Binding of temocillin to plasma proteins in vitro and in vivo: the importance of plasma protein levels in different populations and of co-medications.

BACKGROUND: Temocillin plasma protein binding (PPB) in healthy individuals is reported to be ∼85% but had not been studied in patients. OBJECTIVES: To obtain normative data on temocillin PPB in patients in relation to infection and impact of co-medications widely used in ICU. METHODS: Plasma was obtained from healthy individuals (Group #1), non-ICU patients with UTI (Group #2), ICU patients with suspected/confirmed ventriculitis (Group #3) or with sepsis/septic shock (Group #4). Total and unbound temocillin concentrations were measured in spiked samples from temocillin-naive donors (in vitro) or in plasma from temocillin-treated subjects (in vivo). The impact of diluting plasma, using pharmaceutical albumin, or adding drugs potentially competing for PPB was tested in spiked samples. Data were analysed using a modified Hill-Langmuir equation taking ligand depletion into account. RESULTS: Temocillin PPB was saturable in all groups, both in vitro and in vivo. Maximal binding capacity (Bmax) was 1.2-2-fold lower in patients. At 20 and 200 mg/L (total concentrations), the unbound fraction reached 12%-29%, 23%-42% and 32%-52% in Groups #2, #3, #4. The unbound fraction was inversely correlated with albumin and C-reactive protein concentrations. Binding to albumin was 2-3-fold lower than in plasma and non-saturable. Drugs with high PPB but active at lower molar concentrations than temocillin caused minimal displacement, while fluconazole (low PPB but similar plasma concentrations to temocillin) increased up to 2-fold its unbound fraction. CONCLUSIONS: Temocillin PPB is saturable, 2-4-fold lowered in infected patients in relation to disease severity (ICU admission, hypoalbuminaemia, inflammation) and only partially reproducible with albumin. Competition with other drugs must be considered for therapeutic concentrations to be meaningful.

Large-scale WGS of carbapenem-resistant Acinetobacter baumannii isolates reveals patterns of dissemination of ST clades associated with antibiotic resistance.

OBJECTIVES: To describe the population genetics and antibiotic resistance gene distribution of carbapenem-resistant Acinetobacter baumannii (CRAB) isolates causing infections in three Mediterranean countries. METHODS: Isolates were collected during the 2013-17 AIDA clinical trial in six hospitals in Israel, Greece and Italy. WGS, bioinformatic characterization and antibiotic resistance profiling were performed. RESULTS: In the 247 CRAB isolates characterized in this study, ST distribution varied by country: 29/31 (93.5%) Greek isolates, 34/41 (82.9%) Italian isolates and 70/175 (40.0%) Israeli isolates belonged to ST2. The identified ST2 isolates included eight distinct clades: 2C, 2D and 2H were significantly more common in Italy, while 2F was unique to Greece. The uncommon ST3 was not present among Greek isolates and constituted only 5/41 (12%) Italian isolates. On the other hand, it was much more common among Israeli isolates: 78/175 (44.6%) belonged to ST3. The vast majority of isolates, 240/247 (97.2%), were found to harbour acquired carbapenemases, primarily blaOXA-23. The chromosomal oxaAb (blaOXA-51-like) and ampC genes characteristic of this organism were also ubiquitous. Most (96.4%) ST3 isolates carried a broad-host-range plasmid IncP1α. CONCLUSIONS: The geographical differences in CRAB populations support the theory that clonal spread of CRAB leads to endemicity in hospitals and regions. The close association between antibiotic resistance genes and clades, and between plasmids and STs, suggest that de novo creation of MDR A. baumannii is rare. The clustering of antibiotic resistance genes and plasmids that is unique to each clade/ST, and nearly uniform within clades/STs, suggests that horizontal transmission is rare but crucial to the clade's/ST's success.

European Society of Clinical Microbiology and Infectious Diseases (ESCMID) guidelines for the treatment of infections caused by multidrug-resistant Gram-negative bacilli (endorsed by European society of intensive care medicine).

