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Preview: Journal of Antimicrobial Chemotherapy - current issue

Journal of Antimicrobial Chemotherapy Current Issue





Published: Fri, 20 Jan 2017 00:00:00 GMT

Last Build Date: Mon, 23 Jan 2017 20:45:43 GMT

 



Towards better antimicrobial susceptibility testing: impact of the Journal of Antimicrobial Chemotherapy

2017-01-20

Susceptibility testing of bacteria is one of the most important tests performed in a clinical microbiology laboratory. Improvements in laboratory techniques, especially the move towards standardized susceptibility testing, has provided better consistency and accuracy of testing. When used in conjunction with the most recently developed interpretative criteria, the result is better prediction of the outcome of antimicrobial therapy for infected patients. Throughout the last four decades this Journal has published numerous articles evidencing improvements and new techniques, a valuable source of information for microbiology laboratories.


















Intrinsic rifamycin resistance of Mycobacterium abscessus is mediated by ADP-ribosyltransferase MAB_0591

2016-12-20

Objectives: Rifampicin, a potent first-line TB drug of the rifamycin group, shows only little activity against the emerging pathogen Mycobacterium abscessus. Reportedly, bacterial resistance to rifampicin is associated with polymorphisms in the target gene rpoB or the presence of enzymes that modify and thereby inactivate rifampicin. The aim of this study was to investigate the role of the MAB_0591 (arrMab)-encoded rifampicin ADP-ribosyltransferase (Arr_Mab) in innate high-level rifampicin resistance in M. abscessus.Methods: Recombinant Escherichia coli and Mycobacterium tuberculosis strains expressing MAB_0591 were generated, as was an M. abscessus deletion mutant deficient for MAB_0591. MIC assays were used to study susceptibility to rifampicin and C25 carbamate-modified rifamycin derivatives.Results: Heterologous expression of MAB_0591 conferred rifampicin resistance to E. coli and M. tuberculosis. Rifamycin MIC values were consistently lower for the M. abscessus ΔarrMab mutant as compared with the M. abscessus ATCC 19977 parental type strain. The rifamycin WT phenotype was restored after complementation of the M. abscessus ΔarrMab mutant with arrMab. Further MIC data demonstrated that a C25 modification increases rifamycin activity in WT M. abscessus. However, MIC studies in the M. abscessus ΔarrMab mutant suggest that C25 modified rifamycins are still subject to modification by Arr_Mab.Conclusions: Our findings identify Arr_Mab as the major innate rifamycin resistance determinant of M. abscessus. Our data also indicate that Arr_Mab-mediated rifamycin resistance in M. abscessus can only in part be overcome by C25 carbamate modification.



Gut-sparing treatment of urinary tract infection in patients at high risk of Clostridium difficile infection

2016-12-20

Background: Recipients of faecal microbiota transplantation (FMT) in treatment of recurrent Clostridium difficile infection (RCDI) remain at markedly increased risk of re-infection with C. difficile with new antibiotic provocations. Urinary tract infections (UTIs) are common indications for antibiotics in these patients, often resulting in C. difficile re-infection.Methods: We present a case series of 19 patients treated with parenteral aminoglycosides for UTI following FMT for RCDI. A 3 day outpatient regimen of once-daily intramuscular administration of gentamicin was used to treat 18 consecutive FMT recipients with uncomplicated UTI. One other patient was treated for a complicated UTI with intravenous amikacin. Profiling of 16S rRNA genes was used to track changes in faecal microbial community structure during this regimen in three patients.Results: The protocol was highly effective in treating UTI symptoms. None of the patients suffered a re-infection with C. difficile. The faecal microbial communities remained undisturbed by treatment with intramuscular administration of gentamicin.Conclusions: Despite falling out of favour in recent years, aminoglycoside antibiotics given parenterally have the advantage of minimal penetration into the gut lumen. A brief (3 day) course of parenteral gentamicin was safe and effective in curing UTI in patients at high risk of C. difficile infection without perturbing their gut microbiota.



Relationship between vancomycin tolerance and clinical outcomes in Staphylococcus aureus bacteraemia

2016-12-20

Background: Previous data have demonstrated the clinical importance of vancomycin MIC values in Staphylococcus aureus bacteraemia (SAB); however, the impact of vancomycin tolerance (VT) is unknown.Objectives: To compare the frequency of clinical failure between patients with VT and non-VT isolates in SAB.Methods: This was a retrospective cohort study of patients with SAB, excluding treatment <48 h or polymicrobial bacteraemia. The primary outcome was clinical failure (composite of 30 day mortality, non-resolving signs and symptoms, and 60 day recurrence). Vancomycin MIC and MBC were determined by broth microdilution. The association between VT (MBC/MIC ≥32) and clinical failure was evaluated by multivariable Poisson regression.Results: Of the 225 patients, 26.7% had VT isolates. VT was associated with clinical failure (48.0% overall) in unadjusted analysis [68.3% (n =41/60) versus 40.6% (n =67/165); P <0.001] and this relationship persisted in multivariable analysis (adjusted risk ratio, 1.74; 95% CI, 1.36-2.24; P <0.001). The association between VT and clinical failure was also consistent within strata of methicillin susceptibility [methicillin susceptible (n =125, risk ratio, 1.67; 95% CI, 1.20-2.32; P =0.002); methicillin resistant (n =100, risk ratio, 1.69; 95% CI, 1.14-2.51; P =0.010)]. Among methicillin-susceptible SAB cases treated with β-lactam therapy, VT remained associated with clinical failure (risk ratio, 1.77; 95% CI, 1.19-2.61; P =0.004).Conclusions: VT was associated with clinical failure in SAB, irrespective of methicillin susceptibility or definitive treatment. VT may decrease the effectiveness of cell-wall-active therapy or be a surrogate marker of some other pathogen-specific factor associated with poor outcomes. Future research should evaluate if bactericidal non-cell-wall-active agents improve outcomes in VT SAB.



Development of operationalized intravenous to oral antibiotic switch criteria

2016-12-20

Objectives: Despite huge overlap in suggested criteria for a safe intravenous (iv)-to-oral antibiotic switch, there is considerable variation in their operationalization. The objective of this study was to develop a set of measurable conditions that should be met in adult hospitalized patients for a safe iv-to-oral switch.Methods: A RAND-modified Delphi procedure was performed to develop a set of operationalized iv-to-oral switch criteria. Switch criteria and their accompanying suggested measurable conditions were extracted from the literature and appraised by a multidisciplinary expert panel during two questionnaire rounds with a face-to-face meeting between these two rounds. In a final step, the experts could approve the set of developed operationalized switch criteria.Results: Seven switch criteria and 41 accompanying measurable conditions extracted from the literature were appraised. Sixteen measurable conditions that operationalize six switch criteria were selected: (i) stable systolic blood pressure; and the absence of (ii) fever, (iii) temperature <36°C, (iv) malabsorption syndrome, (v) short bowel syndrome, (vi) severe gastroparesis, (vii) ileus, (viii) continuous nasogastric suction, (ix) vomiting, (x) (severe) sepsis, (xi) fasciitis necroticans, (xii) CNS infection, (xiii) Staphylococcus aureus bacteraemia, and (xiv) endovascular infection. In addition, (xv) the patient should be cooperative and (xvi) adequate antimicrobial concentration should be achievable at the site of infection by oral administration.Conclusions: These operationalized criteria can be used in daily clinical practice. Future use of these criteria in audits and as rules in clinical decision support systems will facilitate the performance and evaluation of iv–oral switch programmes.



Linezolid: a promising option in the treatment of Gram-positives

2016-12-05

Linezolid, an oxazolidinone antimicrobial agent that acts by inhibiting protein synthesis in a unique fashion, is used in the treatment of community-acquired pneumonia, skin and soft-tissue infections and other infections caused by Gram-positive bacteria including VRE and methicillin-resistant staphylococci. Currently, linezolid resistance among these pathogens remains low, commonly <1.0%, although the prevalence of antibiotic resistance is increasing in many countries. Therefore, the development of resistance by clinical isolates should prompt increased attention of clinical laboratories to routinely perform linezolid susceptibility testing for this important agent and should be taken into account when considering its therapeutic use. Considering the importance of linezolid in the treatment of infections caused by Gram-positive bacteria, this review was undertaken to optimize the clinical use of this antibiotic.



