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Journal of Antimicrobial Chemotherapy Current Issue





Published: Fri, 21 Oct 2016 00:00:00 GMT

Last Build Date: Mon, 14 Nov 2016 09:43:37 GMT

 



Contribution of JAC to antimicrobial stewardship

2016-10-21

Abstract
Antimicrobial stewardship programmes are increasingly being used to improve the quality of antimicrobial prescribing, with the dual aim of optimizing clinical outcomes and minimizing the emergence and spread of antimicrobial resistance. The Journal of Antimicrobial Chemotherapy (JAC) is celebrating its 40th anniversary and, as part of activities to commemorate this event, this article highlights the contribution of JAC to antimicrobial stewardship. Papers published in JAC have contributed to the evidence base for stewardship, have highlighted educational and behavioural change initiatives aimed at improving antibiotic prescribing practice, and have actively sought to foster the practice of antimicrobial stewardship amongst its readers.






Population pharmacokinetics and pharmacodynamics of teicoplanin in neonates: making better use of C-reactive protein to deliver individualized therapy

2016-08-19

Objectives
There is uncertainty about the optimal teicoplanin regimens for neonates. The study aim was to determine the population pharmacokinetics (PK) of teicoplanin in neonates, evaluate currently recommended regimens and explore the exposure–effect relationships.
Methods
An open-label PK study was conducted. Neonates from 26 to 44 weeks post-menstrual age were recruited (n = 18). The teicoplanin regimen was a 16 mg/kg loading dose, followed by 8 mg/kg once daily. Therapeutic drug monitoring and dose adjustment were not conducted. A standard two-compartment PK model was developed, followed by models that incorporated weight. A PK/pharmacodynamic (PD) model with C-reactive protein serial measurements as the PD input was fitted to the data. Monte Carlo simulations (n = 5000) were performed using Pmetrics. The AUCs at steady state and the proportion of patients achieving the recommended drug exposures (i.e. Cmin >15 mg/L) were determined. The study was registered in the European Clinical Trials Database Registry (EudraCT: 2012-005738-12).
Results
The PK allometric model best accounted for the observed data. The PK parameters medians were: clearance = 0.435 × (weight/70)0.75 (L/h); volume = 0.765 (L); Kcp = 1.3 (h−1); and Kpc = 0.629 (h−1). The individual time-course of C-reactive protein was well described using the Bayesian posterior estimates for each patient. The simulated median AUC96-120 was 302.3 mg·h/L and the median Cmin at 120 h was 12.9 mg/L; 38.8% of patients attained a Cmin >15 mg/L by 120 h.
Conclusions
Teicoplanin population PK is highly variable in neonates, weight being the best descriptor of PK variability. A low percentage of neonates were able to achieve Cmin >15 mg/L. The routine use of therapeutic drug monitoring and improved knowledge on the PD of teicoplanin is required.



Resistance suppression by high-intensity, short-duration aminoglycoside exposure against hypermutable and non-hypermutable Pseudomonas aeruginosa

2016-08-11

Objectives
Hypermutable bacteria are causing a drastic problem via their enhanced ability to become resistant. Our objectives were to compare bacterial killing and resistance emergence between differently shaped tobramycin concentration–time profiles at a given fAUC/MIC, and determine the tobramycin exposure durations that prevent resistance.
Methods
Static concentration time–kill studies over 24 h used Pseudomonas aeruginosa WT strains (ATCC 27853 and PAO1) and hypermutable PAOΔmutS. fAUC/MIC values of 36, 72 and 168 were assessed at initial inocula of 106 and 104 cfu/mL (all strains) and 101.2 cfu/mL (PAOΔmutS only) in duplicate. Tobramycin was added at 0 h and removed at 1, 4, 10 or 24 h. Proportions of resistant bacteria and MICs were determined at 24 h. Mechanism-based modelling was conducted.
Results
For all strains, high tobramycin concentrations over 1 and 4 h resulted in more rapid and extensive initial killing compared with 10 and 24 h exposures at a given fAUC/MIC. No resistance emerged for 1 and 4 h durations of exposure, although extensive regrowth of susceptible bacteria occurred. The 24 h duration of exposure revealed less regrowth, but tobramycin-resistant populations had completely replaced susceptible bacteria by 24 h for the 106 cfu/mL inoculum. The hypermutable PAOΔmutS showed the highest numbers of resistant bacteria. Total and resistant bacterial counts were described well by novel mechanism-based modelling.
Conclusions
Extensive resistance emerged for 10 and 24 h durations of exposure, but not for shorter durations. The tobramycin concentration–time profile shape is vital for resistance prevention and should aid the introduction of optimized combination regimens.



Improved adipose tissue function with initiation of protease inhibitor-only ART

2016-08-11

Objectives
Use of ART containing HIV PIs has previously been associated with toxicity in subcutaneous adipose tissue (SAT), potentially contributing to the development of lipodystrophy and insulin resistance. However, the effect of PIs on SAT function in ART-naive patients independent of other ART classes is unknown. This study aimed to elucidate the effect of initiating PI-only ART on SAT function in ART-naive subjects.
Methods
In the HIVNAT-019 study, 48 HIV-infected, ART-naive Thai adults commencing PI-only ART comprising lopinavir/ritonavir/saquinavir for 24 weeks underwent assessments of fasting metabolic parameters and body composition. In a molecular substudy, 20 subjects underwent SAT biopsies at weeks 0, 2 and 24 for transcriptional, protein, mitochondrial DNA (mtDNA) and histological analyses. ClinicalTrials.gov registration number: NCT00400738.
Results
Over 24 weeks, limb fat increased (+416.4 g, P = 0.023), coinciding with larger adipocytes as indicated by decreased adipocyte density in biopsies (−32.3 cells/mm2, P = 0.047) and increased mRNA expression of adipogenesis regulator PPARG at week 2 (+58.1%, P = 0.003). Increases in mtDNA over 24 weeks (+600 copies/cell, P = 0.041), decreased NRF1 mRNA expression at week 2 (−33.7%, P < 0.001) and increased COX2/COX4 protein ratio at week 24 (+288%, P = 0.038) indicated improved mitochondrial function. Despite decreased AKT2 mRNA at week 2 (−28.6%, P = 0.002) and increased PTPN1 mRNA at week 24 (+50.3%, P = 0.016) suggesting insulin resistance, clinical insulin sensitivity [by homeostasis model assessment (HOMA-IR)] was unchanged.
Conclusions
Initiation of PI-only ART showed little evidence of SAT toxicity, the changes observed being consistent with a return to health rather than contributing to lipodystrophy.



Understanding the culture of antimicrobial prescribing in agriculture: a qualitative study of UK pig veterinary surgeons

2016-08-11

Objectives
The use of antimicrobials in food-producing animals has been linked with the emergence of antimicrobial resistance in bacterial populations, with consequences for animal and public health. This study explored the underpinning drivers, motivators and reasoning behind prescribing decisions made by veterinary surgeons working in the UK pig industry.
Methods
A qualitative interview study was conducted with 21 veterinary surgeons purposively selected from all UK pig veterinary surgeons. Thematic analysis was used to analyse transcripts.
Results
Ensuring optimum pig health and welfare was described as a driver for antimicrobial use by many veterinary surgeons and was considered a professional and moral obligation. Veterinary surgeons also exhibited a strong sense of social responsibility over the need to ensure that antimicrobial use was responsible. A close relationship between management practices, health and economics was evident, with improvements in management commonly identified as being potential routes to reduce antimicrobial usage; however, these were not always considered economically viable. The relationship with clients was identified as being a source of professional stress for practitioners due to pressure from farmers requesting antimicrobial prescriptions, and concern over poor compliance of antimicrobial administration by some farmers.
Conclusions
The drivers behind prescribing decisions by veterinary surgeons were complex and diverse. A combination of education, improving communication between veterinary surgeons and farmers, and changes in regulations, in farm management and in consumer/retailer demands may all be needed to ensure that antimicrobial prescribing is optimal and to achieve significant reductions in use.