SCOPE: These ESCMID guidelines address the targeted antibiotic treatment of third-generation cephalosporin-resistant Enterobacterales (3GCephRE) and carbapenem-resistant Gram-negative bacteria, focusing on the effectiveness of individual antibiotics and on combination versus monotherapy. METHODS: An expert panel was convened by ESCMID. A systematic review was performed including randomized controlled trials and observational studies, examining different antibiotic treatment regimens for the targeted treatment of infections caused by the 3GCephRE, carbapenem-resistant Enterobacterales, carbapenem-resistant Pseudomonas aeruginosa and carbapenem-resistant Acinetobacter baumannii. Treatments were classified as head-to-head comparisons between individual antibiotics and between monotherapy and combination therapy regimens, including defined monotherapy and combination regimens only. The primary outcome was all-cause mortality, preferably at 30 days and secondary outcomes included clinical failure, microbiological failure, development of resistance, relapse/recurrence, adverse events and length of hospital stay. The last search of all databases was conducted in December 2019, followed by a focused search for relevant studies up until ECCMID 2021. Data were summarized narratively. The certainty of the evidence for each comparison between antibiotics and between monotherapy and combination therapy regimens was classified by the GRADE recommendations. The strength of the recommendations for or against treatments was classified as strong or conditional (weak). RECOMMENDATIONS: The guideline panel reviewed the evidence per pathogen, preferably per site of infection, critically appraising the existing studies. Many of the comparisons were addressed in small observational studies at high risk of bias only. Notably, there was very little evidence on the effects of the new, recently approved, ß-lactam/ß-lactamase inhibitors on infections caused by carbapenem-resistant Gram-negative bacteria. Most recommendations are based on very-low- and low-certainty evidence. A high value was placed on antibiotic stewardship considerations in all recommendations, searching for carbapenem-sparing options for 3GCephRE and limiting the recommendations of the new antibiotics for severe infections, as defined by the sepsis-3 criteria. Research needs are addressed.

Pharmacokinetic/pharmacodynamic analysis of oral fosfomycin against Enterobacterales, Pseudomonas aeruginosa and Enterococcus spp. in an in vitro bladder infection model: impact on clinical breakpoints.

OBJECTIVES: Fosfomycin is an established treatment for uncomplicated urinary tract infections (UTIs), yet evidence supporting susceptibility breakpoints is limited. We examine the UTI susceptibility criteria. METHODS: Fosfomycin susceptibility, heteroresistance and in vitro growth in a bladder infection model, after a single 3 g dose of oral fosfomycin, were bridged to human pharmacokinetics with pharmacokinetic/pharmacodynamic and Monte Carlo analyses. Data from common uropathogens (24 Escherichia coli, 20 Klebsiella pneumoniae, 4 Enterobacter cloacae, 14 Pseudomonas aeruginosa, 8 Enterococcus faecalis and 8 Enterococcus faecium) were compared and analysed to ascertain species-specific PTA. RESULTS: Glucose-6-phosphate (G6P) increased MICs of E. coli, K. pneumoniae and E. cloacae (median 2-fold dilutions 3-5), but not of P. aeruginosa and Enterococcus. Atypical E. coli lacking G6P potentiation were killed in the bladder infection model despite high MICs (32-128 mg/L). Fosfomycin heteroresistance was uncommon in E. coli (MIC > 2 mg/L) but was detected in the majority of K. pneumoniae (MIC > 1 mg/L) and P. aeruginosa (MIC >8 mg/L). For these species, baseline heteroresistance was a strong predictor for treatment failure in the model. No heteroresistance was found in Enterococcus. The fAUC/MIC targets for stasis were 1935, 3393, 9968, 2738 and 283 for typical E. coli, K. pneumoniae, E. cloacae, P. aeruginosa and E. faecalis, respectively (synthetic human urine medium alone promoted a 1 log10 kill in E. faecium). A >95% PTA for stasis was only found at MIC ≤ epidemiological cut-off (ECOFF) for E. coli (4 mg/L). For other species, PTAs were low for WT populations. CONCLUSIONS: With the exception of E. coli, fosfomycin is a poor target for other uropathogen species. A reduction in oral fosfomycin UTI breakpoints is supported.

The synthetic synergistic cinnamon oil CIN-102 is active against Madurella mycetomatis, the most common causative agent of mycetoma.

Mycetoma is a devastating neglected tropical infection of the subcutaneous tissue and most commonly caused by the fungus Madurella mycetomatis. Treatment of mycetoma consists of a combination of a long term antifungal treatment with itraconazole and surgery. However, treatment is associated with low success rates. Therefore, there is a need to identify novel treatments for mycetoma. CIN-102 is a synthetic partial copy of cinnamon oils with activity against many pathogenic bacteria and fungi. In this study we determined the in vitro activity of CIN-102 against 21 M. mycetomatis isolates and its in vivo efficacy in a M. mycetomatis infected Galleria mellonella larval model. In vitro, CIN-102 was active against M. mycetomatis with MICs ranging from 32 µg/mL to 512 µg/mL. 128 µg/mL was needed to inhibit the growth in 50% of tested isolates. In vivo, concentrations below the MIC of 40 mg/kg and 80 mg/kg CIN-102 prolonged larval survival, but higher concentrations of CIN-102 did not.