Alarming increase in pretreatment HIV drug resistance in children living in sub-Saharan Africa: a systematic review and meta-analysis

2016-12-05

Background: Children have an augmented risk of pretreatment HIV drug resistance (PDR) due to exposure to antiretroviral drugs for the prevention of mother-to-child transmission (PMTCT). Paediatric data are essential to evaluate the effectiveness of the restricted number of paediatric regimens currently available, but these data are scarce.Methods: We conducted a systematic review of the literature on PDR in children (median age ≤12 years) in sub-Saharan Africa. We separately extracted the proportion of children with PDR for children with and without prior PMTCT exposure, used random-effects meta-analysis to pool proportions and used meta-regression to assess subgroup differences.Results: We included 19 studies representing 2617 children from 13 countries. The pooled PDR prevalence was 42.7% (95% CI 26.2%–59.1%) among PMTCT-exposed children and 12.7% (95% CI 6.7%–18.7%) among PMTCT-unexposed children (P =0.004). The PDR prevalence in PMTCT-unexposed children increased from 0% in 2004 to 26.8% in 2013 (P =0.009). NNRTI mutations were detected in 32.4% (95% CI 18.7%–46.1%) of PMTCT-exposed children and in 9.7% (95% CI 4.6%–14.8%) of PMTCT-unexposed children; PI mutations were uncommon (<2.5%). PDR was more common in children aged <3 years compared with children aged ≥3 years [40.9% (95% CI 27.6%–54.3%) versus 17.6% (95% CI 8.9%–26.3%), respectively (P =0.025)].Conclusions: The PDR prevalence in African children is high and rapidly increasing. Even in PMTCT-unexposed children, the most recent reports indicate that PDR is present in up to a third of children starting first-line therapy. Our data underscore the importance of initiating PI-based first-line ART in young children (<3 years of age) and suggest that older children may also benefit from this approach.



In vitro activity of gentamicin as an adjunct to penicillin against biofilm group B Streptococcus

2016-12-05

Objectives: Group B Streptococcus (GBS) increasingly causes invasive disease in non-pregnant adults, particularly in elderly persons and those with underlying diseases. Combination therapy with penicillin plus gentamicin has been suggested for periprosthetic joint infection. The postulated synergism of this combination is based on experiments with planktonic bacteria. We aimed to assess the efficacy of this combination against sessile bacteria.Methods: Four different GBS strains were used. We compared results of MICs with those of minimal biofilm eradication concentrations (MBECs), applied chequerboard assays to the MBEC device and calculated the fractional inhibitory concentration index. Synergism was evaluated with time–kill assays against bacteria adherent to cement beads, using penicillin (0.048, 0.2 and 3 mg/L), gentamicin (4 and 12.5 mg/L) and a combination thereof. Results were evaluated via colony counting after sonication of beads and scanning electron microscopy.Results: MBEC/MIC ratios were 2000–4000 for penicillin and 1–4 for gentamicin. In chequerboard assays, synergism was observed in all four isolates. In time–kill assays, penicillin and 12.5 mg/L gentamicin showed synergism in two isolates. In the other two isolates 12.5 mg/L gentamicin alone was as efficient as the combination therapy.Conclusions: These in vitro investigations show activity of 12.5 mg/L gentamicin, alone or as an adjunct to penicillin, against four strains of biofilm GBS. This concentration cannot be achieved in bone with systemic administration, but can be reached if administered locally. The combination of systemic penicillin plus local gentamicin indicates a potential application in orthopaedic-device-associated GBS infections. Studies with a larger number of strains are required to confirm our results.



Synergistic activity of fosfomycin, β-lactams and peptidoglycan recycling inhibition against Pseudomonas aeruginosa

2016-12-05

Objectives: To evaluate the interconnection between peptidoglycan (PG) recycling, fosfomycin susceptibility and synergy between fosfomycin and β-lactams in Pseudomonas aeruginosa.Methods: Fosfomycin MICs were determined by broth microdilution and Etest for a panel of 47 PAO1 mutants defective in several components of PG recycling and/or AmpC induction pathways. PAO1 fosfomycin MICs were also determined in the presence of a 5 mM concentration of the NagZ inhibitor PUGNAc. Population analysis of fosfomycin susceptibility and characterization of the resistant mutants that emerged was also performed for selected strains. Finally, fosfomycin, imipenem and fosfomycin + imipenem killing curves were assessed.Results: Mutants defective in AmpG, NagZ or all three AmpD amidases showed a marked increase in fosfomycin susceptibility (at least two 2-fold dilutions with respect to WT PAO1). Moreover, PAO1 fosfomycin MICs were consistently reduced from 48 to 24 mg/L in the presence of a 5 mM concentration of PUGNAc. Fosfomycin hypersusceptibility of the ampG, nagZ and triple ampD mutants was also clearly confirmed in the performed population analysis, although the emergence of resistant mutants, through GlpT mutations, was not avoided. Synergy between fosfomycin and imipenem was evidenced for the WT strain, the AmpC-hyperproducing strain (triple AmpD mutant) and the NagZ and AmpG mutants in killing curves. Moreover, regrowth of resistant mutants was not evidenced for the combination.Conclusions: PG recycling inhibitors are envisaged as useful adjuvants in the treatment of P. aeruginosa infections with β-lactams and fosfomycin and therefore further development of these molecules is encouraged.



Pharmacological inhibition of p110δ subunit of PI3K confers protection against experimental leishmaniasis

2016-12-05

Objectives: This study aimed to evaluate the immuno-prophylactic and -therapeutic effect of p110δ-specific pharmacological inhibitors (CAL-101 and IC87114), either alone or in combination with amphotericin B, against experimental cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL).Methods: Female BALB/c mice were infected intravenously with Leishmania donovani or subcutaneously with Leishmania major. Prophylactic treatment was initiated 24 h prior to infection, whereas therapeutic treatments with or without amphotericin B were initiated either 1 week or 2 weeks post-infection. At different times post-infection, mice were sacrificed and parasite burden, regulatory T cell (Treg) numbers and cytokine production were assessed in the liver, spleen, draining lymph nodes and footpads. In addition, direct cytolytic effects of the inhibitors on parasite growth in axenic cultures and inside infected and uninfected macrophages were also assessed.Results: Prophylactic and therapeutic administration of p110δ pharmacological inhibitors significantly reduced cutaneous lesion (in CL) and parasite burdens (in VL and CL) in the spleens, livers and footpads of infected mice. The reduction in parasite burden was associated with a concomitant reduction in Treg numbers and cytokine production by liver, spleen and lymph node cells. Combined low-dose CAL-101 and amphotericin B therapy caused complete clearance of parasites in mice infected with L. donovani.Conclusions: Our studies clearly show a novel therapeutic option for leishmaniasis based on CAL-101 monotherapy or CAL-101 and amphotericin B combination therapy. These observations have important and direct implications for antimicrobial immunotherapy and drug/vaccine development against leishmaniasis.



Genotypic susceptibility score (GSS) and CD4+ T cell recovery in HIV-1 patients with suppressed viral load

2016-12-05

Objectives: HIV drug resistance, measured by the genotypic susceptibility score (GSS), has a deleterious effect on the virological outcome of HIV-1-infected patients. However, it is not known if GSS retains any predictive value for CD4 recovery in patients with suppressed viral load. Methods: Four hundred and six patients on virological failure (>500 copies/mL) with GSS<6 months prior to switch therapy who achieved undetectable plasma viral load (<50 copies/mL) within 1 year, remained undetectable >1 year on an unchanged regimen and had CD4 data available during entire follow-up were included. Adjusted and unadjusted analyses of all characteristics at switch related to CD4 recovery were made for three time frames: (i) ‘switch–suppression’; (ii) ‘suppression–1 year’; and (iii) ‘switch–1 year’.Results: Higher GSS was associated with a greater CD4 recovery between ‘switch’ and ‘1 year’ in the unadjusted analysis (P =0.010); however, the effect of GSS was no longer statistically significant after adjusting for pre-switch clinical (CD4 count and plasma viral load) and demographic variables. Furthermore, only a lower pre-switch CD4 count was associated with increased CD4 recovery in the ‘suppression–1 year’ period in both unadjusted and adjusted models. The main CD4 recovery occurred in ‘switch–suppression’ and the variables associated, both unadjusted and adjusted, were CD4 and plasma viral load at switch, maintaining a trend for GSS (P =0.06).Conclusions: In individuals who re-suppressed HIV viraemia after switching therapy, regimens having a higher GSS were associated with improved CD4 recovery only during the period from switch to virological suppression, but, once viral load is re-suppressed, the GSS of the new regimen has no further effect on subsequent CD4 recovery.









