Genomic surveillance and antimicrobial resistance in Neisseria gonorrhoeae isolates in Bangkok, Thailand in 2018.

OBJECTIVES: Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a substantial global public health problem. Gonococcal infections acquired in or from Asia represent most verified ceftriaxone treatment failures, and several ceftriaxone-resistant strains have emerged in Asia and subsequently spread globally. Additionally, in Thailand the gonorrhoea incidence remains high. Herein, we investigate the genomic diversity, AMR and AMR determinants in gonococcal isolates cultured in 2018 in Bangkok, Thailand. METHODS: Gonococcal isolates from males (n = 37) and females (n = 62) were examined by Etest and WGS. AMR determinants and molecular epidemiological STs were characterized. For phylogenomic comparison, raw sequence data were included from China (432 isolates), Japan (n = 270), Vietnam (n = 229), Thailand (n = 3), a global dataset (n = 12 440) and the 2016 WHO reference strains plus WHO Q (n = 15). RESULTS: In total, 88, 66 and 41 different NG-MAST, NG-STAR and MLST STs, respectively, and 31 different NG-STAR clonal complexes were found. A remarkably high frequency (88%) of ß-lactamase TEM genes was detected and two novel TEM alleles were found. The phylogenomic analysis divided the isolates into the previously described lineages A and B, with a large proportion of Thai isolates belonging to the novel sublineage A3. CONCLUSIONS: We describe the first molecular epidemiological study using WGS on gonococcal isolates from Thailand. The high prevalence of AMR and AMR determinants for ciprofloxacin, tetracycline and benzylpenicillin, and some strains belonging to clones/clades especially in sublineage A2 that are prone to develop resistance to extended-spectrum cephalosporins (ESCs) and azithromycin, should prompt continued and strengthened AMR surveillance, including WGS, of N. gonorrhoeae in Thailand.

Flomoxef and fosfomycin in combination for the treatment of neonatal sepsis in the setting of highly prevalent antimicrobial resistance.

BACKGROUND: Neonatal sepsis is a serious bacterial infection of neonates, globally killing up to 680 000 babies annually. It is frequently complicated by antimicrobial resistance, particularly in low- and middle-income country (LMIC) settings with widespread resistance to the WHO's recommended empirical regimen of ampicillin and gentamicin. OBJECTIVES: We assessed the utility of flomoxef and fosfomycin as a potential alternative empirical regimen for neonatal sepsis in these settings. METHODS: We studied the combination in a 16-arm dose-ranged hollow-fibre infection model (HFIM) experiment and chequerboard assays. We further assessed the combination using clinically relevant regimens in the HFIM with six Enterobacterales strains with a range of flomoxef/fosfomycin MICs. RESULTS: Pharmacokinetic/pharmacodynamic modelling of the HFIM experimental output, along with data from chequerboard assays, indicated synergy of this regimen in terms of bacterial killing and prevention of emergence of fosfomycin resistance. Flomoxef monotherapy was sufficient to kill 3/3 strains with flomoxef MICs ≤0.5 mg/L to sterility. Three of three strains with flomoxef MICs ≥8 mg/L were not killed by fosfomycin or flomoxef monotherapy; 2/3 of these were killed with the combination of the two agents. CONCLUSIONS: These data suggest that flomoxef/fosfomycin could be an efficacious and synergistic regimen for the empirical treatment of neonatal sepsis in LMIC settings with prevalent antimicrobial resistance. Our HFIM results warrant further assessment of the flomoxef/fosfomycin combination in clinical trials.

Assessment of flomoxef combined with amikacin in a hollow-fibre infection model for the treatment of neonatal sepsis in low- and middle-income healthcare settings.