Identification of non-PBP2a resistance mechanisms in Staphylococcus aureus after serial passage with ceftaroline: involvement of other PBPs

2016-08-05

Objectives
Ceftaroline (the active metabolite of ceftaroline fosamil) is a cephalosporin that possesses activity against MRSA due to its differentiating high affinity for PBP2a. It is known that PBP2a sequence variations, including some outside of the transpeptidase-binding pocket, impact ceftaroline susceptibility and recent evidence suggests involvement of non-PBP2a mechanisms in ceftaroline resistance. This study evaluated the potential of ceftaroline to select for resistant Staphylococcus aureus clones during serial passage.
Methods
Selection experiments were performed by up to 20 daily passages of three S. aureus isolates (two MRSA and one MSSA) in broth with increasing selective pressure. Mutants that emerged were tested for changes in ceftaroline susceptibility and genetically characterized.
Results
The MSSA isolate developed mutations in PBP2 and PBP3 that increased the ceftaroline MIC by 16-fold and increased the MICs of other β-lactams. A Glu447Lys substitution in the PBP2a transpeptidase pocket in one MRSA isolate elevated the ceftaroline MIC to 8 mg/L. Selective pressure in a ceftaroline-resistant MRSA isolate generated mutations in LytD, as well as changes in the pbp4 promoter previously shown to result in PBP4 overexpression, the one PBP not inhibited by ceftaroline. Elevated ceftaroline MIC was reversed when tested in combination with extremely low levels of methicillin or meropenem that could inhibit the function of PBP4.
Conclusions
These studies demonstrate that resistance to ceftaroline can be manifested through numerous mechanisms. Further, they support a hypothesis where PBP4 can functionally provide the essential transpeptidase activity required for MRSA cell wall biogenesis when PBP2a is inhibited.



Aminoglycoside-associated acute kidney injury in elderly patients with and without shock

2016-08-05

Abstract
Objectives
Multiresistant Gram-negative pathogens pose major healthcare concerns with a limited therapeutic armamentarium. Aminoglycosides (AG) are under-utilized due to nephrotoxicity. We aimed to evaluate AG-associated acute kidney injury (AG-AKI) in elderly inpatients, with and without shock.
Methods
We examined the incidence and predictors of AG-AKI by KDIGO criteria and extended renal dysfunction (ERD) in patients aged >60 years. ERD represented a composite of hospital mortality or absence of renal recovery over 6 months following AG-AKI.
Results
Two hundred and seventy-eight patients (aged 74 ± 8 years) were studied; 43% and 19% received >7 and >10 days of AG therapy, respectively, and 70% gentamicin (versus amikacin). Thirteen per cent had shock and 17% developed AG-AKI. Comparing all patients with shock versus no shock, AG-AKI developed in 33% versus 14%, respectively (P = 0.005); correspondingly among 47 patients with AG-AKI, more with shock had stage 2/3 AKI (92% versus 43%) and dialysis (50% versus 9%) (P < 0.01), but more had other strong AKI confounders than AG therapy alone (83% versus 40%, P = 0.02). Multivariate analyses identified mechanical ventilation, frusemide administration and AG therapy >10 days as predictors of AG-AKI (P < 0.05), whereas shock, pneumonia and frusemide administration predicted more severe stage 2/3 AG-AKI (P < 0.05). Hospital mortality was 30% versus 7% with AG-AKI versus none (P < 0.001). Twenty-three of 211 (11%) patients with extended analysis had ERD, with 47% experiencing renal recovery following AG-AKI. Mechanical ventilation and contrast administration during index hospitalization predicted ERD (P < 0.05).
Conclusions
AG-AKI is common in the elderly, with a significant risk of ERD, but the cause and severity are greatly influenced by critical illness and shock, more so than AG therapy alone.



Pharmacodynamics for antifungal drug development: an approach for acceleration, risk minimization and demonstration of causality

2016-08-05

Abstract
The treatment of invasive fungal diseases constitutes a significant unmet medical need. There are relatively few antifungal agents in clinical development and a paucity of novel targets. Morbidity and mortality remain high and clinical outcomes are compromised by submaximal efficacy, emergence of drug resistance and drug-related toxicity. Thus, new antifungal agents are urgently required. A deep understanding of exposure–response relationships underpins the development of safe and effective clinical regimens of any therapeutic agent. Pharmacokinetics (PK) and pharmacodynamics (PD) is increasingly recognized as a vital tool in the development of new antimicrobial agents and maximizes the probability that the right dose will be studied the first time. There is currently no information or agreement as to what constitutes an adequate PK/PD package for the development of a new antifungal agent. This review provides a summary of the achievements of antifungal PK/PD for the treatment of invasive candidiasis, invasive aspergillosis and cryptococcal meningoencephalitis, and outlines the necessary components of a PK/PD package for a new antifungal agent. Such information is critical for the accelerated and efficient development of new agents and enables improved clinical outcomes to be secured.



Polymyxin B in combination with doripenem against heteroresistant Acinetobacter baumannii : pharmacodynamics of new dosing strategies

2016-08-03

Objectives
Polymyxin B is being increasingly utilized as a last resort against resistant Gram-negative bacteria. We examined the pharmacodynamics of novel dosing strategies for polymyxin B combinations to maximize efficacy and minimize the emergence of resistance and drug exposure against Acinetobacter baumannii.
Methods
The pharmacodynamics of polymyxin B together with doripenem were evaluated in time–kill experiments over 48 h against 108 cfu/mL of two polymyxin-heteroresistant A. baumannii isolates (ATCC 19606 and N16870). Pharmacokinetic/pharmacodynamic relationships were mathematically modelled using S-ADAPT. A hollow-fibre infection model (HFIM) was also used to simulate clinically relevant polymyxin B dosing strategies (traditional, augmented ‘front-loaded’ and ‘burst’ regimens), together with doripenem, against an initial inoculum of 109 cfu/mL of ATCC 19606.
Results
In static time–kill studies, polymyxin B concentrations >4 mg/L in combination with doripenem 25 mg/L resulted in rapid bactericidal activity against both strains with undetectable bacterial counts by 24 h. The mathematical model described the rapid, concentration-dependent killing as subpopulation and mechanistic synergy. In the HFIM, the traditional polymyxin B combination regimen was synergistic, with a >7.5 log10 reduction by 48 h. The polymyxin B ‘front-loaded’ combination resulted in more rapid and extensive initial killing (>8 log10) within 24 h, which was sustained over 10 days. With only 25% of the cumulative drug exposure, the polymyxin B ‘burst’ combination demonstrated antibacterial activity similar to traditional and ‘front-loaded’ combination strategies. The polymyxin B ‘front-loaded’ and ‘burst’ combination regimens suppressed the emergence of resistance.
Conclusions
Early aggressive dosing regimens for polymyxin combinations demonstrate promise for treatment of heteroresistant A. baumannii infections.



Management of skin and soft-tissue infections at a community teaching hospital using a severity-of-illness tool

2016-08-03

Abstract
Objectives
Skin and soft-tissue infections (SSTIs) encompass a diverse range of infections of varying severity. The Clinical Resource Efficiency Support Team (CREST) scoring system stratifies patients into four classes (I = least severe to IV = most severe) based on the Standardized Early Warning Score (SEWS). The objective of this study was to apply CREST to hospitalized patients with SSTIs in order to quantify disease severity and evaluate appropriateness of antibiotic management.
Methods
This was a retrospective, hypothesis-generating, single-centre evaluation of hospitalized patients with SSTIs admitted in 2011. Based on CREST classification, the empirical antimicrobial choices were categorized as appropriate, over-treatment or under-treatment.
Results
A total of 369 patients were screened and 200 met the inclusion criteria. The majority of patients were classified as either CREST class I (n = 68) or class II (n = 102). Over-treatment was more common in the less severe classes (88% and 32% in class I and class II, respectively; P < 0.05). Sixty-three percent of class I (n = 43) were over-treated due to both the use of intravenous antibiotics when oral therapy was sufficient and use of unnecessarily broad-spectrum antibiotics. In contrast, 25% (n = 26) of class II were over-treated due to use of unnecessarily broad-spectrum antibiotics. Overall clinical failure rates remained low with only 1%, 4% and 17% of patients unable to achieve initial response in class II, class III and class IV.
Conclusions
Retrospective application of CREST identified opportunities to improve the management of SSTIs. CREST can be of great value in discriminating less-severe SSTIs, which can be treated on an outpatient basis.