Variability in antibiotic use across Ontario acute care hospitals

2016-11-17

Background: Antibiotic stewardship is a required organizational practice for Canadian acute care hospitals, yet data are scarce regarding the quantity and composition of antibiotic use across facilities. We sought to examine the variability, and risk-adjusted variability, in antibiotic use across acute care hospitals in Ontario, Canada’s most populous province.Methods: Antibiotic purchasing data from IMS Health, previously demonstrated to correlate strongly with internal antibiotic dispensing data, were acquired for 129 Ontario hospitals from January to December 2014 and linked to patient day (PD) denominator data from administrative datasets. Hospital variation in DDDs/1000 PDs was determined for overall antibiotic use, class-specific use and six practices of clinical or ecological significance. Multivariable risk adjustment for hospital and patient characteristics was used to compare observed versus expected utilization.Results: There was 7.4-fold variability in the quantity of antibiotic use across the 129 acute care hospitals, from 253 to 1873 DDDs/1000 PDs. Variation was evident within hospital subtypes, exceeded that explained by hospital and patient characteristics, and included wide variability in proportion of broad-spectrum antibiotics (IQR 36%–48%), proportion of fluoroquinolones among respiratory antibiotics (IQR 40%–62%), proportion of ciprofloxacin among urinary anti-infectives (IQR 44%–60%), proportion of antibiotics with highest risk for Clostridium difficile (IQR 29%–40%), proportion of ‘reserved-use’ antibiotics (IQR 0.8%–3.5%) and proportion of anti-pseudomonal antibiotics among antibiotics with Gram-negative coverage (IQR 26%–40%).Conclusions: There is extensive variability in antibiotic use, and risk-adjusted use, across acute care hospitals. This could motivate, focus and benchmark antibiotic stewardship efforts.



Lack of adherence to SHEA-IDSA treatment guidelines for Clostridium difficile infection is associated with increased mortality

2016-11-14

Objectives
The objective of this study was to determine our institution's compliance with 2010 Society for Healthcare Epidemiology of America and IDSA Clostridium difficile infection (CDI) treatment guidelines and their respective outcomes.
Methods
We collected clinical parameters, laboratory values, antibiotic therapy and clinical outcomes from the electronic medical records for all patients hospitalized at our institution with a diagnosis of CDI from December 2012 to November 2013. We specifically evaluated whether SHEA-IDSA treatment guidelines were followed and evaluated the associations between guideline adherence and severe outcomes including mortality.
Results
We identified 230 patients with CDI meeting inclusion criteria during the study period. Of these, 124 (54%) were appropriately treated, 46 (20%) were under-treated and 60 (26%) were over-treated. All-cause 90 day mortality was 17.4% overall; 43.5% in the under-treated group versus 12.9% in those appropriately treated (P < 0.0001) and 10.9% in those appropriately treated plus over-treated (P < 0.0001). Similarly, 90 day mortality attributed to CDI was 21.7% in those under-treated versus 8.9% in those appropriately treated (P = 0.03) and 8.2% in those either appropriately treated or over-treated (P = 0.015). Severe-complicated CDI occurred in 46 patients. In this subgroup, there was a non-significant trend towards increased mortality in under-treated patients (56.7%) compared with appropriately treated patients (37.5%, P = 0.35). Under-treatment was also associated with a higher rate of CDI-related ICU transfer (17.4% versus 4.8% in those appropriately treated, P = 0.023).
Conclusions
Adherence to CDI treatment guidelines is associated with improved outcomes especially in those with severe disease. Increased emphasis on provision of appropriate, guideline-based CDI treatment appears warranted.



Impact of polypharmacy on antiretroviral prescription in people living with HIV

2016-11-10

Objectives
To evaluate the relationship between polypharmacy and ART, delivered as conventional multi-tablet three-drug regimens, single-tablet regimens or less-drug regimens (simplified mono or dual regimens).
Methods
We conducted a cross-sectional analysis of electronic data from the prospective Modena HIV Metabolic Clinic Cohort Study. We included the last clinical observation for each patient from January 2006 to December 2015. Polypharmacy was defined as the use of five or more medications (excluding ART). Multi-morbidity was classified as the presence of two or more non-infectious comorbidities. Factors associated with different ART regimens were analysed using multivariable multinomial logistic regression analyses with multi-tablet three-drug regimens as the reference.
Results
A total of 2944 patients (33.7% females) were included in the analysis. Multinomial logistic regression analysis identified polypharmacy to be negatively associated with single-tablet regimens [relative risk reduction (RRR) = 0.48, 95% CI = 0.28–0.81] independently from frailty (RRR = 0.68, 95% CI = 0.59–0.78), after correction for age, gender, HIV infection duration, current and nadir CD4 and calendar year. This association was not found comparing multi-tablet three-drug regimens and less-drug regimens.
Conclusions
Single-tablet regimens are less likely to be prescribed in patients with polypharmacy. Single-tablet regimens are perceived to be less flexible in patients with multi-morbidity and at higher risk of drug–drug interaction.



A sampling and metagenomic sequencing-based methodology for monitoring antimicrobial resistance in swine herds

2016-11-08

Objectives
Reliable methods for monitoring antimicrobial resistance (AMR) in livestock and other reservoirs are essential to understand the trends, transmission and importance of agricultural resistance. Quantification of AMR is mostly done using culture-based techniques, but metagenomic read mapping shows promise for quantitative resistance monitoring.
Methods
We evaluated the ability of: (i) MIC determination for Escherichia coli; (ii) cfu counting of E. coli; (iii) cfu counting of aerobic bacteria; and (iv) metagenomic shotgun sequencing to predict expected tetracycline resistance based on known antimicrobial consumption in 10 Danish integrated slaughter pig herds. In addition, we evaluated whether fresh or manure floor samples constitute suitable proxies for intestinal sampling, using cfu counting, qPCR and metagenomic shotgun sequencing.
Results
Metagenomic read-mapping outperformed cultivation-based techniques in terms of predicting expected tetracycline resistance based on antimicrobial consumption. Our metagenomic approach had sufficient resolution to detect antimicrobial-induced changes to individual resistance gene abundances. Pen floor manure samples were found to represent rectal samples well when analysed using metagenomics, as they contain the same DNA with the exception of a few contaminating taxa that proliferate in the extraintestinal environment.
Conclusions
We present a workflow, from sampling to interpretation, showing how resistance monitoring can be carried out in swine herds using a metagenomic approach. We propose metagenomic sequencing should be part of routine livestock resistance monitoring programmes and potentially of integrated One Health monitoring in all reservoirs.



A community survey of antibiotic consumption among children in Madagascar and Senegal: the importance of healthcare access and care quality

2016-11-08

Background
Antibiotic resistance is growing in low-income countries (LICs). Children in LICs are particularly at risk. Information on antibiotic consumption is needed to control the development and spread of resistant bacteria.
Methods
To measure antibiotic consumption and related factors, a community survey was undertaken in two sites in Madagascar (Antananarivo and Moramanga) and in Senegal (Guediawaye) among children under 2. Face-to-face interviews were conducted with parents or caregivers of eligible children. Regression analysis was used to determine variables associated with reported antibiotic consumption. Availability of health structures and health policies were also investigated.
Results
Population estimates for antibiotic consumption in the last 3 months were 37.2% (95% CI 33.4%–41.2%) in Guediawaye, 29.3% (95% CI 25.0%–34.1%) in Antananarivo and 24.6% (95% CI 20.6%–29.1%) in Moramanga. In all sites, the large majority of antibiotics were taken with a prescription (92.2%, 87.0% and 92.0% for Antananarivo, Moramanga and Guediawaye, respectively) and purchased in pharmacies (89.4%, 73.5% and 78.5%, respectively). Living in houses without flushing toilets and baby age were significantly associated with any antibiotic consumption after adjusting for site. A higher density of public health structures was associated with lower antibiotic consumption levels, while a higher density of private pharmacies was associated with higher levels across sites.
Conclusions
These data are crucial for the implementation of local programmes aimed at optimizing antibiotic consumption. Factors such as density of healthcare facilities, prescriber training and national policy must be taken into account when developing strategies to optimize antibiotic consumption in LICs.