BACKGROUND: Annual mortality from neonatal sepsis is an estimated 430 000-680 000 infants globally, most of which occur in low- and middle-income countries (LMICs). The WHO currently recommends a narrow-spectrum ß-lactam (e.g. ampicillin) and gentamicin as first-line empirical therapy. However, available epidemiological data demonstrate high rates of resistance to both agents. Alternative empirical regimens are needed. Flomoxef and amikacin are two off-patent antibiotics with potential for use in this setting. OBJECTIVES: To assess the pharmacodynamics of flomoxef and amikacin in combination. METHODS: The pharmacodynamic interaction of flomoxef and amikacin was assessed in chequerboard assays and a 16-arm dose-ranged hollow-fibre infection model (HFIM) experiment. The combination was further assessed in HFIM experiments mimicking neonatal plasma exposures of clinically relevant doses of both drugs against five Enterobacterales isolates with a range of flomoxef/amikacin MICs. RESULTS: Flomoxef and amikacin in combination were synergistic in bacterial killing in both assays and prevention of emergence of amikacin resistance in the HFIM. In the HFIM assessing neonatal-like drug exposures, the combination killed 3/5 strains to sterility, (including 2/5 that monotherapy with either drug failed to kill) and failed to kill the 2/5 strains with flomoxef MICs of 32 mg/L. CONCLUSIONS: We conclude that the combination of flomoxef and amikacin is synergistic and is a potentially clinically effective regimen for the empirical treatment of neonatal sepsis in LMIC settings and is therefore suitable for further assessment in a clinical trial.

Amikacin Combined with Fosfomycin for Treatment of Neonatal Sepsis in the Setting of Highly Prevalent Antimicrobial Resistance.

Antimicrobial resistance (particularly through extended-spectrum ß-lactamase and aminoglycoside-modifying enzyme production) in neonatal sepsis is a global problem, particularly in low- and middle-income countries, with significant mortality rates. High rates of resistance are reported for the current WHO-recommended first-line antibiotic regimen for neonatal sepsis, i.e., ampicillin and gentamicin. We assessed the utility of fosfomycin and amikacin as a potential alternative regimen to be used in settings of increasingly prevalent antimicrobial resistance. The combination was studied in a 16-arm dose-ranged hollow-fiber infection model (HFIM) experiment. The combination of amikacin and fosfomycin enhanced bactericidal activity and prevented the emergence of resistance, compared to monotherapy with either antibiotic. Modeling of the experimental quantitative outputs and data from checkerboard assays indicated synergy. We further assessed the combination regimen at clinically relevant doses in the HFIM with nine Enterobacterales strains with high fosfomycin and amikacin MICs and demonstrated successful kill to sterilization for 6/9 strains. From these data, we propose a novel combination breakpoint threshold for microbiological success for this antimicrobial combination against Enterobacterales strains, i.e., MICF × MICA < 256 (where MICF and MICA are the fosfomycin and amikacin MICs, respectively). Monte Carlo simulations predict that a standard fosfomycin-amikacin neonatal regimen would achieve >99% probability of pharmacodynamic success for strains with MICs below this threshold. We conclude that the combination of fosfomycin with amikacin is a viable regimen for the empirical treatment of neonatal sepsis and is suitable for further clinical assessment in a randomized controlled trial.

The role of combination therapy in the treatment of severe infections caused by carbapenem resistant gram-negatives: a systematic review of clinical studies.

BACKGROUND: Effective treatment of sepsis due to carbapenem-resistant Gram-negative bacteria (CR-GNB) remains a challenge for clinicians worldwide. In recent years, the combination of antibiotics has become the preferred treatment strategy for CR-GNB infection. However, robust evidence to support this approach is lacking. This systematic review aimed at critically evaluating all available antibiotic options for CR-GNB sepsis with particular focus on combination. METHODS: We systematically searched published literature from January 1945 until December 2018 for observational comparative and non-comparative studies and randomized trials examining any antibiotic option for CR-GNB. Studies were included if reporting microbiologically-confirmed infection caused by Acinetobacter baumannii, Enterobacteriaceae/Klebsiella spp., or Pseudomonas aeruginosa, reporting at least one of the study outcomes, and definitive antibiotic treatment. Carbapenem-resistance was defined as phenotypically-detected in vitro resistance to at least one of the following carbapenems: doripenem, ertapenem, imipenem, meropenem. Each antibiotic regimen was classified as "defined" when at least the molecular class(es) composing the regimen was detailed. Primary outcomes were 30-day and attributable mortality. Bayesian network meta-analysis (NMA) approach was selected for quantitative synthesis to explore feasibility of pooling data on antibiotic regimens. RESULTS: A total of 6306 records were retrieved and 134 studies including 11,546 patients were included: 54 studies were on Acinetobacter, 52 on Enterobacteriaceae/Klebsiella, 21 on mixed Gram-negative, and 7 on Pseudomonas. Nine (7%) were RCTs; 19 prospective cohorts (14%), 89 (66%) retrospective, and 17 (13%) case series. Forty-one studies (31%) were multicentric. Qualitative synthesis showed an heterogeneous and scattered reporting of key-clinical and microbiological variables across studies. Ninety-two distinct antibiotic regimens were identified with 47 of them (51%, 5863 patients) not reporting any details on numbers, type, dosage and in vitro activity of the included antibiotic molecules. The NMAs could not be performed for any of the selected outcome given the presence of too many disconnected components. CONCLUSION: The existing evidence is insufficient to allowing for the formulation of any evidence-based therapeutic recommendation for CR-GNB sepsis. Future studies must provide a standardized definition of antibiotic regimen to drive recommendations for using combination of antibiotics that can be reliably applied to clinical practice.