Tracking inter-institutional spread of NDM and identification of a novel NDM-positive plasmid, pSg1-NDM, using next-generation sequencing approaches

2016-08-02

Objectives
Owing to gene transposition and plasmid conjugation, New Delhi metallo-β-lactamase (NDM) is typically identified among varied Enterobacteriaceae species and STs. We used WGS to characterize the chromosomal and plasmid molecular epidemiology of NDM transmission involving four institutions in Singapore.
Methods
Thirty-three Enterobacteriaceae isolates (collection years 2010–14) were sequenced using short-read sequencing-by-synthesis and analysed. Long-read single molecule, real-time sequencing (SMRTS) was used to characterize genetically a novel plasmid pSg1-NDM carried on Klebsiella pneumoniae ST147.
Results
In 20 (61%) isolates, blaNDM was located on the pNDM-ECS01 plasmid in the background of multiple bacterial STs, including eight K. pneumoniae STs and five Escherichia coli STs. In six (18%) isolates, a novel blaNDM-positive plasmid, pSg1-NDM, was found only in K. pneumoniae ST147. The pSg1-NDM–K. pneumoniae ST147 clone (Sg1-NDM) was fully sequenced using SMRTS. pSg1-NDM, a 90 103 bp IncR plasmid, carried genes responsible for resistance to six classes of antimicrobials. A large portion of pSg1-NDM had no significant homology to any known plasmids in GenBank. pSg1-NDM had no conjugative transfer region. Combined chromosomal–plasmid phylogenetic analysis revealed five clusters of clonal bacterial NDM-positive plasmid transmission, of which two were inter-institution clusters. The largest inter-institution cluster involved six K. pneumoniae ST147-pSg1-NDM isolates. Fifteen patients were involved in transmission clusters, of which four had ward contact, six had hospital contact and five had an unknown transmission link.
Conclusions
A combined sequencing-by-synthesis and SMRTS approach can determine effectively the transmission clusters of blaNDM and genetically characterize novel plasmids. Plasmid molecular epidemiology is important to understanding NDM spread as blaNDM-positive plasmids can conjugate extensively across species and STs.



The impact of a multidisciplinary antimicrobial stewardship team on the timeliness of antimicrobial therapy in patients with positive blood cultures: a randomized controlled trial

2016-08-02

Background
Antimicrobial stewardship teams play an important role in assisting with the optimization of antimicrobial use in acute care settings. We aimed to determine whether a rapid review by a multidisciplinary antimicrobial stewardship team would improve the timeliness of optimal antimicrobial therapy for patients with positive blood cultures.
Methods
This prospective randomized controlled trial was undertaken in two Australian hospitals. Patients received either standard care (a clinical microbiologist, registrar or laboratory scientist communicating the positive blood culture by phone to the treating doctor) or intervention (standard care plus rapid review by a multidisciplinary antimicrobial stewardship team). Outcomes included time to appropriate and/or active antimicrobial therapy and in-hospital mortality. The trial was registered on the Australian New Zealand Clinical Trials Registry (ACTRN12614000258651).
Results
A total of 160 patients were enrolled in this study: 81 in the standard care arm and 79 in the intervention arm. Patients in the intervention arm were commenced earlier on active (HR 8.02, 95% CI: 2.15–29.91) and appropriate antimicrobials (HR 1.95, 95% CI: 1.13–3.38), with a higher proportion of patients allocated to the intervention arm receiving active therapy at 48 h (96% versus 82%) and appropriate therapy at 72 h (70% versus 54%). The majority of patients where the blood culture was a contaminant were not started on antimicrobial therapy, and there were no significant differences in time to cessation of antimicrobial therapy.
Conclusions
Antimicrobial stewardship team review of patients with pathogenic positive blood cultures improved the time to both active and appropriate antimicrobial therapy.



Use of WGS data for investigation of a long-term NDM-1-producing Citrobacter freundii outbreak and secondary in vivo spread of bla NDM-1 to Escherichia coli , Klebsiella pneumoniae and Klebsiella oxytoca

2016-08-01

Objectives
An outbreak of NDM-1-producing Citrobacter freundii and possible secondary in vivo spread of blaNDM-1 to other Enterobacteriaceae were investigated.
Methods
From October 2012 to March 2015, meropenem-resistant Enterobacteriaceae were detected in 45 samples from seven patients at Aalborg University Hospital, Aalborg, Denmark. In silico resistance genes, Inc plasmid types and STs (MLST) were obtained from WGS data from 24 meropenem-resistant isolates (13 C. freundii, 6 Klebsiella pneumoniae, 4 Escherichia coli and 1 Klebsiella oxytoca) and 1 meropenem-susceptible K. oxytoca. The sequences of the meropenem-resistant C. freundii isolates were compared by phylogenetic analyses. In vitro susceptibility to 21 antimicrobial agents was tested. Furthermore, in vitro conjugation and plasmid characterization was performed.
Results
From the seven patients, 13 highly clonal ST18 NDM-1-producing C. freundii were isolated. The ST18 NDM-1-producing C. freundii isolates were only susceptible to tetracycline, tigecycline, colistin and fosfomycin (except for the C. freundii isolates from Patient 2 and Patient 7, which were additionally resistant to tetracycline). The E. coli and K. pneumoniae from different patients belonged to different STs, indicating in vivo transfer of blaNDM-1 in the individual patients. This was further supported by in vitro conjugation and detection of a 154 kb IncA/C2 plasmid with blaNDM-1. Patient screenings failed to reveal any additional cases. None of the patients had a history of recent travel abroad and the source of the blaNDM-1 plasmid was unknown.
Conclusions
To our knowledge, this is the first report of an NDM-1-producing C. freundii outbreak and secondary in vivo spread of an IncA/C2 plasmid with blaNDM-1 to other Enterobacteriaceae.



HIV pre-exposure prophylaxis for women and infants prevents vaginal and oral HIV transmission in a preclinical model of HIV infection

2016-08-01

Background
Approximately 1.5 million HIV-positive women become pregnant annually. Without treatment, up to 45% will transmit HIV to their infants, primarily through breastfeeding. These numbers highlight that HIV acquisition is a major health concern for women and children globally. They also emphasize the urgent need for novel approaches to prevent HIV acquisition that are safe, effective and convenient to use by women and children in places where they are most needed.
Methods
4′-Ethynyl-2-fluoro-2′-deoxyadenosine, a potent NRTI with low cytotoxicity, was administered orally to NOD/SCID/γc−/− mice and to bone marrow/liver/thymus (BLT) humanized mice, a preclinical model of HIV infection. HIV inhibitory activity in serum, cervicovaginal secretions and saliva was evaluated 4 h after administration. 4′-Ethynyl-2-fluoro-2′-deoxyadenosine's ability to prevent vaginal and oral HIV transmission was evaluated using highly relevant transmitted/founder viruses in BLT mice.
Results
Strong HIV inhibitory activity in serum, cervicovaginal secretions and saliva obtained from animals after a single oral dose of 4′-ethynyl-2-fluoro-2′-deoxyadenosine (10 mg/kg) demonstrated efficient drug penetration into relevant mucosal sites. A single daily oral dose of 4′-ethynyl-2-fluoro-2′-deoxyadenosine resulted in efficient prevention of vaginal and oral HIV transmission after multiple high-dose exposures to transmitted/founder viruses in BLT humanized mice.
Conclusions
Our data demonstrated that 4′-ethynyl-2-fluoro-2′-deoxyadenosine efficiently prevents both vaginal and oral HIV transmission. Together with 4′-ethynyl-2-fluoro-2′-deoxyadenosine's relatively low toxicity and high potency against drug-resistant HIV strains, these data support further clinical development of 4′-ethynyl-2-fluoro-2′-deoxyadenosine as a potential pre-exposure prophylaxis agent to prevent HIV transmission in women and their infants.