Excluded versus included patients in a randomized controlled trial of infections caused by carbapenem-resistant Gram-negative bacteria: relevance to external validity.

BACKGROUND: Population external validity is the extent to which an experimental study results can be generalized from a specific sample to a defined population. In order to apply the results of a study, we should be able to assess its population external validity. We performed an investigator-initiated randomized controlled trial (RCT) (AIDA study), which compared colistin-meropenem combination therapy to colistin monotherapy in the treatment of patients infected with carbapenem-resistant Gram-negative bacteria. In order to examine the study's population external validity and to substantiate the use of AIDA study results in clinical practice, we performed a concomitant observational trial. METHODS: The study was conducted between October 1st, 2013 and January 31st, 2017 (during the RCTs recruitment period) in Greece, Israel and Italy. Patients included in the observational arm of the study have fulfilled clinical and microbiological inclusion criteria but were excluded from the RCT due to receipt of colistin for > 96 h, refusal to participate, or prior inclusion in the RCT. Non-randomized cases were compared to randomized patients. The primary outcome was clinical failure at 14 days of infection onset. RESULTS: Analysis included 701 patients. Patients were infected mainly with Acinetobacter baumannii [78.2% (548/701)]. The most common reason for exclusion was refusal to participate [62% (183/295)]. Non-randomized and randomized patients were similar in most of the demographic and background parameters, though randomized patients showed minor differences towards a more severe infection. Combination therapy was less common in non-randomized patients [31.9% (53/166) vs. 51.2% (208/406), p = 0.000]. Randomized patients received longer treatment of colistin [13 days (IQR 10-16) vs. 8.5 days (IQR 0-15), p = 0.000]. Univariate analysis showed that non-randomized patients were more inclined to clinical failure on day 14 from infection onset [82% (242/295) vs. 75.5% (307/406), p = 0.042]. After adjusting for other variables, non-inclusion was not an independent risk factor for clinical failure at day 14. CONCLUSION: The similarity between the observational arm and RCT patients has strengthened our confidence in the population external validity of the AIDA trial. Adding an observational arm to intervention studies can help increase the population external validity and improve implementation of study results in clinical practice. TRIAL REGISTRATION: The trial was registered with ClinicalTrials.gov, number NCT01732250 on November 22, 2012.

The Effect of Antibiotic Restriction Programs on Prevalence of Antimicrobial Resistance: A Systematic Review and Meta-Analysis.

BACKGROUND: In hospital settings, restriction of selected classes of antibiotics is usually believed to contribute to containment of resistance development. We performed a systematic review and meta-analysis to assess the effect of restricting the use of specific antibiotic classes on the prevalence of resistant bacterial pathogens. METHODS: We conducted a systematic literature search in Embase and PubMed/OVID MEDLINE. We included studies until June 4, 2020 in which a restrictive antibiotic policy was applied and prevalence of resistance and use of antibiotics were reported. We calculated the overall effect of antimicrobial resistance between postintervention versus preintervention periods using pooled odds ratios (ORs) from a mixed-effects model. We stratified meta-analysis by antibiotic-pathogen combinations. We assessed heterogeneity between studies using the I2 statistic and sources of heterogeneity using meta-regression. RESULTS: We included 15 individual studies with an overall low quality of evidence. In meta-analysis, significant reductions in resistance were only observed with nonfermenters after restricting fluoroquinolones (OR = 0.77, 95% confidence interval [CI] = 0.62-0.97) and piperacillin-tazobactam (OR = 0.81, 95% CI = 0.72-0.92). High degrees of heterogeneity were observed with studies restricting carbapenem (Enterobacterales, I2 = 70.8%; nonfermenters, I2 = 81.9%), third-generation cephalosporins (nonfermenters, I2 = 63.3%), and fluoroquiolones (nonfermenters, I2 = 64.0%). Results were comparable when excluding studies with fewer than 50 bacteria. There was no evidence of publication bias for any of the antibiotic-pathogen combinations. CONCLUSIONS: We could not confirm that restricting carbapenems or third-generation cephalosporins leads to decrease in prevalence of antibiotic resistance among Enterobacterales, nonfermenters, or Gram-positive bacteria in hospitalized patients. Nevertheless, reducing fluoroquinolone and piperacilline-tazobactam use may decrease resistance in nonfermenters.

Evaluation of the post-antibiotic effect in vivo for the combination of a ß-lactam antibiotic and a ß-lactamase inhibitor: ceftazidime-avibactam in neutropenic mouse thigh and lung infections.