IncFII k plasmid harbouring an amplification of 16S rRNA methyltransferase-encoding gene rmtH associated with mobile element IS CR2

2016-10-28

Objectives
To investigate the resistance mechanisms and genetic support underlying the high resistance level of the Klebsiella pneumoniae strain CMUL78 to aminoglycoside and β-lactam antibiotics.
Methods
Antibiotic susceptibility was assessed by the disc diffusion method and MICs were determined by the microdilution method. Antibiotic resistance genes and their genetic environment were characterized by PCR and Sanger sequencing. Plasmid contents were analysed in the clinical strain and transconjugants obtained by mating-out assays. Complete plasmid sequencing was performed with PacBio and Illumina technology.
Results
Strain CMUL78 co-produced the 16S rRNA methyltransferase (RMTase) RmtH, carbapenemase OXA-48 and ESBL SHV-12. The rmtH- and blaSHV-12-encoding genes were harboured by a novel ∼115 kb IncFIIk plasmid designated pRmtH, and blaOXA-48 by a ∼62 kb IncL/M plasmid related to pOXA-48a. pRmtH plasmid possessed seven different stability modules, one of which is a novel hybrid toxin–antitoxin system. Interestingly, pRmtH plasmid harboured a 4-fold amplification of an rmtH-ISCR2 unit arranged in tandem and inserted within a novel IS26-based composite transposon designated Tn6329.
Conclusions
This is the first known report of the 16S RMTase-encoding gene rmtH in a plasmid. The rmtH-ISCR2 unit was inserted in a composite transposon as a 4-fold tandem repeat, a scarcely reported organization.



Pretreatment HIV-1 drug resistance in Argentina: results from a surveillance study performed according to WHO-proposed new methodology in 2014–15

2016-10-26

Background
In Argentina, current national guidelines recommend starting with NNRTI-based regimens. Recently, there have been some local reports regarding concerning levels of NNRTI-transmitted resistance, but surveillance has never been carried out at a national level.
Objectives
To determine the prevalence of HIV drug resistance in people starting ART in Argentina using a WHO-proposed methodology.
Methods
This was a cross-sectional, nationally representative study. Twenty-five antiretroviral-dispensing sites throughout the country were randomly chosen to enrol at least 330 persons starting ART, to generate a point prevalence estimate of resistance-associated mutations (RAMs) with a 5% CI (for the total population and for those without antiretroviral exposure). All consecutive patients older than 18 years starting or restarting ART in the chosen clinics were eligible. Samples were processed with Trugene and analysed using the Stanford algorithm.
Results
Between August 2014 and March 2015, we obtained 330 samples from people starting ART. The mean ± SD age was 35 ± 11 years, 63.4% were male, 16.6% had prior antiretroviral exposure and the median (IQR) CD4 count was 275 cells/mm3 (106–461). The prevalence of RAMs found was 14% (±4%) for the whole population (3% NRTI-RAMs; 11% NNRTI-RAMs and 2% PI-RAMs) and 13% (±4%) for those without prior antiretroviral exposure (3%, 10% and 2%, respectively). The most common mutation was K103N.
Conclusions
This surveillance study showed concerning levels of HIV drug resistance in Argentina, especially to NNRTIs. Due to this finding, Argentina's Ministry of Health guidelines will change, recommending performing a resistance test for everyone before starting ART. If this is taken up properly, it also might function as a continuing surveillance tool.



Solithromycin, a novel macrolide, does not prolong cardiac repolarization: a randomized, three-way crossover study in healthy subjects

2016-10-26

Background
Macrolide antibiotics may cause QT prolongation.
Objectives
To study the QT effect of a novel macrolide, solithromycin.
Methods
This was a thorough QT study with a three-way crossover design performed in healthy male and female subjects to evaluate the ECG effects of a novel macrolide, solithromycin. Forty-eight subjects were randomized to receive 800 mg of intravenous (iv) solithromycin, 400 mg of oral moxifloxacin and placebo in three separate treatment periods. Continuous 12 lead ECGs were recorded at a pre-dose baseline and serially after drug administration for 24 h.
Results
After the 40 min infusion of 800 mg of solithromycin, the geometric mean solithromycin peak plasma concentration (Cmax) reached 5.9 (SD: 1.30) μg/mL. Solithromycin infusion caused a heart rate increase with a peak effect of 15 bpm immediately after the end of the infusion. The change-from-baseline QTcF (ΔQTcF) was similar after dosing with solithromycin and placebo and the resulting placebo-corrected ΔQTcF (ΔΔQTcF) for solithromycin was therefore small at all timepoints with a peak effect at 4 h of only 2.8 ms (upper bound of the 90% CI: 4.9 ms). Using a linear exposure–response model, a statistically significant, slightly negative slope of −0.86 ms per ng/mL (90% CI: −1.19 to −0.53; P = 0.0001) was observed with solithromycin. The study's ability to detect small QT changes was confirmed by the moxifloxacin response. Solithromycin did not have a clinically meaningful effect on the PR or QRS interval.
Conclusions
The study demonstrated that solithromycin, unlike other macrolide antibiotics, does not cause QT prolongation.



ESBL/AmpC-producing Enterobacteriaceae in households with children of preschool age: prevalence, risk factors and co-carriage

2016-10-26

Objectives
ESBL/AmpC-producing Enterobacteriaceae are an emerging public health concern. As households with preschool children may substantially contribute to the community burden of antimicrobial resistance, we determined the prevalence, risk factors and co-carriage of ESBL/AmpC-producing bacteria in preschool children and their parents.
Methods
From April 2013 to January 2015, each month 2000 preschool children were randomly selected from Dutch population registries. The parents were invited to complete an epidemiological questionnaire and to obtain and send a faecal sample from the selected child and from one parent. Samples were tested for ESBL/AmpC-producing bacteria. Logistic regression was used to identify risk factors for ESBL/AmpC carriage in children and parents, and findings were internally validated by bootstrapping.
Results
In total, 1016 families were included and ESBL/AmpC prevalence was 4.0% (95% CI 3.2%–5.0%); 3.5% (95% CI 2.5%–4.8%) in children and 4.5% (95% CI 3.4%–6.0%) in parents. Attending a daycare centre (DCC) was the only significant risk factor for children (OR 2.1, 95% CI 1.0–4.3). For parents, the only significant risk factor was having one or more children attending DCCs (OR 2.2, 95% CI 1.2–4.8). For parents of ESBL/AmpC-positive children the OR for ESBL/AmpC carriage was 19.7 (95% CI 9.2–42.4). Co-carriage of specific ESBL/AmpC genotypes in child and parent occurred more often than expected by chance (14.6% versus 1.1%, P < 0.001).
Conclusions
In this study, intestinal carriage with ESBL/AmpCs was detected in ∼4% of households with preschool children. DCC attendance was a risk factor in both children and parents and co-carriage of specific genotypes frequently occurred in child–parent pairs. These findings suggest household transmission or/and family-specific exposure to common sources of ESBL/AmpC-producing bacteria.



Clofazimine has delayed antimicrobial activity against Mycobacterium tuberculosis both in vitro and in vivo

2016-10-25

Objectives
The anti-leprosy drug clofazimine has been shown to have antimicrobial activity against Mycobacterium tuberculosis and has been associated with treatment-shortening activity in both clinical and preclinical studies of TB chemotherapy. However, a reported lack of early bactericidal activity (EBA) in TB patients has raised questions regarding the usefulness of clofazimine as an anti-TB drug. Our objective was to systematically evaluate the EBA of clofazimine in vitro and in vivo to provide insight into how and when this drug exerts its antimicrobial activity against M. tuberculosis.
Methods
We evaluated the 14 day EBA of clofazimine (i) in vitro at concentrations ranging from 4 times below to 4 times above the MIC for M. tuberculosis and (ii) in vivo in infected BALB/c mice at doses ranging from 1.5 to 100 mg/kg/day, and serum clofazimine levels were measured. In both experiments, isoniazid was used as the positive control.
Results
In vitro, clofazimine, at any concentration tested, did not exhibit bactericidal activity during the first week of exposure; however, in the second week, it exhibited concentration-dependent antimicrobial activity. In vivo, clofazimine, at any dose administered, did not exhibit bactericidal activity during the first week, and limited antimicrobial activity was observed during the second week of administration. While serum clofazimine levels were clearly dose dependent, the antimicrobial activity was not significantly related to the dose administered.
Conclusions
Our data suggest that clofazimine's delayed antimicrobial activity may be due more to its mechanism of action rather than to host-related factors.