Oral decontamination with aminoglycosides is associated with lower risk of mortality and infections in high-risk patients colonized with colistin-resistant, KPC-producing Klebsiella pneumoniae

2016-07-26

Objectives
Invasive infections caused by KPC-producing Klebsiella pneumoniae (KPCKP) are associated with very high mortality. Because infection is usually preceded by rectal colonization, we investigated if decolonization therapy (DT) with aminoglycosides had a protective effect in selected patients.
Methods
Patients with rectal colonization by colistin-resistant KPCKP who were at high risk of developing infection (because of neutropenia, surgery, previous recurrent KPCKP infections or multiple comorbidities) were followed for 180 days. Cox regression analysis including a propensity score was used to investigate the impact of the use of two intestinal decolonization regimens with oral aminoglycosides (gentamicin and neomycin/streptomycin) on mortality, risk of KPCKP infections and microbiological success. The study was registered with ClinicalTrials.gov (NCT02604849).
Results
The study sample comprised 77 colonized patients, of which 44 (57.1%) received DT. At 180 days of follow-up, decolonization was associated with a lower risk of mortality in multivariate analyses (HR 0.18; 95% CI 0.06–0.55) and a lower risk of KPCKP infections (HR 0.14; 95% CI 0.02–0.83) and increased microbiological success (HR 4.06; 95% CI 1.06–15.6). Specifically, gentamicin oral therapy was associated with a lower risk of crude mortality (HR 0.15; 95% CI 0.04–0.54), a lower risk of KPCKP infections (HR 0.86; 95% CI 0.008–0.94) and increased microbiological response at 180 days of follow-up (HR 5.67; 95% CI 1.33–24.1). Neomycin/streptomycin therapy was only associated with a lower risk of crude mortality (HR 0.22; 95% CI 0.06–0.9).
Conclusions
Intestinal decolonization with aminoglycosides is associated with a reduction in crude mortality and KPCKP infections at 180 days after initiating treatment.



Antimicrobial stewardship in wound care: a Position Paper from the British Society for Antimicrobial Chemotherapy and European Wound Management Association

2016-07-25

Background
With the growing global problem of antibiotic resistance it is crucial that clinicians use antibiotics wisely, which largely means following the principles of antimicrobial stewardship (AMS). Treatment of various types of wounds is one of the more common reasons for prescribing antibiotics.
Objectives
This guidance document is aimed at providing clinicians an understanding of: the basic principles of why AMS is important in caring for patients with infected wounds; who should be involved in AMS; and how to conduct AMS for patients with infected wounds.
Methods
We assembled a group of experts in infectious diseases/clinical microbiology (from the British Society for Antimicrobial Chemotherapy) and wound management (from the European Wound Management Association) who, after thoroughly reviewing the available literature and holding teleconferences, jointly produced this guidance document.
Results
All open wounds will be colonized with bacteria, but antibiotic therapy is only required for those that are clinically infected. Therapy is usually empirical to start, but definitive therapy should be based on results of appropriately collected specimens for culture. When prescribed, it should be as narrowly focused, and administered for the shortest duration, as possible. AMS teams should be interdisciplinary, especially including specialists in infection and pharmacy, with input from administrative personnel, the treating clinicians and their patients.
Conclusions
Available evidence is limited, but suggests that applying principles of AMS to the care of patients with wounds should help to reduce the unnecessary use of systemic or topical antibiotic therapy and ensure the safest and most clinically effective therapy for infected wounds.



Novel chromosome-encoded erm (47) determinant responsible for constitutive MLS B resistance in Helcococcus kunzii

2016-07-25

Objectives
The aim of the study was to identify the determinant responsible for erythromycin resistance in Helcococcus kunzii clinical isolate UCN99 and to characterize the genetic support and environment of this novel gene.
Methods
MICs were determined using the broth microdilution method according to EUCAST guidelines. The entire genome sequence of H. kunzii UCN99 was determined using a 454/Roche GS Junior sequencer. The fragment encompassing the new resistance gene and its own promoter was cloned into the pAT29 shuttle vector and the recombinant plasmid pAT29Ωerm(47) was expressed in both Staphylococcus aureus and Streptococcus agalactiae. The transcription start site (TSS) was experimentally determined by 5′ RACE-PCR.
Results
UCN99 exhibited a constitutive macrolide/lincosamide/streptogramin B (MLSB) resistance phenotype, suggesting the presence of an Erm protein. WGS allowed the identification of a novel gene, named erm(47), encoding a protein sharing 44%–48% amino acid identity with known Erm methylases. In both S. aureus and S. agalactiae, the introduction of pAT29Ωerm(47) conferred a significant increase (≥16-fold) in MICs of all macrolides and lincosamides tested, as well as a 4-fold increase in MICs of quinupristin (streptogramin B), confirming the MLSB resistance. The TSS identification revealed the presence of a short leader peptide, potentially implicated in a translational attenuation mechanism. It was also demonstrated that erm(47) was harboured by a 81 kb genomic island integrated into a chromosomal gene.
Conclusions
This is the first description of a novel MLSB resistance determinant, named erm(47). The prevalence of this gene among Gram-positive cocci must be further investigated to determine its clinical significance.



Molecular characterization of bla ESBL -producing Escherichia coli cultured from pig farms in Ireland

2016-07-25

Abstract
Objectives
To characterize ESBL-encoding Escherichia coli cultured from pigs and their plasmids carrying these genes following conjugation into recipient strains.
Methods
Six ESBL-producing E. coli were recovered from faecal samples taken from pigs along with a further isolate from the environment of a farrowing house on three pig farms in Ireland. These isolates were characterized by phylogenetic grouping, MLST and ESBL genotype analyses. Conjugation experiments were carried out in broth mating assays. S1-nuclease PFGE was used to determine the plasmid profiles. Whole-genome sequences of the seven E. coli were determined and subsequently analysed.
Results
Phylogenetic groups and the corresponding MLST STs identified among the seven tested E. coli isolates included A/ST10, A/ST34, C/ST23 and C/ST1629. All seven isolates carried one or more high-molecular-weight plasmids and demonstrated the ability to transfer their cefotaxime resistance phenotype at high frequencies. Five transmissible plasmid replicon types were detected, including IncK/B (n = 3), IncI1 (n = 2), IncFIA (n = 1), IncFIB (n = 1) and IncN (n = 1). ESBL-encoding genes, including blaCTX-M-14, blaCTX-M-15 and blaTEM-20, were identified.
Conclusions
As the first report from pig sources in Ireland, characterization of these ESBL-encoding isolates and their transmissible plasmids extends our understanding on these resistance markers from porcine E. coli.



Real-time PCR detection of Neisseria gonorrhoeae susceptibility to penicillin

2016-07-25

Objectives
The objective of this study was to develop a real-time PCR assay targeting the gonococcal porB gene (PorB-PCR) for predicting susceptibility of Neisseria gonorrhoeae to penicillin. This complements a previously described PCR assay for detecting penicillinase-producing N. gonorrhoeae (PPNG) developed by our laboratory (PPNG-PCR).
Methods
The PorB-PCR assay was designed using six probes to characterize various combinations of amino acids at positions 101 and 102 of the PorB1b class protein, including the WT G101/A102 and mutant G101K/A102D, G101K/A102N and G101K/A102G sequences, as well as the PorB1a sequence. The ability of these sequences to predict penicillin susceptibility was initially assessed using 2307 N. gonorrhoeae isolates from throughout Australia for which phenotypic susceptibility data were available. The assay was then applied to N. gonorrhoeae-positive clinical specimens (n = 70). Specificity was assessed by testing commensal Neisseria strains (n = 75) and N. gonorrhoeae-negative clinical specimens (n = 171).
Results
Testing of the 2307 N. gonorrhoeae isolates using PorB-PCR to detect G101/A102 and PorB1a sequences identified a total of 78.4% (61.2% and 17.2%, respectively) of penicillin-susceptible isolates with specificities of 97.4% and 99.3% and positive predictive values of 98.8% and 98.9%, where PPNG strains were simultaneously identified and excluded. Similar performance data were obtained when the PorB-PCR assay was applied to the N. gonorrhoeae-positive clinical specimens. No false-positive results were observed for the N. gonorrhoeae-negative samples and no cross-reactions were observed with the non-gonococcal species.
Conclusions
When used in parallel with the previously described PPNG-PCR, the PorB-PCR approach has the potential to facilitate individualized treatment of gonorrhoea using penicillin.