The post-antibiotic effect (PAE) of ceftazidime-avibactam in vivo was evaluated using models of thigh- and lung-infection with Pseudomonas aeruginosa in neutropenic mice. In thigh-infected mice, the PAE was negative (-2.18 to -0.11 h) for three of four strains: caused by a 'burst' of rapid bacterial growth after the drug concentrations had fallen below their pre-specified target values. With lung infection, PAE was positive, and longer for target drug concentrations in ELF (>2 h) than plasma (1.69-1.88 h). The time to the start of regrowth was quantified as a new parameter, PAER, which was positive (0.35-1.00 h) in both thigh- and lung-infected mice. In the context that measurements of the PAE of ß-lactam/ß-lactamase inhibitor combinations in vivo have not previously been reported, it is noted that the negative values were consistent with previous measurements of the PAE of ceftazidime-avibactam in vitro and of ceftazidime alone in vivo.

Activity of Cefepime in Combination with the Novel ß-Lactamase Inhibitor Taniborbactam (VNRX-5133) against Extended-Spectrum-ß-Lactamase-Producing Isolates in In Vitro Checkerboard Assays.

Extended-spectrum-ß-lactamase (ESBL)-producing strains are increasing worldwide, limiting therapeutic options. Taniborbactam (VNRX-5133) is a newly developed ß-lactamase inhibitor with a wide spectrum of activity covering both serine and metallo enzymes. We therefore evaluated cefepime-taniborbactam activity against ESBL-producing isolates and determined the concentrations to be used in MIC determinations in the clinical laboratory. The in vitro activity of cefepime (0.06 to 256 mg liter-1) combined with taniborbactam (0.03 to 32 mg liter-1) against 129 clinically and molecularly well-documented ESBL-producing isolates (42 Escherichia coli, 39 Klebsiella pneumoniae, 28 Pseudomonas aeruginosa, 16 Enterobacter cloacae, 2 Citrobacter freundii, and 2 Enterobacter aerogenes) was tested with a broth microdilution checkerboard method based on the ISO standard. The MICs of cefepime alone and in combination, together with percentage resistance at different concentrations of taniborbactam, were calculated for each species and resistance mechanism. The median (range)/MIC90 of cefepime was 32 (0.125 to 256)/256 mg liter-1 for all Enterobacterales isolates (n = 101), with 72% being resistant, and 32 (8 to 256)/128 mg liter-1 for the 28 P. aeruginosa isolates, with 86% being resistant. The median (range)/90th percentile concentration of taniborbactam required to restore Enterobacterales susceptibility to cefepime (MIC ≤1 mg liter-1) was 0.06 (≤0.03 to 32)/4 mg liter-1 and P. aeruginosa susceptibility to increased exposure to cefepime (MIC ≤8 mg liter-1) 1 (≤0.032 to 32)/32 mg liter-1 At a fixed concentration of 4 mg liter-1 of taniborbactam, cefepime median (range)/MIC90 were reduced to 0.125 (0.06 to 4)/1 mg liter-1 for Enterobacterales with no resistant isolates found, and to 8 (2 to 64)/16 mg liter-1 for P. aeruginosa isolates, where 36% remained resistant. The combination cefepime-taniborbactam demonstrated a potent activity against ESBL isolates, restoring susceptibility of all Enterobacterales and two-thirds of P. aeruginosa isolates.

In-vitro pharmacokinetic/pharmacodynamic model data suggest a potential role of new formulations of posaconazole against Candida krusei but not Candida glabrata infections.

Posaconazole exhibits in-vitro activity against Candida glabrata and Candida krusei. Epidemiological cut-off values set by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI) are 1/1 and 0.5/0.5 mg/L, respectively, but clinical breakpoints have not been established to date. This study explored the pharmacodynamics (PD) of posaconazole in a validated one-compartment in-vitro pharmacokinetic (PK)/PD model, and determined the probability of PK/PD target attainment (PTA) for the available formulations. Five C. glabrata and three C. krusei isolates with posaconazole minimum inhibitory concentrations (MICs) of 0.06-2 and 0.03-0.25 mg/L, respectively, were tested in the PK/PD model simulating different time-concentration profiles of posaconazole. The exposure-effect relationship fAUC0-24/MIC was described for EUCAST/CLSI methods, and PTA was calculated in order to determine PK/PD susceptibility breakpoints for oral solution (400 mg q12h), and intravenous (i.v.)/tablet formulations (300 mg q24h). Fungicidal activity (~2log kill) was found against the most susceptible C. glabrata isolate alone, and against all three C. krusei isolates. The corresponding EUCAST/CLSI PK/PD targets (fAUC0-24/MIC) were 102/79 for C. glabrata and 12/8 for C. krusei. Mean PTA was high (>95%) for C. glabrata isolates with EUCAST/CLSI MICs ≤0.03/≤0.03 mg/L for oral solution and ≤0.125/≤0.125 mg/L for i.v. and tablet formulations for the wild-type population. For C. krusei isolates, mean PTA was high (>95%) for EUCAST/CLSI MICs ≤0.25/≤0.5 mg/L for oral solution and ≤1/≤2 mg/L for i.v. and tablet formulations for the wild-type population. The use of posaconazole to treat C. glabrata infections is questionable. Intravenous and tablet formulations may be therapeutic options for the treatment of C. krusei infections, and oral exposure can be optimized with therapeutic drug monitoring (trough levels >0.6-0.9 mg/L).