Rasch analysis of the Antimicrobial Self-Assessment Toolkit for National Health Service (NHS) Trusts (ASAT v17)

2016-10-25

Objectives
The Antimicrobial Self-Assessment Toolkit for National Health Service (NHS) Trusts (ASAT) was developed to evaluate hospital-based antimicrobial stewardship programmes. Iterative validity investigations of the ASAT were used to produce a 91-item ASAT v17 utilizing qualitative methodology. Rasch analysis was used to generate question (item) behaviour estimates and to investigate the validity of ASAT v17.
Methods
In 2012, the partial credit model (PCM) was used to analyse ASAT responses from 33 NHS Trusts within England. WINSTEPS® outputs such as fit statistics and respondent/item maps were examined to determine unidimensionality, item discrimination and item hierarchy. Ordinary least squares regression modelling was used to determine the predictive validity using NHS Trust ability estimates generated from the PCM and corresponding Clostridium difficile rates.
Results
Each domain contained items that were misfitting the PCM (with INFIT MNSQ <0.7 or >1.3), except Domain 3. Subsequent iterative item removal had a negligible effect on the fit indices within most ASAT domains. Scale analysis demonstrated that most items were productive for measurement (n = 81). Respondent/item maps showed ceiling effects (n = 3) and floor effects (n = 1) within ASAT domains. Ordinary least squares regression modelling identified that there was limited predictive validity due to the small positive correlation between the predictor and outcome variables for participating hospitals (ρ = 0.146; P = 0.418).
Conclusions
Rasch analysis was an effective measurement technique for evaluating the validity of ASAT v17 by providing evidence that each sub-scale and the overall scale demonstrated unidimensionality (construct validity). Improved item targeting may be required to improve item discrimination within the toolkit.



Optimization of the strength of the efavirenz/lamivudine/abacavir fixed-dose combination for paediatric patients

2016-10-24

Background
Child-friendly, low-cost, solid, oral fixed-dose combinations (FDCs) of efavirenz with lamivudine and abacavir are urgently needed to improve clinical management and drug adherence for children.
Methods
Data were pooled from several clinical trials and therapeutic drug monitoring datasets from different countries. The number of children/observations was 505/3667 for efavirenz. Population pharmacokinetic analyses were performed using a non-linear mixed-effects approach. For abacavir and lamivudine, data from 187 and 920 subjects were available (population pharmacokinetic models previously published). Efavirenz/lamivudine/abacavir FDC strength options assessed were (I) 150/75/150, (II) 120/60/120 and (III) 200/100/200 mg. Monte Carlo simulations of the different FDC strengths were performed to determine the optimal dose within each of the WHO weight bands based on drug efficacy/safety targets.
Results
The probability of being within the target efavirenz concentration range 12 h post-dose (1–4 mg/L) varied between 56% and 60%, regardless of FDC option. Option I provided a best possible balance between efavirenz treatment failure and toxicity risks. For abacavir and lamivudine, simulations showed that for option I >75% of subjects were above the efficacy target.
Conclusions
According to simulations, a paediatric efavirenz/lamivudine/abacavir fixed-dose formulation of 150 mg efavirenz, 75 mg lamivudine and 150 mg abacavir provided the most effective and safe concentrations across WHO weight bands, with the flexibility of dosage required across the paediatric population.






Screening of anti-mycobacterial compounds in a naturally infected zebrafish larvae model

2016-10-22

Objectives
Mycobacterium tuberculosis is a deadly human pathogen that causes the lung disease TB. M. tuberculosis latently infects a third of the world's population, resulting in ∼1.5 million deaths per year. Due to the difficulties and expense of carrying out animal drug trials using M. tuberculosis and rodents, infections of the zebrafish Danio rerio with Mycobacterium marinum have become a useful surrogate. However, the infection methods described to date require specialized equipment and a high level of operator expertise.
Methods
We investigated whether zebrafish larvae could be naturally infected with bioluminescently labelled M. marinum by immersion, and whether infected larvae could be used for rapid screening of anti-mycobacterial compounds using bioluminescence. We used rifampicin and a variety of nitroimidazole-based next-generation and experimental anti-mycobacterial drugs, selected for their wide range of potencies against M. tuberculosis, to validate this model for anti-mycobacterial drug discovery.
Results
We observed that five of the six treatments (rifampicin, pretomanid, delamanid, SN30488 and SN30527) significantly reduced the bioluminescent signal from M. marinum within naturally infected zebrafish larvae. Importantly, these same five treatments also retarded the growth of M. tuberculosis in vitro. In contrast, only three of the six treatments tested (rifampicin, delamanid and SN30527) retarded the growth of M. marinum in vitro.
Conclusions
We have demonstrated that zebrafish larvae naturally infected with bioluminescent M. marinum M can be used for the rapid screening of anti-mycobacterial compounds with readily available equipment and limited expertise. The result is an assay that can be carried out by a wide variety of laboratories for minimal cost and without high levels of zebrafish expertise.



Monotherapy or combination therapy of isavuconazole and micafungin for treating murine mucormycosis

2016-10-22

Objectives
Previously we demonstrated the benefit of isavuconazole in treating murine mucormycosis due to Rhizopus. We wanted to determine the efficacy of isavuconazole in treating murine mucormycosis caused by Mucor, the second most common cause of the disease. Furthermore, because we previously determined that Rhizopus possesses the target enzyme for echinocandins and micafungin has activity against murine mucormycosis, we compared the activity of combination therapy (isavuconazole + micafungin) with placebo, either drug alone or standard therapy of liposomal amphotericin B (LAmB) in treating pulmonary murine mucormycosis caused by Rhizopus delemar.
Methods
In vitro susceptibility to isavuconazole of Mucorales was evaluated using the CLSI M38-A2 method. Immunosuppressed mice were intratracheally infected with either Mucor circinelloides or R. delemar. Treatment with isavuconazole (orally), micafungin (intraperitoneally), a combination of both or LAmB (intravenously) was compared, with survival and tissue fungal burden serving as primary and secondary endpoints, respectively.
Results
Isavuconazole was as effective as LAmB in prolonging survival of mice infected with M. circinelloides. Against R. delemar-induced mucormycosis, all monotherapy treatments significantly improved survival of mice versus placebo without showing superiority over one another. However, LAmB was superior in lowering fungal burden in target organs. Although combination therapy of isavuconazole + micafungin did not enhance survival of mice over monotherapy, antagonism was not detected between the two drugs.
Conclusion
Isavuconazole is effective in treating pulmonary murine mucormycosis due to Mucor. In addition, combination therapy of isavuconazole + micafungin does not demonstrate synergy and it is not antagonistic against Rhizopus-induced mucormycosis.



Community carriage of ESBL-producing Escherichia coli is associated with strains of low pathogenicity: a Swedish nationwide study

2016-10-22

Objectives
Community carriage of ESBL-producing Escherichia coli (EPE) is common worldwide and there is a need to understand the connection between carriage and infection. We compared the molecular characteristics of EPE among Swedish community carriers with those of EPE causing invasive infections.
Methods
We collected 2134 faecal samples from randomly selected Swedish inhabitants and examined them for the presence of EPE. All participating volunteers answered a questionnaire about putative risk factors for EPE carriage. Suspected EPE isolates (n = 418) from patients with bloodstream infection (BSI) were collected from Swedish laboratories. Isolates were genotypically and phenotypically characterized.
Results
Our results show that the EPE population found in carriers generally had lower pathogenicity compared with the isolates from BSIs, since carriers had a lower proportion of E. coli belonging to phylogroup B2, ST131 and ST131 subclone H30-Rx. Isolates from carriers also had lower levels of multiresistance. The Swedish carriage rate of EPE was 4.7% (101/2134) among healthy volunteers. Risk factors associated with carriage were travel to countries in Asia (OR = 3.6, 95% CI = 1.4–9.2) and Africa (OR = 3.6, 95% CI = 1.7–7.7) and a diet without pork (OR = 0.5, 95% CI = 0.3–0.8 for pork eaters).
Conclusions
E. coli host factors previously associated with higher pathogenicity were all more common in BSIs compared with carriers. This indicates that the risk of invasive infection with EPE may be relatively modest in many community carriers and that EPE carriage of high-risk strains should be the focus of attention for prevention.