Dose optimization of voriconazole/anidulafungin combination against Aspergillus fumigatus using an in vitro pharmacokinetic/pharmacodynamic model and response surface analysis: clinical implications for azole-resistant aspergillosis

2016-07-25

Background
Combination therapy of voriconazole with an echinocandin is often employed in order to increase the efficacy of voriconazole monotherapy.
Methods
Four clinical Aspergillus fumigatus isolates with different in vitro susceptibilities to voriconazole (MIC 0.125–2 mg/L) and anidulafungin (MEC 0.008–0.016 mg/L) were tested in an in vitro pharmacokinetic/pharmacodynamic model simulating human serum concentrations of standard dosages of voriconazole and anidulafungin. Fungal growth was assessed using galactomannan production and quantitative PCR. Drug concentrations were determined with bioassays. Pharmacodynamic interactions were assessed using Bliss independence analysis (BI) and Loewe additivity-based canonical mixture response-surface non-linear regression analysis (LA). Probability of target attainment (PTA) was estimated with Monte Carlo analysis for different doses of anidulafungin (25, 50 and 100 mg) and azole resistance rates (5%–25%).
Results
Synergy [BI 51% (8%–80%), LA 0.63 (0.38–0.79)] was found at low anidulafungin (fCmax/MEC <10) and voriconazole (fAUC/MIC <10) exposures, whereas antagonism [BI 12% (5%–18%, LA 1.12 (1.04–4.6)] was found at higher drug exposures. The largest increase in PTA was found with 25 mg of anidulafungin and voriconazole MIC distributions with high (>10%) resistance rates. PTAs for isolates with voriconazole MICs of 1, 2 and 4 mg/L was 78%, 12% and 0% with voriconazole monotherapy and 96%–100%, 68%–82% and 9%–20% with combination therapy, respectively. Optimal activity was associated with a voriconazole tCmin/MIC ratio of 1.5 for monotherapy and 0.75 for combination therapy.
Conclusions
The present study indicated that the combination of voriconazole with low-dose anidulafungin may increase the efficacy and reduce the cost and potential toxicity of antifungal therapy, particularly against azole-resistant A. fumigatus isolates and in patients with subtherapeutic serum levels. This hypothesis warrants further in vivo verification.






High efficacy of first-line ART in a West African cohort, assessed by dried blood spot virological and pharmacological measurements

2016-07-20

Objectives
The objectives of this study were to determine the rate of viral success in HIV-infected patients on first-line ART by the assessment of dried blood spot (DBS) viral load (VL) and to assess the performance of DBS sampling for VL measurement, genotypic resistance and antiretroviral concentration determinations.
Methods
HIV-infected patients treated for >1 year with first-line ART in Niamey, Niger were included. VL based on nucleic acid sequence-based amplification (NASBA) assay (limit of quantification <800 copies/mL) was measured on DBS capillary samples. Resistance genotype was assessed for all detectable VLs (limit of detection >100 copies/mL); antiretroviral concentrations were interpreted using standard plasma cut-offs after extrapolation of blood to plasma results. Median (IQR) results are presented.
Results
Two hundred and eighteen patients (61% women), aged 41 (34–46) years, with 138 (56–235) CD4 cells/mm3 at baseline were included. After 4 (2–6) years of follow-up under therapy, CD4 gain was +197 (98–372) cells/mm3; 81% had VL <800 copies/mL. Antiretroviral concentrations were adequate in 87% of patients and nevirapine/efavirenz concentrations were related to viral success (P < 0.001). DBS genotypic resistance amplification succeeded in 71% of failing patients: NRTI drug resistance mutations were identified in 73% including resistance to lamivudine/emtricitabine (67%), abacavir (30%) and tenofovir (21%); and NNRTI drug resistance mutations were identified in 82% including resistance to rilpivirine (39%) and etravirine (15%).
Conclusions
This study demonstrated a good response after 4 years of first-line ART in Niger. Adherence was high, according to antiretroviral concentrations, and the majority of failures were explained by selection of drug resistance mutations detected in the DBS genotype. Using DBS might improve the assessment of ART failure in HIV-infected patients in low-income countries.



A feasibility service evaluation of screening and treatment of group A streptococcal pharyngitis in community pharmacies

2016-07-20

Objectives
The UK 5 year antimicrobial resistance strategy recognizes the role of point-of-care diagnostics to identify where antimicrobials are required, as well as to assess the appropriateness of the diagnosis and treatment. A sore throat test-and-treat service was introduced in 35 community pharmacies across two localities in England during 2014–15.
Methods
Trained pharmacy staff assessed patients presenting with a sore throat using the Centor scoring system and patients meeting three or all four of the criteria were offered a throat swab test for Streptococcus pyogenes, Lancefield group A streptococci. Patients with a positive throat swab test were offered antibiotic treatment.
Results
Following screening by pharmacy staff, 149/367 (40.6%) patients were eligible for throat swab testing. Of these, only 36/149 (24.2%) were positive for group A streptococci. Antibiotics were supplied to 9.8% (n = 36/367) of all patients accessing the service. Just under half of patients that were not showing signs of a bacterial infection (60/123, 48.8%) would have gone to their general practitioner if the service had not been available.
Conclusions
This study has shown that it is feasible to deliver a community-pharmacy-based screening and treatment service using point-of-care testing. This type of service has the potential to support the antimicrobial resistance agenda by reducing unnecessary antibiotic use and inappropriate antibiotic consumption.






Impact on antiviral resistance of E119V, I222L and R292K substitutions in influenza A viruses bearing a group 2 neuraminidase (N2, N3, N6, N7 and N9)

2016-07-17

Objectives
While subtype-specific substitutions linked to neuraminidase (NA) inhibitor resistance are well described in human N1 and N2 influenza NAs, little is known about other NA subtypes. The aim of this study was to determine whether the R292K and E119V ± I222L substitutions could be associated with oseltamivir resistance in all group 2 NAs and had an impact on virus fitness.
Methods
Reassortant viruses with WT NA or variant N2, N3, N6, N7 or N9 NAs, bearing R292K or E119V ± I222L substitutions, were produced by reverse genetics. The antiviral susceptibility, activity, Km of the NA, mutation stability and in vitro virus fitness in MDCK cells were determined.
Results
NA activities could be ranked as follows regardless of the substitution: N3 ≥ N6 > N2 ≥ N9 > N7. Using NA inhibitor resistance interpretation criteria used for human N1 or N2, the NA-R292K substitution conferred highly reduced inhibition by oseltamivir and the N6- or N9-R292K substitution conferred reduced inhibition by zanamivir and laninamivir. Viruses with the N3- or N6-E119V substitution showed normal inhibition by oseltamivir, while those with the N2-, N7- or N9-E119V substitution showed reduced inhibition by oseltamivir. Viruses with NA-E119V + I222L substitutions showed reduced inhibition (N3 and N6) or highly reduced inhibition (N2, N7 and N9) by oseltamivir. Viruses bearing the NA-R292K substitution had lower affinity and viruses bearing the NA-E119V substitution had higher affinity for the MUNANA substrate than viruses with corresponding WT NA.
Conclusions
NA-R292K and E119V + I222L substitutions conferred reduced inhibition by oseltamivir for all group 2 NAs. Surveillance of NA inhibitor resistance for zoonotic and human influenza viruses and the development of novel antiviral agents with different targets should be continued.



The novel 2016 WHO Neisseria gonorrhoeae reference strains for global quality assurance of laboratory investigations: phenotypic, genetic and reference genome characterization

2016-07-17

Objectives
Gonorrhoea and MDR Neisseria gonorrhoeae remain public health concerns globally. Enhanced, quality-assured, gonococcal antimicrobial resistance (AMR) surveillance is essential worldwide. The WHO global Gonococcal Antimicrobial Surveillance Programme (GASP) was relaunched in 2009. We describe the phenotypic, genetic and reference genome characteristics of the 2016 WHO gonococcal reference strains intended for quality assurance in the WHO global GASP, other GASPs, diagnostics and research worldwide.
Methods
The 2016 WHO reference strains (n = 14) constitute the eight 2008 WHO reference strains and six novel strains. The novel strains represent low-level to high-level cephalosporin resistance, high-level azithromycin resistance and a porA mutant. All strains were comprehensively characterized for antibiogram (n = 23), serovar, prolyliminopeptidase, plasmid types, molecular AMR determinants, N. gonorrhoeae multiantigen sequence typing STs and MLST STs. Complete reference genomes were produced using single-molecule PacBio sequencing.
Results
The reference strains represented all available phenotypes, susceptible and resistant, to antimicrobials previously and currently used or considered for future use in gonorrhoea treatment. All corresponding resistance genotypes and molecular epidemiological types were described. Fully characterized, annotated and finished references genomes (n = 14) were presented.
Conclusions
The 2016 WHO gonococcal reference strains are intended for internal and external quality assurance and quality control in laboratory investigations, particularly in the WHO global GASP and other GASPs, but also in phenotypic (e.g. culture, species determination) and molecular diagnostics, molecular AMR detection, molecular epidemiology and as fully characterized, annotated and finished reference genomes in WGS analysis, transcriptomics, proteomics and other molecular technologies and data analysis.



