of experimental M. pneumoniae respiratory infection.
macrolides; cytokines; asthma; pharmacokinetics; pharmacodynamics
To investigate the β-lactamase background of ertapenem non-susceptible isolates for the presence of the most commonly detected carbapenemase genes, blaKPC, blaOXA-48 and blaVIM, and the newly described blaNDM-1.
Two hundred and thirty-five ertapenem-non-susceptible (MIC ≥0.5 mg/L) isolates of Enterobacteriaceae from the worldwide Study for Monitoring Antimicrobial Resistance Trends (SMART) 2009 programme were screened using a multiplex PCR for the presence of blaKPC, blaOXA-48, blaVIM and blaNDM-1 genes. Extended-spectrum β-lactamase (ESBL) and AmpC genes (blaESBL and blaAmpC) were identified using the Check-MDR CT101 microarray. DNA sequencing was performed to identify the blaESBL, blaKPC and blaNDM-1 genes. Molecular typing was also performed to genetically characterize these isolates.
Sixty-six isolates (28%) had a carbapenemase gene, with blaNDM-1 identified in 33 isolates including 2 isolates carrying both blaNDM-1 and blaOXA-48; other carbapenemase genes found included blaKPC (n = 23), blaVIM (n = 7) and blaOXA-48 (n = 3). All blaNDM-1-carrying isolates were from patients in India and comprised five different species. With the exception of one isolate carrying only blaNDM-1, all blaNDM-1 carbapenemase-possessing isolates carried additional β-lactamases in various combinations: blaESBL and blaAmpC (n = 18); blaESBL (n = 10); blaESBL, blaAmpC and blaOXA-48 (n = 2); and blaAmpC (n = 2). Except for blaOXA-48-carrying isolates, novel multilocus sequence types or enterobacterial repetitive intergenic consensus PCR patterns were observed along with clonal dissemination within and among sites.
A range of carbapenemase genes, associated with diverse ESBLs and/or AmpC backgrounds, were found among Enterobacteriaceae isolated during the study. Many of these ertapenem non-susceptible strains were clonally related and carried various combinations of β-lactamases.
carbapenemases; Enterobacteriaceae; microarray analysis
To identify the genes responsible for tetracycline resistance in a strain of Streptococcus australis isolated from pooled saliva from healthy volunteers in France. S. australis is a viridans Streptococcus, originally isolated from the oral cavity of children in Australia, and subsequently reported in the lungs of cystic fibrosis patients and as a cause of invasive disease in an elderly patient.
Agar containing 2 mg/L tetracycline was used for the isolation of tetracycline-resistant organisms. A genomic library in Escherichia coli was used to isolate the tetracycline resistance determinant. In-frame deletions and chromosomal repair were used to confirm function. Antibiotic susceptibility was determined by agar dilution and disc diffusion assay.
The tetracycline resistance determinant from S. australis FRStet12 was isolated from a genomic library in E. coli and DNA sequencing showed two open reading frames predicted to encode proteins with similarity to multidrug resistance-type ABC transporters. Both genes were required for tetracycline resistance (to both the naturally occurring and semi-synthetic tetracyclines) and they were designated tetAB(46).
This is the first report of a predicted ABC transporter conferring tetracycline resistance in a member of the oral microbiota.
antibiotic resistance; oral microflora; oral streptococci
This study describes parasite kinetics in the blood of visceral leishmaniasis patients treated with liposomal amphotericin B (L-AmB) or a preformed fat emulsion of amphotericin B (ApL) using real-time quantitative PCR (qPCR).
Forty-six patients were treated with a single dose (15 mg/kg of body weight) of either L-AmB (n = 13) or ApL (n = 33). qPCR was used to estimate parasite kinetics by detection of Leishmania donovani DNA using kinetoplast DNA-specific primers in peripheral blood samples using an absolute quantification method.
The mean parasite load decreased from baseline (day 0) values of 894.07 and 980.48 to 71.72 and 211.52 parasite genomes/mL at day 7 in L-AmB and ApL groups, respectively, and at day 30 these further declined to 8.30 and 133.98 parasite genomes/mL, respectively. At day 30 post-treatment evaluation, the decline in parasite load was significantly greater (P = 0.024) with L-AmB compared with ApL. Four of 33 patients in the ApL group failed treatment (1 primary failure and 3 relapses) with the presence of parasites, whereas all patients in the L-AmB group were cured at 6 month follow-up.
qPCR can be a tool to measure parasite dynamics accurately and provide a marker to measure the efficacy of various drugs. It can be used as a test of cure, allowing us to do away with invasive and risky methods such as splenic or bone marrow aspiration.
efficacy; fat emulsion of amphotericin B; liposomal amphotericin B; Leishmania donovani; qPCR; VL
The mechanism of action of, and resistance to, metronidazole in the anaerobic (or micro-aerotolerant) protozoan parasite Giardia lamblia has long been associated with the reduction of ferredoxin (Fd) by the enzyme pyruvate:ferredoxin oxidoreductase (PFOR) and the subsequent activation of metronidazole by Fd to toxic radical species. Resistance to metronidazole has been associated with down-regulation of PFOR and Fd. The aim of this study was to determine whether the PFOR/Fd couple is the only pathway involved in metronidazole activation in Giardia.
PFOR and Fd activities were measured in extracts of highly metronidazole-resistant (MTRr) lines and activities of recombinant G. lamblia thioredoxin reductase (GlTrxR) and NADPH oxidase were assessed for their involvement in metronidazole activation and resistance.