The Role of New Posaconazole Formulations in the Treatment of Candida albicans Infections: Data from an In Vitro Pharmacokinetic-Pharmacodynamic Model.

Posaconazole is more active than fluconazole against Candida albicansin vitro and is approved for the treatment of oropharyngeal candidiasis but not for that of invasive candidiasis (IC). Here, we explored the efficacy of posaconazole against C. albicans in an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model of IC and determined the probability of pharmacodynamic target attainment for the oral solution and intravenous (i.v.)/tablet formulations. Three clinical C. albicans isolates (posaconazole MICs, 0.008 to 0.25 mg/liter) were studied in the in vitro PK/PD dilution model simulating steady-state posaconazole PK. The in vitro exposure-effect relationship, area under the 24-h free drug concentration curve (fAUC0-24)/MIC, was described and compared with in vivo outcome in animals with IC. PK/PD susceptibility breakpoints and trough levels required for optimal treatment were determined for EUCAST and CLSI 24-h/48-h (CLSI24h/CLSI48h) methods using the fAUC0-24/MIC associated with half-maximal activity (EI50) and Monte Carlo simulation analysis for oral solution (400 mg every 12 hours [q12h]) and i.v./tablet formulations (300 mg q24h). The in vitro mean (95% confidence interval [CI]) EI50 was 330 (183 to 597) fAUC0-24/MIC for CLSI24h and 169 (92 to 310) for EUCAST/CLSI48h methods, which are close to the near-stasis in vivo effect. The probability of target attainment for EI50 was estimated; for the wild-type isolates (MIC ≤ 0.06 mg/liter), it was low for the oral solution and higher than 95% for the i.v./tablet formulations for the EUCAST/CLSI48h methods but not for the CLSI 24-h method. Non-wild-type isolates with EUCAST/CLSI48h MICs of 0.125 and 0.25 mg/liter would require trough levels of >1.2 and >2.4 mg/liter, respectively. Posaconazole i.v./tablet formulations may have a role in the therapy of invasive infections by wild-type C. albicans isolates, provided that a steady state is reached quickly. A PK/PD susceptibility breakpoint at the epidemiological cutoff (ECV/ECOFF) of 0.06 mg/liter was determined.

Multicentre testing of the EUCAST broth microdilution reference method for MIC determination on Mycobacterium tuberculosis.

OBJECTIVES: The first objective of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) subcommittee for antimycobacterial susceptibility testing (AMST), launched in 2016, was to set a reference method for determining the MICs of antituberculous agents, since many protocols are used worldwide and a consensus one is needed for the determination of microbiological breakpoints. METHODS: During 2017 and 2018, MIC determination protocols were evaluated prospectively in a multicentre study within the four AMST laboratories. MIC results were obtained for isoniazid, levofloxacin and amikacin on the reference strain Mycobacterium tuberculosis H37Rv ATCC 27294. Broth microdilution (BMD) in Middlebrook 7H9 and solid medium dilution (SMD) in Middlebrook 7H10 were performed using two inoculum concentrations. MICs were interpreted with regard to visual and 99% inhibition after 7, 14 or 21 days of incubation for BMD and 21 days for SMD. RESULTS: Following the EUCAST reference protocol, intra- and inter-assay agreements were within ±1 MIC dilution for >95% of the observations for the three drugs in both methods. MIC values, presented as MIC mode (range) for BMD and SMD respectively, were: 0.03 (0.015-0.06) mg/L and 0.12 (0.06-0.25) mg/L for isoniazid, 0.25 mg/L (0.25-0.5) and 0.5 mg/L (0.12-0.5) for levofloxacin, and 0.5 mg/L (0.5-1.0) and 0.5 mg/L (0.5-1.0) for amikacin. CONCLUSIONS: Both SMD and BMD were reproducible and eligible as a reference method for MIC determination of the Mycobacterium tuberculosis complex (MTBC). BMD was finally selected as the EUCAST reference method. From now on it will be used to set epidemiological cut-off values and clinical breakpoints of new and old antituberculous agents.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis complex isolates - the EUCAST broth microdilution reference method for MIC determination.