WGS analysis and molecular resistance mechanisms of azithromycin-resistant (MIC >2 mg/L) Neisseria gonorrhoeae isolates in Europe from 2009 to 2014

2016-07-17

Objectives
To elucidate the genome-based epidemiology and phylogenomics of azithromycin-resistant (MIC >2 mg/L) Neisseria gonorrhoeae strains collected in 2009–14 in Europe and clarify the azithromycin resistance mechanisms.
Methods
Seventy-five azithromycin-resistant (MIC 4 to >256 mg/L) N. gonorrhoeae isolates collected in 17 European countries during 2009–14 were examined using antimicrobial susceptibility testing and WGS.
Results
Thirty-six N. gonorrhoeae multi-antigen sequence typing STs and five phylogenomic clades, including 4–22 isolates from several countries per clade, were identified. The azithromycin target mutation A2059G (Escherichia coli numbering) was found in all four alleles of the 23S rRNA gene in all isolates with high-level azithromycin resistance (n = 4; MIC ≥256 mg/L). The C2611T mutation was identified in two to four alleles of the 23S rRNA gene in the remaining 71 isolates. Mutations in mtrR and its promoter were identified in 43 isolates, comprising isolates within the whole azithromycin MIC range. No mutations associated with azithromycin resistance were found in the rplD gene or the rplV gene and none of the macrolide resistance-associated genes [mef(A/E), ere(A), ere(B), erm(A), erm(B), erm(C) and erm(F)] were identified in any isolate.
Conclusions
Clonal spread of relatively few N. gonorrhoeae strains accounts for the majority of the azithromycin resistance (MIC >2 mg/L) in Europe. The four isolates with high-level resistance to azithromycin (MIC ≥256 mg/L) were widely separated in the phylogenomic tree and did not belong to any of the main clades. The main azithromycin resistance mechanisms were the A2059G mutation (high-level resistance) and the C2611T mutation (low- and moderate-level resistance) in the 23S rRNA gene.



HIV-1-RNA in seminal plasma correlates with detection of HIV-1-DNA in semen cells, but not with CMV shedding, among MSM on successful antiretroviral regimens

2016-07-17

Abstract
Objectives
Intermittent seminal HIV-RNA detection can occur in MSM despite concomitant plasma virological control on combined ART (cART). We undertook the present study to determine if seminal HIV detection was associated with seminal cytomegalovirus (CMV) detection or detection of HIV-infected cells in semen.
Methods
Longitudinal semen samples from HIV-1-infected MSM on successful cART enrolled in the EVARIST ANRS EP 49 study were analysed. We first conducted a case–control analysis (ratio 1 : 3) to assess HIV-DNA detection in semen cells in the 20 patients with detectable HIV-RNA in seminal plasma (cases) matched with 60 participants with undetectable HIV-RNA (controls) based on total HIV-DNA load in blood cells. Second, we measured CMV-DNA in all seminal plasma samples.
Results
HIV-1-DNA in semen cells was detected on at least one sample visit in 12/20 cases and 11/60 controls. Detection of HIV-RNA in seminal plasma was associated significantly with the detection of HIV-DNA in semen cells [OR, 7.6 (95% CI, 2.1–28.4); P = 0.002] when adjusted on total HIV-DNA in blood cells. CMV-DNA was detected in 107/273 seminal plasma samples with a median value of 3.62 log10 copies/mL (IQR, 2.83–4.38), yielding a prevalence of 39.2%. Seminal CMV-DNA shedding [OR, 1.5 (95% CI, 0.6–3.6); P = 0.343] was not associated with the risk of detection of HIV-RNA in seminal plasma.
Conclusions
The presence of HIV-DNA in semen cells was predictive of HIV-RNA detection, suggesting that viral particles arise through local HIV replication by infected semen cells. Despite virological control, compartmentalization of HIV in the genital tract might act in residual replication and transmission.



Maraviroc/raltegravir simplification strategy following 6 months of quadruple therapy with tenofovir/emtricitabine/maraviroc/raltegravir in treatment-naive HIV patients

2016-07-17

Objective
We assessed the virological efficacy of a 6 month maraviroc/raltegravir simplification strategy following 6 months of quadruple therapy combining tenofovir disoproxil fumarate/emtricitabine with maraviroc/raltegravir.
Methods
HIV-1-infected naive patients were enrolled in an open label, single-arm, Phase 2 trial. All patients received maraviroc 300 mg twice daily, raltegravir 400 mg twice daily and tenofovir/emtricitabine for 24 weeks. Patients with stable HIV-RNA <50 copies/mL stopped tenofovir/emtricitabine at week (W) 24 and pursued maraviroc/raltegravir until W48. The primary endpoint was the virological response defined by HIV-RNA <50 copies/mL at W48.
Results
Thirty-three patients were analysed. Patients were mostly male (94%), Caucasians (91%), MSM (82%); their median age was 42 years. At baseline, median CD4 cell count was 453 cells/mm3 and HIV-RNA was 4.3 log copies/mL. All patients had CCR5-tropic viruses by genotropism and phenotropism assays. All but one patient had an HIV-RNA < 50 copies/mL at W24 and entered the simplification phase. Virological success was maintained at W48 in 88% (90% CI 79%–97%) of patients. N155H mutation was detected at failure in one patient. No tropism switch was observed. Raltegravir and maraviroc plasma exposure were satisfactory in 92% and 79% of 41 samples from 21 patients. Five severe adverse events (SAEs) were observed up to W48; none was related to the study drugs. Four patients presented grade 3 AEs; none was related to the study. No grade 4 AE was observed. No patient died.
Conclusions
Maraviroc/raltegravir maintenance therapy following a 6 month induction phase with maraviroc/raltegravir/tenofovir/emtricitabine was well tolerated and maintained virological efficacy in these carefully selected patients.



Exploring the coverage of antimicrobial stewardship across UK clinical postgraduate training curricula

2016-07-17

Objectives
Antimicrobial resistance (AMR) is a global political and patient safety issue. With ongoing strategic interventions to improve the shape of UK postgraduate clinical training, ensuring that all clinicians have appropriate knowledge and practical skills in the area of AMR is essential. To assess this, a cross-sectional analysis was undertaken of the coverage and quality of antimicrobial stewardship (AMS)/AMR within UK postgraduate clinical training curricula.
Methods
UK clinical specialty training curricula were identified. Topics and individual learning points relating to AMS or AMR were extracted for each specialty. Learning points were quality assessed against the expected level of clinical competence. Inter-specialty analysis was performed.
Results
Overall 37 specialties were assessed, equating to 2318 topics and 42 527 learning points. Of these, 8/2318 (0.3%) topics and 184/42 527 (0.4%) learning points were related to AMS/AMR. Infectious diseases represented all eight topics and 43/184 (23%) of the learning points. In contrast, primary care, which is responsible for the highest proportion of antimicrobial usage, had no topics and only 2/1368 (0.15%) of the AMS/AMR learning points. This paucity of representation was reflected across most of the remaining specialties. On quality assessment, the majority of learning points (111/184; 60%) required knowledge only, with no demonstration of behaviour in clinical practice required.
Conclusions
Coverage of AMS/AMR is poor across the majority of UK postgraduate clinical training curricula, with little depth of learning required. Given the threat of AMR, and evolving changes in clinical training pathways, we call for cross-specialty action to address this current lack of engagement.