We demonstrated that several lines of highly MTRr G. lamblia have fully functional PFOR and Fd indicating that PFOR/Fd-independent mechanisms are involved in metronidazole activation and resistance in these cells. Flavin-dependent GlTrxR, like TrxR of other anaerobic protozoa, reduces 5-nitroimidazole compounds including metronidazole, although expression of TrxR is not decreased in MTRr Giardia. However, reduction of flavins is suppressed in highly MTRr cells, as evidenced by as much as an 80% decrease in NADPH oxidase flavin mononucleotide reduction activity. This suppression is consistent with generalized impaired flavin metabolism in highly MTRr Trichomonas vaginalis.
These data add to the mounting evidence against the dogma that PFOR/Fd is the only couple with a low enough redox potential to reduce metronidazole in anaerobes and point to the multi-factorial nature of metronidazole resistance.
metronidazole; ronidazole; tinidazole; Blastocystis; NADPH oxidase
The development of daptomycin resistance in Staphylococcus aureus is associated with clinical treatment failures. The mechanism(s) of such resistance have not been clearly defined.
We studied an isogenic daptomycin-susceptible (DAPS) and daptomycin-resistant (DAPR) S. aureus strain pair (616; 701) from a patient with relapsing endocarditis during daptomycin treatment, using comparative transcriptomic and proteomic techniques.
Minor differences in the genome content were found between strains by DNA hybridization. Transcriptomic analyses identified a number of genes differentially expressed in important functional categories: cell division; metabolism of bacterial envelopes; and global regulation. Of note, the DAPR isolate exhibited reduced expression of the major cell wall autolysis gene coincident with the up-regulation of genes involved in cell wall teichoic acid production. Using quantitative (q)RT–PCR on the gene cadre putatively involved in cationic peptide resistance, we formulated a putative regulatory network compatible with microarray data sets, mainly implicating bacterial envelopes. Of interest, qRT–PCR of this same gene cadre from two distinct isogenic DAPS/DAPR clinical strain pairs revealed evidence of other strain–dependent networks operative in the DAPR phenotype. Comparative proteomics of 616 versus 701 revealed a differential abundance of proteins in various functional categories, including cell wall-associated targets and biofilm formation proteins. Phenotypically, strains 616 and 701 showed major differences in their ability to develop bacterial biofilms in the presence of the antibacterial lipid, oleic acid.
Compatible with previous in vitro observations, in vivo-acquired DAPR in S. aureus is a complex, multistep phenomenon involving: (i) strain-dependent phenotypes; (ii) transcriptome adaptation; and (iii) modification of the lipid and protein contents of cellular envelopes.
cell wall metabolism; antibiotic resistance; biofilms; δ-haemolysis; oleic acid; microarrays; virulence; quantitative proteomics
The stationary phase of Clostridium difficile, which is associated with the symptoms of the diarrhoeal disease, is refractory to antibiotic killing. The aim of this study was to explore whether probiotic-derived reutericyclin and related synthetic analogues could kill stationary phase C. difficile at concentrations achievable in the gastrointestinal tract.
The bactericidal activities of reutericyclin and lead compound derivatives were examined against logarithmic and stationary phase cultures of different C. difficile strains. The absorption of compounds across the intestinal epithelia was tested using the Caco-2 permeability model.
Unlike vancomycin and metronidazole, reutericyclins demonstrated concentration-dependent killing, being rapidly bactericidal against both logarithmic and stationary phase cells, at low concentrations (0.09–2 mg/L). The intestinal absorption of unmodified reutericyclin was poor and comparable to that of vancomycin. However, this property varied significantly for the synthetic reutericyclin analogues, ranging from well absorbed to non-absorbed. The non-absorbable compounds were highly effluxed, suggesting this parameter could be modulated to obtain agents with superior efficacy.
Reutericyclins showed excellent potency against the lethal non-growing stage of C. difficile at concentrations that may be attained in the gastrointestinal tract. Since these agents represent novel potential treatments for C. difficile infection, further development of this compound class is warranted.
anti-C. difficile; stationary phase; probiotic metabolite; reutericyclins
The UK Department of Health has made recommendations on safe and appropriate prescribing of anti-infectives. In response, we reviewed our anti-infective policies to ensure they were in line with best practice. As a result, a new adult anti-infective policy was launched. To help facilitate its implementation, a quality improvement programme was established, with the aim of achieving >90% compliance with the new policy.
Patients under the care of the medical admissions teams who had been prescribed one or more systemic anti-infectives between January and November 2008 were included in the study. Study pharmacists collected data daily on all patients, including the anti-infective(s) prescribed and indication(s) documented on either the patient's drug prescription chart or health records. A definition of compliance was developed, which required documented indication(s) and associated anti-infectives to match the anti-infective policy. A baseline compliance level was established; we then implemented a series of interventions using the plan-do-study-act (‘PDSA’) approach to monitor and improve compliance. Three overlapping intervention phases were retrospectively identified: raising awareness; education; and weekly feedback of results in the form of run charts distributed to medical teams.
Over the 11 month study period, compliance with the policy increased from 30% to 71%. Since 2008, we have seen the average compliance increase year-on-year to over 90% in 2010 using a sustainable once weekly data collection model.
This study shows that it is possible to use quality improvement methodology to support antimicrobial stewardship within existing resources and suggests that an improvement in policy compliance can be both achieved and sustained.
antibiotic stewardship; anti-infectives; QIP; prescribing