SCOPE: Several methods are used worldwide for antibiotic susceptibility testing (AST) for the Mycobacterium tuberculosis complex (MTBC). The variability in the results obtained with these methods hampers setting epidemiological cut-off (ECOFF) values and clinical breakpoints according to EUCAST guidelines. Methods for susceptibility testing and determination of the minimal inhibitory concentrations (MICs) need to be standardized for MTBC isolates for old and new agents. Our objective was to establish a standardized reference method for MIC determination for MTBC. METHODS: The EUCAST antimycobacterial susceptibility testing subcommittee (AMST) compared protocols of MIC determination with regard to medium, inoculum preparation, antituberculous agent preparation, incubation, reading of the results and interpretation. RECOMMENDATIONS: The EUCAST reference method of MIC determination for MTBC is the broth microdilution method in Middlebrook 7H9-10% OADC medium. The final inoculum is a 105 CFU/mL suspension, obtained from a 10-2 dilution of a 0.5 McFarland suspension prepared after vortexing bacterial colonies with glass beads before suspending them in sterile water. The culture is maintained in a U-shaped 96-well polystyrene microtitre sterile plate with a lid incubated at 36° ± 1°C. Reading is done using an inverted mirror as soon as the 1:100 diluted control (i.e. 103 CFU/mL suspension) shows visual growth. The MIC, expressed in mg/L, is the lowest concentration that inhibits visual growth. Mycobacterium tuberculosis H37Rv ATCC 27294 is used as the reference strain and its targeted MIC values are within the range 0.03-0.12 for isoniazid, 0.12-0.5 for levofloxacin and 0.25-1 mg/L for amikacin. CONCLUSIONS: The EUCAST reference method for MTBC was endorsed by EUCAST after public consultation and will from now on be used to define EUCAST ECOFFs and clinical breakpoints. This reference method is not primarily intended to be used under routine conditions and the AST methods will need to be calibrated against this reference method to be used with EUCAST breakpoints.

Guide-free Cas9 from pathogenic Campylobacter jejuni bacteria causes severe damage to DNA.

CRISPR-Cas9 systems are enriched in human pathogenic bacteria and have been linked to cytotoxicity by an unknown mechanism. Here, we show that upon infection of human cells, Campylobacter jejuni secretes its Cas9 (CjeCas9) nuclease into their cytoplasm. Next, a native nuclear localization signal enables CjeCas9 nuclear entry, where it catalyzes metal-dependent nonspecific DNA cleavage leading to cell death. Compared to CjeCas9, native Cas9 of Streptococcus pyogenes (SpyCas9) is more suitable for guide-dependent editing. However, in human cells, native SpyCas9 may still cause some DNA damage, most likely because of its ssDNA cleavage activity. This side effect can be completely prevented by saturation of SpyCas9 with an appropriate guide RNA, which is only partially effective for CjeCas9. We conclude that CjeCas9 plays an active role in attacking human cells rather than in viral defense. Moreover, these unique catalytic features may therefore make CjeCas9 less suitable for genome editing applications.

Study protocol for an international, multicentre stepped-wedge cluster randomised trial to evaluate the impact of a digital antimicrobial stewardship smartphone application.

INTRODUCTION: With the widespread use of electronic health records and handheld electronic devices in hospitals, informatics-based antimicrobial stewardship interventions hold great promise as tools to promote appropriate antimicrobial drug prescribing. However, more research is needed to evaluate their optimal design and impact on quantity and quality of antimicrobial prescribing. METHODS AND ANALYSIS: Use of smartphone-based digital stewardship applications (apps) with local guideline directed empirical antimicrobial use by physicians will be compared with antimicrobial prescription as per usual as primary outcome in three hospitals in the Netherlands, Sweden and Switzerland. Secondary outcomes will include antimicrobial use metrics, clinical and process outcomes. A multicentre stepped-wedge cluster randomised trial will randomise entities defined as wards or specialty regarding time of introduction of the intervention. We will include 36 hospital entities with seven measurement periods in which the primary outcome will be measured in 15 participating patients per time period per cluster. At participating wards, patients of at least 18 years of age using antimicrobials will be included. After a baseline period of 2-week measurements, six periods of 4 weeks will follow in which the intervention is introduced in 6 wards (in three hospitals) until all 36 wards have implemented the intervention. Thereafter, we allow use of the app by everyone, and evaluate the sustainability of the app use 6 months later. ETHICS AND DISSEMINATION: This protocol has been approved by the institutional review board of each participating centre. Results will be disseminated via media, to healthcare professionals via professional training and meetings and to researchers via conferences and publications. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov registry (NCT03793946). Stage; pre-results.