Pharmacodynamics of zoliflodacin plus doxycycline combination therapy against Neisseria gonorrhoeae in a gonococcal hollow-fiber infection model.

Antimicrobial resistance in the sexually transmitted bacterium Neisseria gonorrhoeae is compromising the management and control of gonorrhea globally. Optimized use and enhanced stewardship of current antimicrobials and development of novel antimicrobials are imperative. The first in class zoliflodacin (spiropyrimidinetrione, DNA Gyrase B inhibitor) is a promising novel antimicrobial in late-stage clinical development for gonorrhea treatment, i.e., the phase III randomized controlled clinical trial (ClinicalTrials.gov Identifier: NCT03959527) was recently finalized, and zoliflodacin showed non-inferiority compared to the recommended ceftriaxone plus azithromycin dual therapy. Doxycycline, the first-line treatment for chlamydia and empiric treatment for non-gonococcal urethritis, will be frequently given together with zoliflodacin because gonorrhea and chlamydia coinfections are common. In a previous static in vitro study, it was indicated that doxycycline/tetracycline inhibited the gonococcal killing of zoliflodacin in 6-h time-kill curve analysis. In this study, our dynamic in vitro hollow-fiber infection model (HFIM) was used to investigate combination therapies with zoliflodacin and doxycycline. Dose-range experiments using the three gonococcal strains WHO F (susceptible to relevant therapeutic antimicrobials), WHO X (extensively drug-resistant, including ceftriaxone-resistant; zoliflodacin-susceptible), and SE600/18 (zoliflodacin-susceptible strain with GyrB S467N substitution) were conducted simulating combination therapy with a single oral dose of zoliflodacin 0.5-4 g combined with a doxycycline daily oral dose of 200 mg administered as 100 mg twice a day, for 7 days (standard dose for chlamydia treatment). Comparing combination therapy of zoliflodacin (0.5-4 g single dose) plus doxycycline (200 mg divided into 100 mg twice a day orally, for 7 days) to zoliflodacin monotherapy (0.5-4 g single dose) showed that combination therapy was slightly more effective than monotherapy in the killing of N. gonorrhoeae and suppressing emergence of zoliflodacin resistance. Accordingly, WHO F was eradicated by only 0.5 g single dose of zoliflodacin in combination with doxycycline, and WHO X and SE600/18 were both eradicated by a 2 g single dose of zoliflodacin in combination with doxycycline; no zoliflodacin-resistant populations occurred during the 7-day experiment when using this zoliflodacin dose. When using suboptimal (0.5-1 g) zoliflodacin doses together with doxycycline, gonococcal mutants with increased zoliflodacin MICs, due to GyrB D429N and the novel GyrB T472P, emerged, but both the mutants had an impaired biofitness. The present study shows the high efficacy of zoliflodacin plus doxycycline combination therapy using a dynamic HFIM that more accurately and comprehensively simulate gonococcal infection and their treatment, i.e., compared to static in vitro models, such as short-time checkerboard experiments or time-kill curve analysis. Based on our dynamic in vitro HFIM work, zoliflodacin plus doxycycline for the treatment of both gonorrhea and chlamydia can be an effective combination.

