Epidemiological trends and susceptibility patterns of bloodstream infections caused by Enterococcus spp. in six German university hospitals: a prospectively evaluated multicentre cohort study from 2016 to 2020 of the R-Net study group.

PURPOSE: To analyse recent epidemiological trends of bloodstream infections (BSI) caused by Enterococcus spp. In adult patients admitted to tertiary care centres in Germany. METHODS: Epidemiological data from the multicentre R-NET study was analysed. Patients presenting with E. faecium or E. faecalis in blood cultures in six German tertiary care university hospitals between October 2016 and June 2020 were prospectively evaluated. In vancomycin-resistant enterococci (VRE), the presence of vanA/vanB was confirmed via molecular methods. RESULTS: In the 4-year study period, 3001 patients with BSI due to Enterococcus spp. were identified. E. faecium was detected in 1830 patients (61%) and E. faecalis in 1229 patients (41%). Most BSI occurred in (sub-) specialties of internal medicine. The pooled incidence density of enterococcal BSI increased significantly (4.0-4.5 cases per 10,000 patient days), which was primarily driven by VRE BSI (0.5 to 1.0 cases per 10,000 patient days). In 2020, the proportion of VRE BSI was > 12% in all study sites (range, 12.8-32.2%). Molecular detection of resistance in 363 VRE isolates showed a predominance of the vanB gene (77.1%). CONCLUSION: This large multicentre study highlights an increase of BSI due to E. faecium, which was primarily driven by VRE. The high rates of hospital- and ICU-acquired VRE BSI point towards an important role of prior antibiotic exposure and invasive procedures as risk factors. Due to limited treatment options and high mortality rates of VRE BSI, the increasing incidence of VRE BSI is of major concern.

Early treatment with fluvoxamine, bromhexine, cyproheptadine, and niclosamide to prevent clinical deterioration in patients with symptomatic COVID-19: a randomized clinical trial.

Background: Repurposed drugs with host-directed antiviral and immunomodulatory properties have shown promise in the treatment of COVID-19, but few trials have studied combinations of these agents. The aim of this trial was to assess the effectiveness of affordable, widely available, repurposed drugs used in combination for treatment of COVID-19, which may be particularly relevant to low-resource countries. Methods: We conducted an open-label, randomized, outpatient, controlled trial in Thailand from October 1, 2021, to June 21, 2022, to assess whether early treatment within 48-h of symptoms onset with combinations of fluvoxamine, bromhexine, cyproheptadine, and niclosamide, given to adults with confirmed mild SARS-CoV-2 infection, can prevent 28-day clinical deterioration compared to standard care. Participants were randomly assigned to receive treatment with fluvoxamine alone, fluvoxamine + bromhexine, fluvoxamine + cyproheptadine, niclosamide + bromhexine, or standard care. The primary outcome measured was clinical deterioration within 9, 14, or 28 days using a 6-point ordinal scale. This trial is registered with ClinicalTrials.gov (NCT05087381). Findings: Among 1900 recruited, a total of 995 participants completed the trial. No participants had clinical deterioration by day 9, 14, or 28 days among those treated with fluvoxamine plus bromhexine (0%), fluvoxamine plus cyproheptadine (0%), or niclosamide plus bromhexine (0%). Nine participants (5.6%) in the fluvoxamine arm had clinical deterioration by day 28, requiring low-flow oxygen. In contrast, most standard care arm participants had clinical deterioration by 9, 14, and 28 days. By day 9, 32.7% (110) of patients in the standard care arm had been hospitalized without requiring supplemental oxygen but needing ongoing medical care. By day 28, this percentage increased to 37.5% (21). Additionally, 20.8% (70) of patients in the standard care arm required low-flow oxygen by day 9, and 12.5% (16) needed non-invasive or mechanical ventilation by day 28. All treated groups significantly differed from the standard care group by days 9, 14, and 28 (p < 0.0001). Also, by day 28, the three 2-drug treatments were significantly better than the fluvoxamine arm (p < 0.0001). No deaths occurred in any study group. Compared to standard care, participants treated with the combination agents had significantly decreased viral loads as early as day 3 of treatment (p < 0.0001), decreased levels of serum cytokines interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1ß) as early as day 5 of treatment, and interleukin-8 (IL-8) by day 7 of treatment (p < 0.0001) and lower incidence of post-acute sequelae of COVID-19 (PASC) symptoms (p < 0.0001). 23 serious adverse events occurred in the standard care arm, while only 1 serious adverse event was reported in the fluvoxamine arm, and zero serious adverse events occurred in the other arms. Interpretation: Early treatment with these combinations among outpatients diagnosed with COVID-19 was associated with lower likelihood of clinical deterioration, and with significant and rapid reduction in the viral load and serum cytokines, and with lower burden of PASC symptoms. When started very soon after symptom onset, these repurposed drugs have high potential to prevent clinical deterioration and death in vaccinated and unvaccinated COVID-19 patients. Funding: Ped Thai Su Phai (Thai Ducks Fighting Danger) social giver group.