Analysis of linezolid and tigecycline as candidates for local prophylaxis via antibiotic-loaded bone cement

2016-10-21

Objectives
To assess the Gram-positive-specific antibiotic linezolid and the broad-spectrum antibiotic tigecycline for use in local antibiotic delivery via antibiotic-loaded bone cement.
Methods
Linezolid and tigecycline were added to Biomet bone cement at varying concentrations. Antibiotic elution over 1 week was quantified by HPLC-MS. The effect of wear on elution over 51 h was determined using a modified TE-66 wear tester. Eluted antibiotics were used to determine the MICs for a panel of clinically relevant bacteria. The impact strength of antibiotic-loaded samples was determined using a Charpy-type impact testing apparatus. Cytotoxicity of eluted antibiotics against MG-63 cells was evaluated using an MTT assay.
Results
Linezolid and tigecycline eluted from bone cement to clinically relevant levels within 1 h and retained activity over 1 week. Mechanical wear significantly reduced elution of tigecycline, but had little effect on elution of linezolid. Linezolid showed low cytotoxicity towards MG-63 cells with ≤300 mg/mL resulting in >50% cell activity. Cytotoxicity of tigecycline was higher, with an IC50 of 5–10 mg/L.
Conclusions
Linezolid and tigecycline retain activity after elution from bone cement. The concentration of tigecycline may need to be carefully controlled due to cytotoxicity. The effect of wear on bone cement may need to be considered if tigecycline is to be used for local delivery. Up to 10% linezolid can be added without affecting the impact strength of the bone cement. These results are promising indications for future investigation of these antibiotics for use in local antibiotic delivery strategies.



Etest ® versus broth microdilution for ceftaroline MIC determination with Staphylococcus aureus : results from PREMIUM, a European multicentre study

2016-10-20

Objectives
To compare the concordance of ceftaroline MIC values by reference broth microdilution (BMD) and Etest (bioMérieux, France) for MSSA and MRSA isolates obtained from PREMIUM (D372SL00001), a European multicentre study.
Methods
Ceftaroline MICs were determined by reference BMD and by Etest for 1242 MSSA and MRSA isolates collected between February and May 2012 from adult patients with community-acquired pneumonia or complicated skin and soft tissue infections; tests were performed across six European laboratories. Selected isolates with ceftaroline resistance in broth (MIC >1 mg/L) were retested in three central laboratories to confirm their behaviour.
Results
Overall concordance between BMD and Etest was good, with >97% essential agreement and >95% categorical agreement. Nevertheless, 12 of the 26 MRSA isolates found resistant by BMD scored as susceptible by Etest, with MICs ≤1 mg/L, thus counting as very major errors, whereas only 5 of 380 MRSA isolates found ceftaroline susceptible in BMD were miscategorized as resistant by Etest. Twenty-one of the 26 isolates with MICs of 2 mg/L by BMD were then retested twice by each of three central laboratories: BMD MICs of 2 mg/L were consistently found for 19 of the 21 isolates. Among 147 Etest results for these 21 isolates (original plus six repeats per isolate) 112 were >1 mg/L.
Conclusions
BMD and Etest have good overall agreement for ceftaroline against Staphylococcus aureus; nevertheless, reliable Etest-based discrimination of the minority of ceftaroline-resistant (MIC 2 mg/L) MRSA is extremely challenging, requiring careful reading of strips, ideally with duplicate testing.



Phenotypic detection of the cfiA metallo-β-lactamase in Bacteroides fragilis with the meropenem–EDTA double-ended Etest and the ROSCO KPC/MBL Confirm Kit

2016-10-20

Objectives
To investigate the performance of the meropenem and imipenem double-ended Etest ± EDTA and the tablet-based (meropenem and meropenem + dipicolinic acid) KPC/MBL Confirm Kit to detect cfiA metallo-β-lactamase (MBL) in Bacteroides fragilis.
Methods
Well-characterized B. fragilis isolates, most from previously published studies, harbouring the cfiA gene and covering a wide range of meropenem MICs were included (n = 21).
Results
The imipenem double-ended Etest showed an indeterminate result in 95% of the included isolates with the cfiA gene (20 of 21), whereas the meropenem double-ended Etest gave an MIC ratio ≥8 (positive test) with all the isolates. All isolates that were meropenem intermediate or resistant had a zone diameter difference ≥6 mm with the KPC/MBL Confirm Kit.
Conclusions
The meropenem double-ended Etest and not imipenem should be preferred for phenotypic detection of MBLs in B. fragilis. The KPC/MBL Confirm Kit could be an alternative with isolates that are meropenem intermediate or resistant (MIC >2 mg/L).



Daclatasvir 30 mg/day is the correct dose for patients taking atazanavir/cobicistat

2016-10-20

Background
Atazanavir is boosted with the cytochrome P450 (CYP) 3A4 inhibitor ritonavir. When combined with the CYP3A4 substrate daclatasvir, the daclatasvir dosage should be reduced from 60 to 30 mg once daily. Recently, cobicistat was licensed as a CYP3A booster and used with atazanavir.
Objectives
To determine whether the fixed-dose combination of atazanavir/cobicistat has an influence on daclatasvir pharmacokinetics comparable to that of the separate agents atazanavir and ritonavir.
Methods
A prospective, open-label, two-period, randomized, cross-over trial was performed in 16 healthy subjects (NCT02565888). Treatment consisted of 300/100 mg of atazanavir/ritonavir plus 30 mg of daclatasvir once daily (reference) and a second period of 300/150 mg of atazanavir/cobicistat plus 30 mg of daclatasvir once daily (test). A 24 h pharmacokinetic, steady-state curve was recorded for all drugs. Geometric mean ratios (GMRs) with 90% CI were calculated for daclatasvir and atazanavir AUCτ and Cmax to compare the effect of both treatments (test versus reference). Laboratory safety and adverse events were evaluated throughout the trial.
Results
All 16 healthy subjects completed the study. Median (range) age and BMI were 48.5 (21–55) years and 24.5 (19.0–29.2) kg/m2, respectively. Pharmacokinetic parameters of ritonavir and cobicistat were comparable to those in the literature. The GMRs (90% CI) of daclatasvir AUCτ and Cmax (test versus reference) were 101% (92%–111%) and 97% (89%–106%), respectively. Atazanavir GMRs (90% CI) of AUCτ and Cmax were 82% (75%–79%) and 74% (68%–81%), respectively. No serious adverse events were reported.
Conclusions
Atazanavir/cobicistat and atazanavir/ritonavir had a similar influence on daclatasvir pharmacokinetics in healthy volunteers. Daclatasvir at 30 mg once daily is the correct dose when combined with atazanavir/cobicistat.



Impact of antibiotic de-escalation on clinical outcomes in community-acquired pneumococcal pneumonia

2016-10-20

Background
Although antibiotic de-escalation is regarded as a measure that reduces selection pressure, adverse drug effects and costs, evidence supporting this practice in community-acquired pneumococcal pneumonia (CAPP) is lacking.
Methods
We carried out a retrospective analysis of prospectively collected data of a cohort of hospitalized adults with CAPP. Pneumococcal aetiology was established in patients with one or more positive cultures for Streptococcus pneumoniae obtained from blood, sterile fluids or sputum, and/or a positive urinary antigen test. De-escalation therapy was considered when the initial antibiotic therapy was narrowed to penicillin, amoxicillin or amoxicillin/clavulanate within the first 72 h after admission. The primary outcomes were 30 day mortality and length of hospital stay (LOS). Adjustment for confounders was performed with multivariate and propensity score analyses.
Results
Of 1410 episodes of CAPP, antibiotic de-escalation within the first 72 h after admission was performed in 166 cases. After adjustment, antibiotic de-escalation was not associated with a higher risk of mortality (OR = 0.83, 95% CI = 0.24–2.81), but it was found to be a protective factor for prolonged LOS (above the median) (OR = 0.46, 95% CI = 0.30–0.70). Similar results were found in patients classified into high-risk pneumonia severity index classes (IV–V), those with clinical instability and those with bacteraemia. No significant differences were documented in adverse drug reactions or readmission (<30 days).
Conclusions
Antibiotic de-escalation seems to be safe and effective in reducing the duration of LOS, and did not adversely affect outcomes of patients with CAPP, even those with bacteraemia and severe disease, and those who were clinically unstable.



Antibiotic resistance among Ureaplasma spp. isolates: cause for concern?