Pharmacodynamic Evaluation of Zoliflodacin Treatment of Neisseria gonorrhoeae Strains With Amino Acid Substitutions in the Zoliflodacin Target GyrB Using a Dynamic Hollow Fiber Infection Model.

Novel antimicrobials for effective treatment of uncomplicated gonorrhea are essential, and the first-in-class, oral spiropyrimidinetrione DNA gyrase B inhibitor zoliflodacin appears promising. Using our newly developed Hollow Fiber Infection Model (HFIM), the pharmacodynamics of zoliflodacin was examined. A clinical zoliflodacin-susceptible N. gonorrhoeae strain, SE600/18 (harbouring a GyrB S467N amino acid substitution; MIC = 0.25 mg/L), and SE600/18-D429N (zoliflodacin-resistant mutant with a second GyrB substitution, D429N, selected in the HFIM experiments; zoliflodacin MIC = 2 mg/L), were examined. Dose-range experiments, simulating zoliflodacin single oral dose regimens of 0.5, 1, 2, 3, and 4 g, were performed for SE600/18. For SE600/18-D429N, dose-range experiments, simulating zoliflodacin single oral 2, 3, 4, and 6 g doses, and zoliflodacin oral dose-fractionation experiments with 4, 6, and 8 g administered as q12 h were performed. Both strains grew well in the untreated HFIM growth control arms and mostly maintained growth at 1010-1011 CFU/ml for 7 days. Zoliflodacin 3 and 4 g single dose oral regimens successfully eradicated SE600/18 and no growth was recovered during the 7-days experiments. However, the single oral 0.5, 1, and 2 g doses failed to eradicate SE600/18, and zoliflodacin-resistant populations with a GyrB D429N substitution were selected with all these doses. The zoliflodacin-resistant SE600/18-D429N mutant was not eradicated with any examined treatment regimen. However, this in vitro-selected zoliflodacin-resistant mutant was substantially less fit compared to the zoliflodacin-susceptible SE600/18 parent strain. In conclusion, the rare clinical gonococcal strains with GyrB S467N substitution are predisposed to develop zoliflodacin resistance and may require treatment with zoliflodacin ≥3 g. Future development may need to consider the inclusion of diagnostics directed at identifying strains resistant or predisposed to resistance development at a population level and to strengthen surveillance (phenotypically and genetically), and possibly also at the patient level to guide treatment.

Pharmacodynamic evaluation of lefamulin in the treatment of gonorrhea using a hollow fiber infection model simulating Neisseria gonorrhoeae infections.

The emergence and spread of antimicrobial resistance in Neisseria gonorrhoeae is seriously threatening the treatment and control of gonorrhea globally. Novel treatment options are essential, coupled with appropriate methods to pharmacodynamically examine the efficacy and resistance emergence of these novel drugs. Herein, we used our dynamic in vitro hollow fiber infection model (HFIM) to evaluate protein-unbound lefamulin, a semisynthetic pleuromutilin, against N. gonorrhoeae. Dose-range and dose-fractionation experiments with N. gonorrhoeae reference strains: WHO F (susceptible to all relevant antimicrobials), WHO X (extensively drug-resistant, including ceftriaxone resistance), and WHO V (high-level azithromycin resistant, and highest gonococcal MIC of lefamulin (2 mg/l) reported), were performed to examine lefamulin gonococcal killing and resistance development during treatment. The dose-range experiments, simulating a single oral dose of lefamulin based on human plasma concentrations, indicated that ≥1.2 g, ≥2.8 g, and ≥9.6 g of lefamulin were required to eradicate WHO F, X, and V, respectively. Dose-fractionation experiments, based on human lefamulin plasma concentrations, showed that WHO X was eradicated with ≥2.8 g per day when administered as q12 h (1.4 g twice a day) and with ≥3.6 g per day when administered as q8 h (1.2 g thrice a day), both for 7 days. However, when simulating the treatment with 5-10 times higher concentrations of free lefamulin in relevant gonorrhea tissues (based on urogenital tissues in a rat model), 600 mg every 12 h for 5 days (approved oral treatment for community-acquired bacterial pneumonia) eradicated all strains, and no lefamulin resistance emerged in the successful treatment arms. In many arms failing single or multiple dose treatments for WHO X, lefamulin-resistant mutants (MIC = 2 mg/l), containing an A132V amino acid substitution in ribosomal protein L3, were selected. Nevertheless, these lefamulin-resistant mutants demonstrated an impaired biofitness. In conclusion, a clinical study is warranted to elucidate the clinical potential of lefamulin as a treatment option for uncomplicated gonorrhea (as well as several other bacterial STIs).