Efficacy of single and multiple oral doses of fosfomycin against Pseudomonas aeruginosa urinary tract infections in a dynamic in vitro bladder infection model.

OBJECTIVES: We used a dynamic bladder infection in vitro model with synthetic human urine (SHU) to examine fosfomycin exposures to effectively kill, or prevent emergence of resistance, among Pseudomonas aeruginosa isolates. METHODS: Dynamic urinary fosfomycin concentrations after 3 g oral fosfomycin were simulated, comparing single and multiple (daily for 7 days) doses. Pharmacodynamic response of 16 P. aeruginosa (MIC range 1 to >1024 mg/L) were examined. Baseline disc diffusion susceptibility, broth microdilution MIC and detection of heteroresistance were assessed. Pathogen kill and emergence of resistance over 72 h following a single dose, and over 216 h following daily dosing for 7 days, were investigated. The fAUC0-24/MIC associated with stasis and 1, 2 and 3 log10 kill were determined. RESULTS: Pre-exposure high-level resistant (HLR) subpopulations were detected in 11/16 isolates after drug-free incubation in the bladder infection model. Five of 16 isolates had >2 log10 kill after single dose, reducing to 2/16 after seven doses. Post-exposure HLR amplification occurred in 8/16 isolates following a single dose and in 11/16 isolates after seven doses. Baseline MIC ≥8 mg/L with an HLR subpopulation predicted post-exposure emergence of resistance following the multiple doses. A PK/PD target of fAUC0-24/MIC >5000 was associated with 3 log10 kill at 72 h and 7 day-stasis. CONCLUSIONS: Simulated treatment of P. aeruginosa urinary tract infections with oral fosfomycin was ineffective, despite exposure to high urinary concentrations and repeated daily doses for 7 days. Emergence of resistance was observed in the majority of isolates and worsened following prolonged therapy. Detection of a baseline resistant subpopulation predicted treatment failure.

Exploring the Interplay of Resistance Nodulation Division Efflux Pumps, AmpC and OprD in Antimicrobial Resistance of Burkholderia cepacia Complex in Clinical Isolates.

Aim: This study aimed at investigating the association of gene expression of multidrug efflux pumps (MexA, MexC, MexE, and MexX), the outer membrane porin OprD, and the ß-lactamase AmpC with the antimicrobial susceptibility among 44 clinical isolates of Burkholderia cepacia complex (Bcc). Results: Increased expression of ampC gene showed significant association with reduced susceptibility to chloramphenicol. In fact, reduced susceptibility to chloramphenicol was correlated with overexpression of most genes (ampC, mexC, mexE, and mexX) studied here in majority (>95%) of the Bcc isolates. Increased mexA expression showed significant association with reduced susceptibility to ß-lactam antimicrobials (ceftazidime, piperacillin-tazobactam, and meropenem) and co-trimoxazole. Reduced susceptibility to meropenem also showed significant correlation with overexpression of mexC and mexX, whereas reduced susceptibility to ceftazidime was also associated with mexE overexpression. Reduced susceptibility to levofloxacin was significantly associated with overexpression of mexX. The involvement of the efflux pumps in levofloxacin and ceftazidime resistance was further inferred from the finding that the efflux pump inhibitor, carbonyl cyanide m-chlorophenylhydrazone reduced minimum inhibitory concentrations for both the antimicrobials. Conclusions: To conclude, this study explored the high-level expression of mexC, mexE, and mexX efflux pumps genes and ampC in the clinical isolates of Bcc, which can be targeted at treating infections caused by Bcc.

Single-dose pharmacokinetics of temocillin in plasma and soft tissues of healthy volunteers after intravenous and subcutaneous administration: a randomized crossover microdialysis trial.