Genomic Surveillance Uncovers a 10-Year Persistence of an OXA-24/40 Acinetobacter baumannii Clone in a Tertiary Hospital in Northern Spain.

Infections caused by carbapenem-resistant Acinetobacter baumannii are a global threat causing a high number of fatal infections. This microorganism can also easily acquire antibiotic resistance determinants, making the treatment of infections a big challenge, and has the ability to persist in the hospital environment under a wide range of conditions. The objective of this work was to study the molecular epidemiology and genetic characteristics of two blaOXA24/40Acinetobacter baumannii outbreaks (2009 and 2020-21) at a tertiary hospital in Northern Spain. Thirty-six isolates were investigated and genotypically screened by Whole Genome Sequencing to analyse the resistome and virulome. Isolates were resistant to carbapenems, aminoglycosides and fluoroquinolones. Multi-Locus Sequence Typing analysis identified that Outbreak 1 was mainly produced by isolates belonging to ST3Pas/ST106Oxf (IC3) containing blaOXA24/40, blaOXA71 and blaADC119. Outbreak 2 isolates were exclusively ST2Pas/ST801Oxf (IC2) blaOXA24/40, blaOXA66 and blaADC30, the same genotype seen in two isolates from 2009. Virulome analysis showed that IC2 isolates contained genes for capsular polysaccharide KL32 and lipooligosacharide OCL5. A 8.9 Kb plasmid encoding the blaOXA24/40 gene was common in all isolates. The persistance over time of a virulent IC2 clone highlights the need of active surveillance to control its spread.

Exploring indoor and outdoor dust as a potential tool for detection and monitoring of COVID-19 transmission.

This study investigated the potential of using SARS-CoV-2 viral concentrations in dust as an additional surveillance tool for early detection and monitoring of COVID-19 transmission. Dust samples were collected from 8 public locations in 16 districts of Bangkok, Thailand, from June to August 2021. SARS-CoV-2 RNA concentrations in dust were quantified, and their correlation with community case incidence was assessed. Our findings revealed a positive correlation between viral concentrations detected in dust and the relative risk of COVID-19. The highest risk was observed with no delay (0-day lag), and this risk gradually decreased as the lag time increased. We observed an overall decline in viral concentrations in public places during lockdown, closely associated with reduced human mobility. The effective reproduction number for COVID-19 transmission remained above one throughout the study period, suggesting that transmission may persist in locations beyond public areas even after the lockdown measures were in place.

A global view on carbapenem-resistant Acinetobacter baumannii.

To give an update on the molecular epidemiology and global distribution of carbapenemase encoding genes, we subjected 313 carbapenem-resistant Acinetobacter baumannii isolated from 114 study centers in 47 countries in five world regions, Africa, Asia, Europe, Latin America, and North America, to whole genome sequencing. Numbers of isolates investigated were proportional to the population size of the contributing countries. Molecular epidemiology was investigated using seven-loci and core genome multilocus sequence typing, whole-genome single nucleotide polymorphism phylogenies, and the intrinsic blaOXA-51-like variant. Carbapenemase encoding genes were identified by multiplex PCR and ResFinder. Among the total of 313 isolates, 289 (92.3%) were assigned to A. baumannii international clones (IC) IC1-IC8. IC2 predominated with 196 isolates (62.6%) and was spread worldwide, followed by IC5 with 44 isolates (14.1%) mainly confined to Latin America. Six isolates (1.9%) originating from Belgium, Egypt, Italy, and Pakistan represent the novel IC9. Acquired OXA-type carbapenemase genes were found in 300 (96%) isolates with blaOXA-23-like and blaOXA-40-like predominating, which constitutes a significant increase compared to our findings from 2010. Metallo-beta-lactamases were rare with seven isolates (2.2%). The distribution of ICs and carbapenemase determinants can vary widely among different geographical regions. IMPORTANCE Carbapenem-resistant Acinetobacter baumannii are of increasing public health importance, as they are resistant to last-line antibiotics. International clones with well-characterized resistance genes dominate globally; however, locally, other lineages with different properties may be of importance to consider. This study investigated isolates from a broad geographic origin from 114 hospitals in 47 countries and from five world regions ensuring the greatest possible diversity in an organism known for its propensity for clonal epidemic spread and reflecting the current global epidemiology of carbapenem-resistant A. baumannii. In Latin America, a lineage different from other geographic regions circulates, with a different resistance gene profile. This knowledge is important to adjust local infection prevention measures. In a global world with migration and increasing use of antimicrobials, multidrug-resistant bacteria will continue to adapt and challenge our healthcare systems worldwide.