Clinical management of severe infections caused by carbapenem-resistant gram-negative bacteria: a worldwide cross-sectional survey addressing the use of antibiotic combinations.

OBJECTIVES: Optimal treatment of carbapenem-resistant Gram-negative bacteria (CR-GNB) infections is uncertain because of the lack of good-quality evidence and the limited effectiveness of available antibiotics. The aim of this survey was to investigate clinicians' prescribing strategies for treating CR-GNB infections worldwide. METHODS: A 36-item questionnaire was developed addressing the following aspects of antibiotic prescribing: respondent's background, diagnostic and therapeutic availability, preferred antibiotic strategies and rationale for selecting combination therapy. Prescribers were recruited following the snowball sampling approach, and a post-stratification correction with inverse proportional weights was used to adjust the sample's representativeness. RESULTS: A total of 1012 respondents from 95 countries participated in the survey. Overall, 298 (30%) of the respondents had local guidelines for treating CR-GNB at their facility and 702 (71%) had access to Infectious Diseases consultation, with significant discrepancies according to country economic status: 85% (390/502) in high-income countries versus 59% (194/283) in upper-medium-income countries and 30% (118/196) in lower-middle-income countries/lower-income-countries). Targeted regimens varied widely, ranging from 40 regimens for CR-Acinetobacter spp. to more than 100 regimens for CR-Enterobacteriaceae. Although the majority of respondents acknowledged the lack of evidence behind this choice, dual combination was the preferred treatment scheme and carbapenem-polymyxin was the most prescribed regimen, irrespective of pathogen and infection source. Respondents noticeably disagreed around the meaning of 'combination therapy' with 20% (150/783) indicating the simple addition of multiple compounds, 42% (321/783) requiring the presence of in vitro activity and 38% (290/783) requiring in vitro synergism. CONCLUSIONS: Management of CR-GNB infections is far from being standardized. Strategic public health focused randomized controlled trials are urgently required to inform evidence-based treatment guidelines.

Pharmacodynamic Evaluation of Dosing, Bacterial Kill, and Resistance Suppression for Zoliflodacin Against Neisseria gonorrhoeae in a Dynamic Hollow Fiber Infection Model.

Antimicrobial resistance in Neisseria gonorrhoeae is threatening the treatment and control of gonorrhea globally, and new treatment options are imperative. Utilizing our dynamic in vitro hollow fiber infection model (HFIM), we examined the pharmacodynamics of the first-in-class spiropyrimidinetrione (DNA gyrase B inhibitors), zoliflodacin, against the N. gonorrhoeae reference strains World Health Organization F (susceptible to all relevant antimicrobials) and WHO X (extensively drug resistant, including resistance to ceftriaxone) over 7 days. Dose-range experiments with both strains, simulating zoliflodacin single oral dose regimens of 0.5-8 g, and dose-fractionation experiments with WHO X, simulating zoliflodacin oral dose therapy with 1-4 g administered as q12 h and q8 h for 24 h, were performed. A kill-rate constant that reflected a rapid bacterial kill during the first 6.5 h for both strains and all zoliflodacin doses was identified. In the dose-range experiments, the zoliflodacin 2-8 g single-dose treatments successfully eradicated both WHO strains, and resistance to zoliflodacin was not observed. However, zoliflodacin as a single 0.5 g dose failed to eradicate both WHO strains, and a 1 g single dose failed to eradicate WHO X in one of two experiments. The zoliflodacin 1 g/day regimen also failed to eradicate WHO X when administered as two and three divided doses given at q12 h and q8 h in the dose-fractionation studies, respectively. All failed regimens selected for zoliflodacin-resistant mutants. In conclusion, these data demonstrate that zoliflodacin should be administered at >2 g as a single oral dose to provide effective killing and resistance suppression of N. gonorrhoeae. Future studies providing pharmacokinetic data for zoliflodacin (and other gonorrhea therapeutic antimicrobials) in urogenital and extragenital infection sites, particularly in the pharynx, and evaluation of gonococcal strains with different gyrB mutations would be important.