2016-10-20

There is growing global concern regarding the rise of antibiotic-resistant organisms. Many of these reports have focused on various Gram-positive and Gram-negative pathogens, with little attention to the genus Ureaplasma. Ureaplasma spp. are associated with numerous infectious diseases affecting pregnant women, neonates and the immunocompromised. Treatment options are extremely limited due to high levels of intrinsic resistance resulting from the unique physiology of these organisms and further restricted in cases of the developing fetus or neonate, often limiting therapeutic options to predominantly macrolides or rarely fluoroquinolones. The increasing presence of macrolide- and fluoroquinolone-resistant strains among neonatal infections may result in pan-drug resistance and potentially untreatable conditions. Here, we review the requirements for accurate measurement of antimicrobial susceptibility, provide a comprehensive review of the antimicrobial resistance (AMR) for Ureaplasma species in the literature and contextualize these results relative to some investigators' reliance on commercial kits that are not CLSI compliant when determining AMR. The dramatic variation in the resistance patterns and impact of high levels of AMR amongst neonatal populations suggests the need for continued surveillance. Commercial kits represent an excellent tool for initial antibiotic susceptibility determination and screening. However, AMR reporting must utilize internationally standardized methods, as high-titre samples, or Mycoplasma hominis-contaminated samples routinely give false AMR results. Furthermore, there is a requirement for future reports to determine the underlying AMR mechanisms and determine whether expanding AMR is due to spontaneous mutation, transmission of resistance genes on mobile elements or selection and expansion of resistant clones.



Mechanisms of action and therapeutic efficacies of the lipophilic antimycobacterial agents clofazimine and bedaquiline

2016-10-20

Drug-resistant (DR)-TB is the major challenge confronting the global TB control programme, necessitating treatment with second-line anti-TB drugs, often with limited therapeutic efficacy. This scenario has resulted in the inclusion of Group 5 antibiotics in various therapeutic regimens, two of which promise to impact significantly on the outcome of the therapy of DR-TB. These are the ‘re-purposed’ riminophenazine, clofazimine, and the recently approved diarylquinoline, bedaquiline. Although they differ structurally, both of these lipophilic agents possess cationic amphiphilic properties that enable them to target and inactivate essential ion transporters in the outer membrane of Mycobacterium tuberculosis. In the case of bedaquiline, the primary target is the key respiratory chain enzyme F1/F0-ATPase, whereas clofazimine is less selective, apparently inhibiting several targets, which may underpin the extremely low level of resistance to this agent. This review is focused on similarities and differences between clofazimine and bedaquiline, specifically in respect of molecular mechanisms of antimycobacterial action, targeting of quiescent and metabolically active organisms, therapeutic efficacy in the clinical setting of DR-TB, resistance mechanisms, pharmacodynamics, pharmacokinetics and adverse events.






MRSA infections among patients in the emergency department: a European multicentre study

2016-10-17

Background
MRSA is a therapeutic concern worldwide, and a major agent of community-acquired skin and soft tissue infections (CA-SSTIs). While the US epidemiology of MRSA in CA-SSTIs is well described and reports the high prevalence of the USA300 clone, data on the European situation are lacking.
Objectives
To determine the prevalence and clonal characteristics of MRSA in CA-SSTIs in seven European emergency departments.
Patients and methods
From April to June 2015, patients presenting to the tertiary hospital emergency department with a Staphylococcus aureus CA-SSTI were prospectively enrolled. S. aureus isolates were characterized by antimicrobial susceptibility testing, detection of Panton–Valentine leucocidin encoding genes and spa-typing, MLST and/or DNA microarray.
Results
Two-hundred and five cases of S. aureus-associated CA-SSTIs were included, comprising folliculitis, furuncles, abscesses, paronychia, impetigo, carbuncles and cellulitis. Of the 205 cases, we report an MRSA prevalence rate of 15.1%, with a north (0%) to south (29%) increasing gradient. Fifty-one isolates were Panton–Valentine leucocidin-positive (24.9%), whether MSSA or MRSA, with a heterogeneous distribution between countries. Clonal distribution of MSSA and MRSA showed high diversity, with no predominant circulating clone and no archetypical USA300 CA-MRSA clone.
Conclusions
This original prospective multicentre study highlights stark differences in European MRSA epidemiology compared with the USA, and that the USA300 CA-MRSA clone is not predominant among community-infected patients in Europe.



In vitro evaluation of the effect of linezolid and levofloxacin on Bacillus anthracis toxin production, spore formation and cell growth

2016-10-17

Background
Owing to its ability to form spores and toxins, Bacillus anthracis is considered a bioterror agent. Although current therapeutic strategies can be effective, treatment does not prevent sporulation and toxin production.
Objectives
To quantify the combined effect of a protein synthesis inhibitor and a bactericidal agent on B. anthracis toxin production, sporulation and cell growth.
Methods
Susceptibility and synergy titrations were conducted on B. anthracis Sterne and 03-0191 strains using linezolid and levofloxacin. The effect of antibiotic exposure on cell viability was evaluated using a continuous medium replacement model. In vitro static models were used to study the effect of linezolid and levofloxacin on sporulation and toxin production. Spores were quantified using the heat shock method. Toxin was quantified via commercial ELISA.
Results
Synergy titrations indicated that the combination was synergistic or indifferent; however, in all models antagonism was observed. In the spore model, linezolid resulted in the lowest sporulation rates, while combination therapy resulted in the highest. In the toxin model, linezolid prevented toxin production altogether.
Conclusions
This study advances our understanding of the effects of combination therapy on B. anthracis infection. Used alone, linezolid therapy abolishes toxin production and reduces sporulation. These results suggest that studies using a step-wise approach using linezolid initially to stop sporulation and toxin production followed by levofloxacin to rapidly kill vegetative B. anthracis can be recommended.









Efficacy and safety of 3 day versus 7 day cefditoren pivoxil regimens for acute uncomplicated cystitis: multicentre, randomized, open-label trial

2016-10-11

Background
Fluoroquinolone-non-susceptible Escherichia coli isolated from patients with acute uncomplicated cystitis are a matter of increasing concern. Cefditoren pivoxil is an oral, β-lactamase-stable, extended-spectrum cephalosporin that is effective against fluoroquinolone-non-susceptible bacteria.
Objectives
To evaluate the clinical and microbiological efficacies of cefditoren pivoxil against acute uncomplicated cystitis and to determine the optimal duration of cefditoren pivoxil treatment.
Methods
We compared 3 and 7 day regimens of cefditoren pivoxil in a multicentre, randomized, open-label study.
Results
A total of 104 female patients with acute uncomplicated cystitis were enrolled and randomized into 3 day (n = 51) or 7 day (n = 53) treatment groups. At first visit, 94 bacterial strains were isolated from the 104 participants of which 81.7% (85/104) were E. coli. Clinical and microbiological efficacies were evaluated 5–9 days following administration of the final dose of cefditoren pivoxil. The clinical efficacies of the 3 and 7 day groups were 90.9% (40/44) and 93.2% (41/44), respectively (P =1.000). The microbiological efficacies of the 3 and 7 day groups were 82.5% (33/40) and 90.2% (37/41), respectively (P =0.349). There were no adverse events due to cefditoren pivoxil treatment, with the exception of a mild allergic reaction in one patient, after which the cefditoren pivoxil was exchanged for another antimicrobial.
Conclusions
Cefditoren pivoxil is safe and effective for uncomplicated cystitis, with no significant differences in clinical and microbiological efficacies between 3 and 7 day regimens.



Mixed gonococcal infections in a high-risk population, Sydney, Australia 2015: implications for antimicrobial resistance surveillance?

2016-10-05

Objectives
Previous studies have shown that mixed-strain gonococcal infections can occur. However, it remains unclear whether such infections impact upon the reliability of Neisseria gonorrhoeae antimicrobial resistance (AMR) surveillance. In this study, we aimed to resolve this question by intensively sampling isolates from gonorrhoea-positive specimens in a high-risk population in Sydney, Australia.
Methods
A total of 615 N. gonorrhoeae isolates, originating from 63 clinical samples (31 rectal swabs and 32 throat swabs), were characterized. All isolates were subject to N. gonorrhoeae identification, antimicrobial susceptibility testing and genotyping by SNP-based MLST.
Results
Only 2 of the 63 (3.2%) samples provided evidence of mixed-strain infections. These comprised two rectal swabs that harboured isolates of different SNP-based MLST genotypes; however, the AMR susceptibility profiles of the different genotypes from these samples were indistinguishable. Within-sample differences in the AMR susceptibility profiles were observed for a further seven samples; however, the differences were not considered significant; MIC values were typically within a 2-fold difference or were close to test breakpoints.
Conclusions
Results of this study provide further evidence that mixed-strain gonococcal infections do occur, although at low prevalence. Our data indicate that at a population level such infections are unlikely to impact significantly upon N. gonorrhoeae AMR surveillance.