BACKGROUND: The antibiotic temocillin has recently been rediscovered as a promising therapeutic option against MDR Gram-negative bacteria. However, some aspects of the pharmacokinetic (PK) profile of the drug are still to be elucidated: subcutaneous administration of temocillin might be of interest as an alternative to the intravenous route in selected patients. Similarly, information on the penetration of temocillin into human soft tissues is lacking. OBJECTIVES: To investigate the feasibility and plasma PK of subcutaneous dosing as well as soft tissue PK of temocillin after intravenous administration to healthy volunteers. METHODS: Eight healthy volunteers received 2 g of temocillin both as intravenous and subcutaneous infusion in a randomized two-period crossover study. Concentration-time profiles of total temocillin in plasma (after both routes) and of unbound temocillin in plasma, muscle and subcutis (only after intravenous dosing) were determined up to 12 h post-dose. RESULTS: Subcutaneous dosing caused some infusion site discomfort but resulted in sustained drug concentrations over time with only slightly decreased overall exposure compared with intravenous dosing. Plasma protein binding of temocillin showed concentration-dependent behaviour and was higher than previously reported. Still, unbound drug concentrations in muscle and subcutis determined by microdialysis markedly exceeded those in plasma, suggesting good tissue penetration of temocillin. CONCLUSIONS: The subcutaneous administration of temocillin is a valid and feasible alternative to intravenous dosing. With the description of plasma protein binding and soft tissue PK of temocillin in healthy volunteers, this study provides important information that adds to the ongoing characterization of the PK profile of temocillin and might serve as input for PK/PD considerations.

Trends, seasonality and the association between outpatient antibiotic use and antimicrobial resistance among urinary bacteria in the Netherlands.

OBJECTIVES: To determine trends, seasonality and the association between community antibiotic use and antimicrobial resistance (AMR) in Escherichia coli and Klebsiella pneumoniae in urinary tract infections. METHODS: We analysed Dutch national databases from January 2008 to December 2016 regarding antibiotic use and AMR for nitrofurantoin, trimethoprim, fosfomycin and ciprofloxacin. Antibiotic use was expressed as DDD/1000 inhabitant-days (DID) and AMR was expressed as the percentage of resistance from total tested isolates. Temporal trends and seasonality were analysed with autoregressive integrated moving average (ARIMA) models. Each antibiotic use-resistance combination was cross-correlated with a linear regression of the ARIMA residuals. RESULTS: The trends of DID increased for ciprofloxacin, fosfomycin and nitrofurantoin, but decreased for trimethoprim. Similar trends were found in E. coli and K. pneumoniae resistance to the same antibiotics, except for K. pneumoniae resistance to ciprofloxacin, which decreased. Resistance levels peaked in winter/spring, whereas antibiotic use peaked in summer/autumn. In univariate analysis, the strongest and most significant cross-correlations were approximately 0.20, and had a time delay of 3-6 months between changes in antibiotic use and changes in resistance. In multivariate analysis, significant effects of nitrofurantoin use and ciprofloxacin use on resistance to these antibiotics were found in E. coli and K. pneumoniae, respectively. There was a significant association of nitrofurantoin use with trimethoprim resistance in K. pneumoniae after adjusting for trimethoprim use. CONCLUSIONS: We found a relatively low use of antibiotics and resistance levels over a 9 year period. Although the correlations were weak, variations in antibiotic use for these four antibiotics were associated with subsequent variations in AMR in urinary pathogens.

Colistin plus meropenem for carbapenem-resistant Gram-negative infections: in vitro synergism is not associated with better clinical outcomes.

OBJECTIVES: In vitro models showing synergism between polymyxins and carbapenems support combination treatment for carbapenem-resistant Gram-negative (CRGN) infections. We tested the association between the presence of in vitro synergism and clinical outcomes in patients treated with colistin plus meropenem. METHODS: This was a secondary analysis of AIDA, a randomized controlled trial comparing colistin with colistin-meropenem for severe CRGN infections. We tested in vitro synergism using a checkerboard assay. Based on the fractional inhibitory concentration (ΣFIC) index for each colistin-meropenem combination, we categorized results as synergistic, antagonistic or additive/indifferent. The primary outcome was clinical failure at 14 days. Secondary outcomes were 14- and 28-day mortality and microbiological failure. RESULTS: The sample included 171 patients with infections caused by carbapenem-resistant Acinetobacter baumannii (n = 131), Enterobacteriaceae (n = 37) and Pseudomonas aeuruginosa (n = 3). In vitro testing showed synergism for 73 isolates, antagonism for 20 and additivism/indifference for 78. In patients who received any colistin plus meropenem, clinical failure at 14 days was 59/78 (75.6%) in the additivism/indifference group (reference category), 54/73 (74.0%) in the synergism group (adjusted odds ratio (aOR) 0.76, 95% CI 0.31-1.83), and 11/20 (55%) in the antagonism group (aOR 0.77, 95% CI 0.22-2.73). There was no significant difference between groups for any secondary outcome. Comparing the synergism group to patients treated with colistin monotherapy, synergism was not protective against 14-day clinical failure (aOR 0.52, 95% CI 0.26-1.04) or 14-day mortality (aOR1.09, 95% CI 0.60-1.96). DISCUSSION: In vitro synergism between colistin and meropenem via checkerboard method did not translate into clinical benefit.