Tracing the transmission of mpox through wastewater surveillance in Southeast Asia.

High population density and tourism in Southeast Asia increase the risk of mpox due to frequent interpersonal contacts. Our wastewater surveillance in six Southeast Asian countries revealed positive signals for Monkeypox virus (MPXV) DNA, indicating local transmission. This alerts clinicians and helps allocate resources like testing, vaccines and therapeutics in resource-limited countries.

In Vitro Activity of Cefiderocol against a Global Collection of Carbapenem-Resistant Acinetobacter baumannii Isolates.

Background: Cefiderocol is a novel siderophore cephalosporin with potent activity against multi-drug-resistant Gram-negative pathogens including carbapenem-resistant Acinetobacter baumannii (CRAB). Methods: The susceptibility of 313 non-duplicate CRAB isolates with defined carbapenem resistance mechanisms from a global collection to cefiderocol, ceftazidime, ceftazidime/avibactam, ceftolozane/tazobactam, ciprofloxacin, colistin, imipenem/relebactam, meropenem, meropenem/vaborbactam, minocycline, and piperacillin/tazobactam was determined using the broth microdilution method. Isolates were obtained from various body sites from patients in 47 countries in five world regions between 2012 and 2016. The identification of carbapenem resistance mechanisms and assignment to A. baumannii international clonal lineages were based on whole genome sequencing. Results: Cefiderocol showed greater activity than comparator antimicrobials of the ß-lactam class, including novel ß-lactams combined with ß-lactamase inhibitors, ciprofloxacin, and minocycline. Cefiderocol MIC50 and MIC90 values were 0.5 mg/L and 4 mg/L, respectively, while colistin had comparable activity with a higher MIC50 at 1 mg/L and a lower MIC90 value of 2 mg/L. Many isolates with elevated cefiderocol MICs ≥ 4 mg/L represented A. baumannii international clone (IC) 1 and harbored a metallo-ß-lactamase. Conclusions: While cefiderocol is a useful addition to the limited armamentarium of drugs targeting this problematic pathogen, a considerable part of CRAB isolates had elevated MIC values in a range of 4 -> 32 mg/L, including all isolates with a metallo-ß-lactamase.

Ca-EDTA restores the activity of ceftazidime-avibactam or aztreonam against carbapenemase-producing Klebsiellapneumoniae infections.

Developing an effective therapy to overcome carbapenemase-positive Klebsiella pneumoniae (CPKp) is an important therapeutic challenge that must be addressed urgently. Here, we explored a Ca-EDTA combination with aztreonam or ceftazidime-avibactam in vitro and in vivo against diverse CPKp clinical isolates. The synergy testing of this study demonstrated that novel aztreonam-Ca-EDTA or ceftazidime-avibactam-Ca-EDTA combination was significantly effective in eliminating planktonic and mature biofilms in vitro, as well as eradicating CPKp infections in vivo. Both combinations revealed significant therapeutic efficacies in reducing bacterial load in internal organs and protecting treated mice from mortality. Conclusively, this is the first in vitro and in vivo study to demonstrate that novel aztreonam-Ca-EDTA or ceftazidime-avibactam-Ca-EDTA combinations provide favorable efficacy and safety for successful eradication of carbapenemase-producing Klebsiella pneumoniae planktonic and biofilm infections.

The Acinetobacter baumannii website (Ab-web): a multidisciplinary knowledge hub, communication platform, and workspace.

Acinetobacter baumannii is a Gram-negative bacterium increasingly implicated in hospital-acquired infections and outbreaks. Effective prevention and control of such infections are commonly challenged by the frequent emergence of multidrug-resistant strains. Here we introduce Ab-web (https://www.acinetobacterbaumannii.no), the first online platform for sharing expertise on A. baumannii. Ab-web is a species-centric knowledge hub, initially with 10 articles organized into two main sections, 'Overview' and 'Topics', and three themes, 'epidemiology', 'antibiotic resistance', and 'virulence'. The 'workspace' section provides a spot for colleagues to collaborate, build, and manage joint projects. Ab-web is a community-driven initiative amenable to constructive feedback and new ideas.