Potential Antibiotics for the Treatment of Neonatal Sepsis Caused by Multidrug-Resistant Bacteria.

Neonatal sepsis causes up to an estimated 680,000 deaths annually worldwide, predominantly in low- and middle-income countries (LMICs). A significant and growing proportion of bacteria causing neonatal sepsis are resistant to multiple antibiotics, including the World Health Organization-recommended empiric neonatal sepsis regimen of ampicillin/gentamicin. The Global Antibiotic Research and Development Partnership is aiming to develop alternative empiric antibiotic regimens that fulfil several criteria: (1) affordable in LMIC settings; (2) activity against neonatal bacterial pathogens, including extended-spectrum ß-lactamase producers, gentamicin-resistant Gram-negative bacteria, and methicillin-resistant Staphylococcus aureus (MRSA); (3) a licence for neonatal use or extensive experience of use in neonates; and (4) minimal toxicities. In this review, we identify five antibiotics that fulfil these criteria: amikacin, tobramycin, fosfomycin, flomoxef, and cefepime. We describe the available characteristics of each in terms of mechanism of action, resistance mechanisms, clinical pharmacokinetics, pharmacodynamics, and toxicity profile. We also identify some knowledge gaps: (1) the neonatal pharmacokinetics of cefepime is reliant on relatively small and limited datasets, and the pharmacokinetics of flomoxef are also reliant on data from a limited demographic range and (2) for all reviewed agents, the pharmacodynamic index and target has not been definitively established for both bactericidal effect and emergence of resistance, with many assumed to have an identical index/target to similar class molecules. These five agents have the potential to be used in novel combination empiric regimens for neonatal sepsis. However, the data gaps need addressing by pharmacokinetic trials and pharmacodynamic characterisation.

Systematic review and meta-analysis of in vitro efficacy of antibiotic combination therapy against carbapenem-resistant Gram-negative bacilli.

The superiority of combination therapy for carbapenem-resistant Gram-negative bacilli (CR-GNB) infections remains controversial. In vitro models may predict the efficacy of antibiotic regimens against CR-GNB. A systematic review and meta-analysis was performed including pharmacokinetic/pharmacodynamic (PK/PD) and time-kill (TK) studies examining the in vitro efficacy of antibiotic combinations against CR-GNB [PROSPERO registration no. CRD42019128104]. The primary outcome was in vitro synergy based on the effect size (ES): high, ES ≥ 0.75, moderate, 0.35 < ES < 0.75; low, ES ≤ 0.35; and absent, ES = 0). A network meta-analysis assessed the bactericidal effect and re-growth rate (secondary outcomes). An adapted version of the ToxRTool was used for risk-of-bias assessment. Over 180 combination regimens from 136 studies were included. The most frequently analysed classes were polymyxins and carbapenems. Limited data were available for ceftazidime/avibactam, ceftolozane/tazobactam and imipenem/relebactam. High or moderate synergism was shown for polymyxin/rifampicin against Acinetobacter baumannii [ES = 0.91, 95% confidence interval (CI) 0.44-1.00], polymyxin/fosfomycin against Klebsiella pneumoniae (ES = 1.00, 95% CI 0.66-1.00) and imipenem/amikacin against Pseudomonas aeruginosa (ES = 1.00, 95% CI 0.21-1.00). Compared with monotherapy, increased bactericidal activity and lower re-growth rates were reported for colistin/fosfomycin and polymyxin/rifampicin in K. pneumoniae and for imipenem/amikacin or imipenem/tobramycin against P. aeruginosa. High quality was documented for 65% and 53% of PK/PD and TK studies, respectively. Well-designed in vitro studies should be encouraged to guide the selection of combination therapies in clinical trials and to improve the armamentarium against carbapenem-resistant bacteria.

In vivo studies on antibiotic combination for the treatment of carbapenem-resistant Gram-negative bacteria: a systematic review and meta-analysis protocol.