In vitro ‘time-to-kill’ assay to assess the cidal activity dynamics of current reference drugs against Leishmania donovani and Leishmania infantum

2016-10-05

Objectives
Despite a continued search for novel antileishmanial drugs, treatment options remain restricted to a few standard drugs, e.g. antimonials, miltefosine, amphotericin B and paromomycin. Although these drugs have now been used for several decades, their mechanism of action still remains partly hypothetical and their dynamics of cidal action and time-to-kill are still poorly documented.
Methods
An in vitro time-to-kill assay on intracellular amastigotes of the laboratory reference strains Leishmania donovani (MHOM/ET/67/L82) and Leishmania infantum [MHOM/MA(BE)/67/ITMAP263] evaluated the cidal action dynamics of the listed reference drugs at three different concentrations: at IC50, 2 × IC50 and the near cytotoxic dose level (CC90: determined on MRC-5 cells). This assay focused on identifying the minimal exposure time needed to completely eliminate viable intracellular amastigotes, using the standard microscopic Giemsa assay and the promastigote back-transformation assay.
Results
While 100% reduction was microscopically apparent for most drugs, the promastigote back-transformation assay clearly demonstrated a concentration- and time-dependent cidal mechanism. The time-to-kill at 2 × IC50 was ≥240 h for pentavalent antimony (77 μg eq./mL), 96 h for trivalent antimony (44 μg eq./mL), 168 to >240 h for miltefosine (10 μM), 168 h for paromomycin (100 μM) and >240 h for amphotericin B (2 μM). No differences were noted between both Leishmania species.
Conclusions
Evaluation of the concentration- and time-dependent cidal activity using the promastigote back-transformation assay revealed striking differences in efficacy of the different antileishmanial reference drugs. This assay should allow in-depth pharmacodynamic evaluation of novel drug leads in comparison with the existing antileishmanial drug repertoire.



Carbapenemase-producing Enterobacteriaceae in the UK: a national study (EuSCAPE-UK) on prevalence, incidence, laboratory detection methods and infection control measures

2016-09-29

Objectives
To estimate UK prevalence and incidence of clinically significant carbapenemase-producing Enterobacteriaceae (CPE), and to determine epidemiological characteristics, laboratory methods and infection prevention and control (IPC) measures in acute care facilities.
Methods
A 6 month survey was undertaken in November 2013–April 2014 in 21 sentinel UK laboratories as part of the European Survey on Carbapenemase-Producing Enterobacteriaceae (EuSCAPE) project. Up to 10 consecutive, non-duplicate, clinically significant and carbapenem-non-susceptible isolates of Escherichia coli or Klebsiella pneumoniae were submitted to a reference laboratory. Participants answered a questionnaire on relevant laboratory methods and IPC measures.
Results
Of 102 isolates submitted, 89 (87%) were non-susceptible to ≥1 carbapenem, and 32 (36%) were confirmed as CPE. CPE were resistant to most antibiotics, except colistin (94% susceptible), gentamicin (63%), tigecycline (56%) and amikacin (53%). The prevalence of CPE was 0.02% (95% CI = 0.01%–0.03%). The incidence of CPE was 0.007 per 1000 patient-days (95% CI = 0.005–0.010), with north-west England the most affected region at 0.033 per 1000 patient-days (95% CI = 0.012–0.072). Recommended IPC measures were not universally followed, notably screening high-risk patients on admission (applied by 86%), using a CPE ‘flag’ on patients’ records (70%) and alerting neighbouring hospitals when transferring affected patients (only 30%). Most sites (86%) had a laboratory protocol for CPE screening, most frequently using chromogenic agar (52%) or MacConkey/CLED agars with carbapenem discs (38%).
Conclusions
The UK prevalence and incidence of clinically significant CPE is currently low, but these MDR bacteria affect most UK regions. Improved IPC measures, vigilance and monitoring are required.









Genetic characterization of mcr-1 -bearing plasmids to depict molecular mechanisms underlying dissemination of the colistin resistance determinant

2016-09-28

Objectives
To analyse and compare mcr-1-bearing plasmids from animal Escherichia coli isolates, and to investigate potential mechanisms underlying dissemination of mcr-1.
Methods
Ninety-seven ESBL-producing E. coli strains isolated from pig farms in China were screened for the mcr-1 gene. Fifteen mcr-1-positive strains were subjected to molecular characterization and bioinformatic analysis of the mcr-1-bearing plasmids that they harboured.
Results
Three major types of mcr-1-bearing plasmids were recovered: IncX4 (∼33 kb), IncI2 (∼60 kb) and IncHI2 (∼216–280 kb), among which the IncX4 and IncI2 plasmids were found to harbour the mcr-1 gene only, whereas multiple resistance elements including blaCTX-M, blaCMY, blaTEM, fosA, qnrS, floR and oqxAB were detected, in various combinations, alongside mcr-1 in the IncHI2 plasmids. The profiles of mcr-1-bearing plasmids in the test strains were highly variable, with coexistence of two mcr-1-bearing plasmids being common. However, the MIC of colistin was not affected by the number of mcr-1-carrying plasmids harboured. Comparative analysis of the plasmids showed that they contained an mcr-1 gene cassette with varied structures (mcr-1-orf, ISApl1-mcr-1



In vitro activity of daptomycin combined with dalbavancin and linezolid, and dalbavancin with linezolid against MRSA strains

2016-09-28

Objectives
Combination therapies have a distinct advantage over monotherapies in terms of their broad spectrum, synergistic effect and prevention of the emergence of drug resistance. In the present study, the in vitro antibacterial activity of daptomycin combinations with linezolid and dalbavancin, and dalbavancin with linezolid were evaluated against 30 clinical MRSA strains.
Methods
The MICs of all antibiotics were determined using microbroth dilution as described by the CLSI. The in vitro activities of antibiotics in combination were assessed by using a microbroth ‘chequerboard’ assay. The MIC values of all antibiotics determined were evaluated in accordance with the recommendations of the CLSI for daptomycin and linezolid, and the FDA for dalbavancin.
Results
All strains (100%) were found to be susceptible to daptomycin, dalbavancin and linezolid. The MIC50, MIC90 and MICrange values of these antibiotics were determined to be 1, 1 and 0.5–1 mg/L, 0.12, 0.12 and 0.03–0.12 mg/L, and 1, 2 and 1–2 mg/L, respectively. The rates of synergistic effects were 67% for daptomycin combined with dalbavancin and with linezolid, and 60% for dalbavancin combined with linezolid.
Conclusions
The results of this study show that in vitro combinations of these new antimicrobials will be effective in the therapy of MRSA infections.



Single-dose pharmacokinetics and pharmacodynamics of oral tenofovir and emtricitabine in blood, saliva and rectal tissue: a sub-study of the ANRS IPERGAY trial

2016-09-28

Objectives
In the ANRS IPERGAY pre-exposure prophylaxis (PrEP) trial, a single dose of tenofovir disoproxil fumarate and emtricitabine was taken orally 2–24 h before sexual intercourse. A sub-study was conducted to assess the pharmacokinetics of tenofovir and emtricitabine in blood, saliva and rectal tissue following this initial oral intake.
Methods
Plasma, PBMC, saliva and rectal tissue sampling was performed over 24 h in 12 seronegative men before enrolment in the ANRS IPERGAY trial, following a single dose of 600 mg tenofovir disoproxil fumarate/400 mg emtricitabine. Ex vivo HIV infectibility of rectal biopsies was also assessed.
Results
The median plasma Tmax of tenofovir (median Cmax: 401 μg/L) and emtricitabine (median Cmax: 2868 μg/L) was obtained 1 h (range: 0.5–4) and 2 h (range: 1–4) after dosing, respectively. The median C24 of tenofovir and emtricitabine was 40 and 63 μg/L, respectively. The median PBMC tenofovir diphosphate and emtricitabine triphosphate levels were 12.2 and 16.7 fmol/106 cells and 2800 and 2000 fmol/106 cells at 2 and 24 h after dosing, respectively. Saliva/plasma AUC0–24 ratios were 2% and 17% for tenofovir and emtricitabine, respectively. Emtricitabine was detected in rectal tissue 30 min after dosing, whereas tenofovir was only detectable at 24 h. Ex vivo HIV infectibility assays of rectal biopsies showed partial protection after dosing (P < 0.07).
Discussion
A single high dose of oral tenofovir disoproxil fumarate/emtricitabine provides rapid and high blood levels of tenofovir and emtricitabine, with rapid diffusion of emtricitabine in saliva and rectal tissue.