Therapeutic drug monitoring of vancomycin: a guideline of the Division of Therapeutic Drug Monitoring, Chinese Pharmacological Society

2016-07-11

Background
Guideline development should be based on the quality of evidence, balance of benefits and harms, economic evaluation and patients’ views and preferences. Therefore, these factors were considered in the development of a new guideline for therapeutic drug monitoring (TDM) of vancomycin.
Objectives
To develop an evidence-based guideline for vancomycin TDM and to promote standardized vancomycin TDM in clinical practice in China.
Methods
We referred to the WHO Handbook for Guideline Development and used the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system to rate the quality of evidence and grade the strength of recommendations, according to economic evaluation and patients’ views and preferences. We used the GRADE Grid method to formulate the recommendations.
Results
The guideline presents recommendations about who should receive vancomycin TDM, how to monitor vancomycin efficacy and renal safety, therapeutic trough concentrations, time to start initial vancomycin TDM, loading dose and how to administer and adjust the vancomycin dose.
Conclusions
We developed an evidence-based guideline for vancomycin TDM, which provides recommendations for clinicians and pharmacists to conduct vancomycin TDM in China.



Cluster-dependent colistin hetero-resistance in Enterobacter cloacae complex

2016-07-11

Abstract
Objectives
Aims of this study were to: (i) evaluate whether the cluster membership could have an impact on hetero-resistance phenotype to colistin in the Enterobacter cloacae complex (ECC); and (ii) determine the genetic mechanism of colistin hetero-resistance in ECC.
Methods
A collection of 124 clinical isolates belonging to 13 clusters were used to analyse the hetero-resistance phenotype (MICs were determined using the broth microdilution method, Etest and population analysis profiling). Different mutants (ΔphoP, ΔphoQ, ΔphoPQ, ΔpmrA, ΔpmrB, ΔpmrAB, ΔarnE, ΔarnF and ΔarnBCADTEF) were constructed and tested for their colistin hetero-resistance phenotype.
Results
Based on broth microdilution and Etest results, it was shown that the hetero-resistance to colistin depended on the cluster: strains from clusters I, II, IV, VII, IX, X, XI and XII were usually hetero-resistant, whereas those from clusters III, V, VI, VIII and XIII were categorized as susceptible. However, for some cluster V and VIII strains, a small proportion (<10−7) of cells appeared resistant when tested by population analysis profiling. From a mechanistic point of view, analysis of mutants revealed that the mechanism of hetero-resistance was mainly due to the expression of the arn operon and the phoP/phoQ two-component regulatory system.
Conclusions
Because the colistin hetero-resistance appeared cluster-dependent in the ECC, it should be advocated to determine the cluster of the strain associated with the infection in parallel with the MIC of colistin. The resistance mechanism may not be similar to other Enterobacteriaceae since only the two-component regulatory system PhoP/PhoQ (and not PmrA/PmrB) seemed to play a role in resistance regulation.



Impact of AAC(6′)-Ib-cr in combination with chromosomal-mediated mechanisms on clinical quinolone resistance in Escherichia coli

2016-07-11

Objectives
aac(6′)-Ib-cr is the most prevalent plasmid-mediated fluoroquinolone (FQ) resistance mechanism in Enterobacteriaceae. We aimed to analyse the interplay between this plasmid-mediated gene and chromosomal-mediated quinolone resistance mechanisms on both FQ resistance and bacterial fitness in Escherichia coli.
Methods
E. coli ATCC 25922 and derived isogenic strains carrying chromosomal-mediated quinolone resistance modifications (Ser83Leu–Asp87Asn in GyrA, Ser80Arg in ParC and/or a marR gene deletion) were electroporated with a pBK-CMV vector encoding AAC(6′)-Ib-cr. The MICs of FQs were determined by microdilution and bactericidal activity was determined using time–kill curves. A peritoneal sepsis murine model was used to evaluate the in vivo impact. Bacterial fitness was analysed using growth curves and competition assays.
Results
The presence of the aac(6′)-Ib-cr gene increased the MICs of ciprofloxacin and norfloxacin 4–8-fold for all E. coli genotypes, independently of the initial resistance level. Combination of the aac(6′)-Ib-cr gene with three or four chromosomal mechanisms was necessary to reach MIC values above the susceptible category. Killing curve assays showed a clear selective advantage for survival in strains harbouring the aac(6′)-Ib-cr gene (up to 7 log10 cfu/mL after 24 h). AAC(6′)-Ib-cr significantly reduced the ciprofloxacin efficacy in vivo. In terms of bacterial fitness cost, maximal OD was significantly lower for all strains harbouring the aac(6′)-Ib-cr gene, independently of chromosomal mutations associated.
Conclusions
The aac(6′)-Ib-cr gene, in spite of producing low-level resistance by itself, plays a relevant role in acquisition of a clinical level of ciprofloxacin and norfloxacin resistance, when combined with three or four chromosomal mutations, both in vitro and in vivo.



Tissue pharmacokinetics of telavancin in healthy volunteers: a microdialysis study

2016-07-11

Abstract
Background
Telavancin is a novel lipoglycoprotein antibiotic with MRSA activity. To date, tissue pharmacokinetics (PK) and plasma protein binding of the drug are insufficiently described.
Objectives
To investigate tissue PK and plasma protein binding of telavancin in healthy volunteers.
Methods
Eight male healthy subjects received a single dose of 10 mg/kg of body weight of telavancin as an intravenous infusion over 1 h. At defined timepoints before and up to 24 h after treatment, total telavancin concentrations were measured in plasma. Additionally, unbound telavancin levels were determined in plasma, muscle and subcutis by means of microdialysis.
Results
Key PK parameters of total telavancin in plasma were in good agreement with previously described values. Mean ± SD Cmax and calculated AUC0-24 of free telavancin in plasma were 13.8 ± 7.8 mg/L and 82.9 ± 34.3 mg·h/L, respectively. Unbound drug levels in plasma ranged from 13.2% to 24.8% of corresponding total telavancin. Mean ± SD Cmax and AUC0-24 of unbound telavancin were 4.3 ± 1.5 mg/L and 61.5 ± 27.1 mg·h/L for muscle and 3.4 ± 1.8 and 50.0 ± 29.8 mg·h/L for subcutis, respectively. Relevant PK/pharmacodynamic indices were calculated for total and unbound drug.
Conclusions
This study provides important information on soft tissue PK and plasma protein binding of telavancin in healthy volunteers. Unbound plasma concentrations above levels assumed from previously available data and sustained free drug exposure in soft tissues support the current mode of administration.



Levels of bone markers in a population of infants exposed in utero and during breastfeeding to tenofovir within an Option B+ programme in Malawi

2016-07-11

Objectives
No data are available on bone metabolism in infants exposed to tenofovir during breastfeeding. We investigated bone metabolism markers in the first year of life in infants from mothers who received tenofovir, lamivudine and efavirenz during pregnancy and 12 months of breastfeeding in a national Option B+ programme in Malawi.
Methods
Serum samples collected at 6 and 12 months in tenofovir-exposed infants and in a small sample of tenofovir-unexposed infants from the same clinical centre were analysed in batches for levels of bone-specific alkaline phosphatase (BAP; marker of bone formation) and of C-terminal telopeptide of type I collagen (CTX; marker of bone resorption).
Results
Overall, 136 tenofovir-exposed infants were evaluated. No infant had at either timepoint CTX values above the upper normal limit, while most of them had at 6 and 12 months levels of BAP above the upper normal limit for the age range. Levels of bone markers showed no differences by gender and no association with growth parameters. Tenofovir-unexposed and -exposed children had similar mean levels of bone markers at 6 months (CTX: 0.62 versus 0.55 ng/mL, P = 0.122; BAP: 384 versus 362 U/L, P = 0.631).
Conclusions
No significant association between treatment with tenofovir and CTX or BAP levels was found. The high levels of BAP, coupled to the normal levels observed for CTX, might reflect primarily skeletal growth. Potential negative effects of prolonged exposure to tenofovir through breastfeeding cannot however be excluded and longitudinal studies that evaluate bone mineralization status in children enrolled in Option B+ programmes are warranted.