OBJECTIVE: There is poor evidence to determine the superiority of combination regimens versus monotherapy against infections due to carbapenem-resistant (CR) Gram-negative bacteria. In vivo models can simulate the pathophysiology of infections in humans and assess antibiotic efficacy. We aim to investigate in vivo effects of antibiotic combination on mortality and disease burden for infections due to CR Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacteriaceae and provide an unbiased overview of existing knowledge. The results of the study can help prioritising future research on the most promising therapies against CR bacteria. METHODS AND ANALYSIS: This protocol was formulated using the Systematic Review Protocol for Animal Intervention Studies (SYRCLE) Checklist. Publications will be collected from PubMed, Scopus, Embase and Web of Science. Quality checklists adapted by Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies and SYRCLE's risk of bias tool will be used. If the meta-analysis seems feasible, the ES and the 95% CI will be analysed. The heterogeneity between studies will be assessed by I2 test. Subgroup meta-analysis will be performed when possible to assess the impact of the studies on efficacy of the treatments. Funnel plotting will be used to evaluate the risk of publication bias. DISSEMINATION: This systematic review and meta-analysis is part of a wider research collaboration project, the COmbination tHErapy to treat sepsis due to carbapenem-Resistant bacteria in adult and paediatric population: EvideNCE and common practice (COHERENCE) study that includes also the analyses of in vitro and human studies. Data will be presented at international conferences and the results will be published in peer-reviewed journals. PROSPERO REGISTRATION NUMBER: CRD42019128104(available at: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42019128104).

Pharmacokinetic/pharmacodynamic considerations for new and current therapeutic drugs for uncomplicated gonorrhoea-challenges and opportunities.

BACKGROUND: Increasing multidrug resistance rates in Neisseria gonorrhoeae have raised concerns and an urgent call for new antibiotics for treatment of gonorrhoea. Several decades of subdued drug development in this field and the recent failures of two new antibiotics to show non-inferiority compared with the current first-line antibiotics ceftriaxone plus azithromycin highlight the need for improved preclinical tools to predict clinical outcome of new drugs in the development process. OBJECTIVES: To summarize current pharmacokinetic/pharmacodynamic (PK/PD) knowledge and dose-finding strategies for antibiotics against gonorrhoea. SOURCES: Literature review of published papers and discussions by global experts at a special workshop on this topic. CONTENT: We review current knowledge of gonococcal specific PK/PD principles and provide an update on new in vitro and in vivo models to correlate drug exposure with clinical outcome, and identify challenges and gaps in gonococcal therapeutic research. IMPLICATIONS: Identifying the ideal antimicrobial agent and dose for treating uncomplicated urogenital and pharyngeal gonococcal disease requires appropriate validated non-clinical PK/PD models. Recent advances in adapting in vitro and in vivo models for use in gonorrhoea are an important step for enabling the development of new drugs with reduced risk of failure in Phase 3 clinical development and diminish the risk of emergence of resistance.

In vitro activities of the Rx-01 oxazolidinones against hospital and community pathogens.

Rx-01_423 and Rx-01_667 are two members of the family of oxazolidinones that were designed using a combination of computational and medicinal chemistry and conventional biological techniques. The compounds have a two- to eightfold-improved potency over linezolid against serious gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant streptococci, and vancomycin-resistant enterococci. This enhanced potency extends to the coverage of linezolid-resistant gram-positive microbes, especially multidrug-resistant enterococci and pneumococci. Compounds from this series expand the spectrum compared with linezolid to include fastidious gram-negative organisms like Haemophilus influenzae and Moraxella catarrhalis. Like linezolid, the Rx-01 compounds are bacteriostatic against MRSA and enterococci but are generally bactericidal against S. pneumoniae and H. influenzae.

R chi-01, a new family of oxazolidinones that overcome ribosome-based linezolid resistance.

New and improved antibiotics are urgently needed to combat the ever-increasing number of multidrug-resistant bacteria. In this study, we characterized several members of a new oxazolidinone family, R chi-01. This antibiotic family is distinguished by having in vitro and in vivo activity against hospital-acquired, as well as community-acquired, pathogens. We compared the 50S ribosome binding affinity of this family to that of the only marketed oxazolidinone antibiotic, linezolid, using chloramphenicol and puromycin competition binding assays. The competition assays demonstrated that several members of the R chi-01 family displace, more effectively than linezolid, compounds known to bind to the ribosomal A site. We also monitored binding by assessing whether R chi-01 compounds protect U2585 (Escherichia coli numbering), a nucleotide that influences peptide bond formation and peptide release, from chemical modification by carbodiimide. The R chi-01 oxazolidinones were able to inhibit translation of ribosomes isolated from linezolid-resistant Staphylococcus aureus at submicromolar concentrations. This improved binding corresponds to greater antibacterial activity against linezolid-resistant enterococci. Consistent with their ribosomal A-site targeting and greater potency, the R chi-01 compounds promote nonsense suppression and frameshifting to a greater extent than linezolid. Importantly, the gain in potency does not impact prokaryotic specificity as, like linezolid, the members of the R chi-01 family show translation 50% inhibitory concentrations that are at least 100-fold higher for eukaryotic than for prokaryotic ribosomes. This new family of oxazolidinones distinguishes itself from linezolid by having greater intrinsic activity against linezolid-resistant isolates and may therefore offer clinicians an alternative to overcome linezolid resistance. A member of the R chi-01 family of compounds is currently undergoing clinical trials.

Crystal structure of the oxazolidinone antibiotic linezolid bound to the 50S ribosomal subunit.

The oxazolidinone antibacterials target the 50S subunit of prokaryotic ribosomes. To gain insight into their mechanism of action, the crystal structure of the canonical oxazolidinone, linezolid, has been determined bound to the Haloarcula marismortui 50S subunit. Linezolid binds the 50S A-site, near the catalytic center, which suggests that inhibition involves competition with incoming A-site substrates. These results provide a structural basis for the discovery of improved oxazolidinones active against emerging drug-resistant clinical strains.

Structure-based drug design meets the ribosome.

The high-resolution structures of the bacterial ribosomal subunits and those of their complexes with antibiotics have advanced significantly our understanding of small-molecule interactions with RNA. The wealth of RNA structural data generated by these structures has allowed computational chemists to employ a drug discovery paradigm focused on RNA-based targets. The structures also show how target-based resistance affects antibiotics acting at the level of the ribosome. Not only are the sites pinpointed where different classes of antibiotics inhibit protein synthesis, but their orientations, relative dispositions, and unique mechanisms of action are also revealed at the atomic level. Both the 30S and the 50S ribosomal subunits have been shown to be "targets of targets", offering several adjacent, functionally relevant binding pockets for antibiotics. It is the detailed knowledge of these validated locations, or ribofunctional loci, plus the mapping of the resistance hot-spots that allow the rational design of next-generation antibacterials. When the structural information is combined with a data-driven computational toolkit able to describe and predict molecular properties appropriate for bacterial cell penetration and drug-likeness, a structure-based drug design approach for novel antibacterials shows great promise.

Structural basis for the antibiotic activity of ketolides and azalides.

The azalide azithromycin and the ketolide ABT-773, which were derived by chemical modifications of erythromycin, exhibit elevated activity against a number of penicillin- and macrolide-resistant pathogenic bacteria. Analysis of the crystal structures of the large ribosomal subunit from Deinococcus radiodurans complexed with azithromycin or ABT-773 indicates that, despite differences in the number and nature of their contacts with the ribosome, both compounds exert their antimicrobial activity by blocking the protein exit tunnel. In contrast to all macrolides studied so far, two molecules of azithromycin bind simultaneously to the tunnel. The additional molecule also interacts with two proteins, L4 and L22, implicated in macrolide resistance. These studies illuminated and rationalized the enhanced activity of the drugs against specific macrolide-resistant bacteria.

Accelerating global innovation to address antibacterial resistance: introducing CARB-X.

A global response to the chronic shortfall in antibiotic innovation is urgently needed to combat antimicrobial resistance. Here, we introduce CARB-X, a new global public-private partnership that will invest more than US$350 million in the next 5 years to accelerate the progression of a diverse portfolio of innovative antibacterial products into clinical trials.