Bacteriophage LM33_P1, a fast-acting weapon against the pandemic ST131-O25b:H4 Escherichia coli clonal complex

2016-07-07

Objectives
Amongst the highly diverse Escherichia coli population, the ST131-O25b:H4 clonal complex is particularly worrisome as it is associated with a high level of antibiotic resistance. The lack of new antibiotics, the worldwide continuous increase of infections caused by MDR bacteria and the need for narrow-spectrum antimicrobial agents have revived interest in phage therapy. In this article, we describe a virulent bacteriophage, LM33_P1, which specifically infects O25b strains, and provide data related to its therapeutic potential.
Methods
A large panel of E. coli strains (n = 283) was used to assess both the specificity and the activity of bacteriophage LM33_P1. Immunology, biochemistry and genetics-based methods confirmed this specificity. Virology methods and sequencing were used to characterize this bacteriophage in vitro, while three relevant mouse models were employed to show its in vivo efficacy.
Results
Bacteriophage LM33_P1 exclusively infects O25b E. coli strains with a 70% coverage on sequence types associated with high antibiotic resistance (ST131 and ST69). This specificity is due to an interaction with the LPS mediated by an original tail fibre. LM33_P1 also has exceptional intrinsic properties with a high adsorption constant and produces over 300 virions per cell in <10 min. Using animal pneumonia, septicaemia and urinary tract infection models, we showed the in vivo efficacy of LM33_P1 to reduce the bacterial load in several organs.
Conclusions
Bacteriophage LM33_P1 represents the first weapon that specifically and quickly kills O25b E. coli strains. Therapeutic approaches derived from this bacteriophage could be developed to stop or slow down the spread of the ST131-O25b:H4 drug-resistant clonal complex in humans.



Positive regulation of the Candida albicans multidrug efflux pump Cdr1p function by phosphorylation of its N-terminal extension

2016-07-07

Objectives
Overexpression of ATP-binding cassette (ABC) transporters is a frequent cause of multidrug resistance in cancer cells and pathogenic microorganisms. One example is the Cdr1p transporter from the human fungal pathogen Candida albicans that belongs to the pleiotropic drug resistance (PDR) subfamily of ABC transporters found in fungi and plants. Cdr1p is overexpressed in several azole-resistant clinical isolates, causing azole efflux and treatment failure. Cdr1p appears as a doublet band in western blot analyses, suggesting that the protein is post-translationally modified. We investigated whether Cdr1p is phosphorylated and the function of this modification.
Methods
Phosphorylated residues were identified by MS. Their function was investigated by site-directed mutagenesis and expression of the mutants in a C. albicans endogenous system that exploits a hyperactive allele of the Tac1p transcription factor to drive high levels of Cdr1p expression. Fluconazole resistance was measured by microtitre plate and spot assays and transport activity by Nile red accumulation.
Results
We identified a cluster of seven phosphorylated amino acids in the N-terminal extension (NTE) of Cdr1p. Mutating all seven sites to alanine dramatically diminished the ability of Cdr1p to confer fluconazole resistance and transport Nile red, without affecting Cdr1p localization. Conversely, a Cdr1p mutant in which the seven amino acids were replaced by glutamate was able to confer high levels of fluconazole resistance and to export Nile red.
Conclusions
Our results demonstrate that the NTE of Cdr1p is phosphorylated and that NTE phosphorylation plays a major role in regulating Cdr1p and possibly other PDR transporter function.



IFN-free therapy for HIV/HCV-coinfected patients within the liver transplant setting

2016-07-07

Objectives
IFN-based therapy against hepatitis C recurrence after liver transplantation (LT) has poor effectiveness and tolerability. In HIV/HCV-coinfected liver transplant recipients, the results are even poorer. Here, we report our experience using direct antiviral agents (DAAs) in 11 consecutive coinfected patients within the LT setting.
Methods
Four patients with compensated cirrhosis and hepatocellular carcinoma were treated while awaiting LT and seven patients received antiviral therapy due to severe hepatitis C recurrence after LT [fibrosing cholestatic hepatitis (n = 1), fibrosis stage ≥F3 (n = 2) and decompensated cirrhosis (n = 4)]. Patients were treated with different sofosbuvir-based regimens with or without ribavirin for 12 or 24 weeks.
Results
Sustained virological response (SVR) was achieved in all patients. Two of the four patients treated while awaiting LT reached the time of transplant with undetectable HCV-RNA that remained undetectable 12 weeks after LT, one patient had detectable HCV-RNA at the time of transplant but achieved SVR after completing 12 weeks of therapy after LT and the last patient is still on the waiting list. Seven patients with severe post-LT hepatitis C recurrence were treated within 11–120 months after LT. In these patients, viral eradication was associated with an improvement in liver function and clinical decompensation. Tolerance to antiviral therapy was good and only four patients reported mild adverse events.
Conclusions
IFN-free regimens are effective and well tolerated in HIV/HCV-coinfected patients within the LT setting, but more data are needed to confirm our promising results and to establish the best treatment option in this population.



Virological efficacy of PI monotherapy for HIV-1 in clinical practice

2016-07-07

Background
Clinical trials of PI monotherapy indicate that most participants maintain viral suppression and emergent protease resistance is rare. However, outcomes among patients receiving PI monotherapy for clinical reasons, such as toxicity or adherence issues, are less well studied.
Methods
An observational study of patients attending an HIV treatment centre in London, UK, who had received PI monotherapy between 2004 and 2013, was conducted using prospectively collected clinical data and genotypic resistance reports. Survival analysis techniques were used to examine the times to virological failure and treatment discontinuation.
Results
Ninety-five patients had PI monotherapy treatment for a median duration of 126 weeks. Virological failure occurred during 64% of episodes and 8% of patients developed emergent protease mutations. We estimate failure occurs in half of episodes within 2 years following initiation. Where PI monotherapy was continued following virological failure, 68% of patients achieved viral re-suppression. Despite a high incidence of virological failure, many patients continued PI monotherapy and 79% of episodes were ongoing at the end of the study. The type of PI used, the presence of baseline protease mutations and the plasma HIV RNA at initiation did not have a significant impact on treatment outcomes.
Conclusions
There was a higher incidence of virological failure and emerging resistance in our UK clinical setting than described in PI monotherapy clinical trials and other European observational studies. Despite this, many patients continued PI monotherapy and regained viral suppression, indicating this strategy remains a viable option in certain individuals following careful clinical evaluation.






Impact of amoxicillin therapy on resistance selection in patients with community-acquired lower respiratory tract infections: a randomized, placebo-controlled study

2016-06-26

Objectives
To determine the effect of amoxicillin treatment on resistance selection in patients with community-acquired lower respiratory tract infections in a randomized, placebo-controlled trial.
Methods
Patients were prescribed amoxicillin 1 g, three times daily (n = 52) or placebo (n = 50) for 7 days. Oropharyngeal swabs obtained before, within 48 h post-treatment and at 28–35 days were assessed for proportions of amoxicillin-resistant (ARS; amoxicillin MIC ≥2 mg/L) and -non-susceptible (ANS; MIC ≥0.5 mg/L) streptococci. Alterations in amoxicillin MICs and in penicillin-binding-proteins were also investigated. ITT and PP analyses were conducted.
Results
ARS and ANS proportions increased 11- and 2.5-fold, respectively, within 48 h post-amoxicillin treatment compared with placebo [ARS mean increase (MI) 9.46, 95% CI 5.57–13.35; ANS MI 39.87, 95% CI 30.96–48.78; P < 0.0001 for both]. However, these differences were no longer significant at days 28–35 (ARS MI −3.06, 95% CI −7.34 to 1.21; ANS MI 4.91, 95% CI −4.79 to 14.62; P > 0.1588). ARS/ANS were grouped by pbp mutations. Group 1 strains exhibited significantly lower amoxicillin resistance (mean MIC 2.8 mg/L, 95% CI 2.6–3.1) than group 2 (mean MIC 9.3 mg/L, 95% CI 8.1–10.5; P < 0.0001). Group 2 strains predominated immediately post-treatment (61.07%) and although decreased by days 28–35 (30.71%), proportions remained higher than baseline (18.70%; P = 0.0004).
Conclusions
By utilizing oropharyngeal streptococci as model organisms this study provides the first prospective, experimental evidence that resistance selection in patients receiving amoxicillin is modest and short-lived, probably due to ‘fitness costs’ engendered by high-level resistance-conferring mutations. This evidence further supports European guidelines that recommend amoxicillin when an antibiotic is indicated for community-acquired lower respiratory tract infections.