COVID-19 monitoring with sparse sampling of sewered and non-sewered wastewater in urban and rural communities.

Equitable SARS-CoV-2 surveillance in low-resource communities lacking centralized sewers is critical as wastewater-based epidemiology (WBE) progresses. However, large-scale studies on SARS-CoV-2 detection in wastewater from low-and middle-income countries is limited because of economic and technical reasons. In this study, wastewater samples were collected twice a month from 186 urban and rural subdistricts in nine provinces of Thailand mostly having decentralized and non-sewered sanitation infrastructure and analyzed for SARS-CoV-2 RNA variants using allele-specific RT-qPCR. Wastewater SARS-CoV-2 RNA concentration was used to estimate the real-time incidence and time-varying effective reproduction number (Re). Results showed an increase in SARS-CoV-2 RNA concentrations in wastewater from urban and rural areas 14-20 days earlier than infected individuals were officially reported. It also showed that community/food markets were "hot spots" for infected people. This approach offers an opportunity for early detection of transmission surges, allowing preparedness and potentially mitigating significant outbreaks at both spatial and temporal scales.

Characterisation of carbapenemase-producing Acinetobacter baumannii isolates from danish patients 2014-2021: detection of a new international clone - IC11.

OBJECTIVES: This study aimed to characterise carbapenemase-producing Acinetobacter baumannii (A. baumannii) isolates from Danish patients using whole genome sequencing (WGS). It also compared typing and epidemiological data for further investigation of the spread and origin of the carbapenemase-producing A. baumannii isolates. METHODS: From 1 January 2014 to 30 September 2021, 141 carbapenemase-producing A. baumannii isolates, received at the national reference laboratory at Statens Serum Institut, were investigated using WGS. Multilocus sequence typing (MLST) and cgMLST data, obtained by SeqSphere+ software, were linked to data related to source of isolation, patient age and sex, hospital admission and travel history. RESULTS: Most of the carbapenemase-producing A. baumannii isolates were from males (n = 100, 71%). Most patients (n = 88, 63%) had travelled outside Scandinavia before admission to a Danish hospital. The most prevalent carbapenemase gene was blaOXA-23 (n = 124). Isolates belonging to the dominating international clone IC2 accounted for 78% of all isolates. A new international ST164/OXA-91 clone, proposed to be named IC11, was recognised and described. The cgMLST analysis revealed 17 clusters, reflecting both sporadic travel to similar geographical areas and confirmed outbreaks in Danish hospitals. CONCLUSIONS: The occurrence of carbapenemase-producing A. baumannii in Denmark was still low; however, isolates belonging to major international clones with a high potential to spread within hospitals, mainly IC2, dominated. OXA-23 was by far the most prevalent carbapenemase detected. Sporadic and travel-related introductions to Danish hospitals, also intra-hospital transmission, could be confirmed, emphasising the need for continuing vigilance.

In Vitro Activity of Cefiderocol against Clinical Gram-Negative Isolates Originating from Germany in 2016/17.

Antimicrobial resistance poses a global threat to public health. Of great concern are Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacterales with resistance to carbapenems or third-generation cephalosporins. The aim of the present study was to investigate the in vitro activity of the novel siderophore cephaloporin cefiderocol (CID) and four comparator ß-lactam-ß-lactamase-inhibitor combinations and to give insights into the genetic background of CID-resistant isolates. In total, 301 clinical Enterobacterales and non-fermenting bacterial isolates were selected for this study, including randomly chosen isolates (set I, n = 195) and challenge isolates (set II, n = 106; enriched with ESBL and carbapenemase producers, as well as colistin-resistant isolates). Isolates displayed CID MIC50/90 values of 0.12/0.5 mg/L (set I) and 0.5/1 mg/L (set II). Overall, the CID activity was superior to the comparators against A. baumannii, Stenotrophomonas maltophilia and set II isolates of P. aeruginosa. There were eight CID-resistant isolates detected (MIC > 2 mg/L): A. baumannii (n = 1), E. cloacae complex (n = 5) and P. aeruginosa (n = 2). Sequencing analyses of these isolates detected the acquired ß-lactamase (bla) genes blaNDM-1,blaSHV-12 and naturally occurring blaOXA-396, blaACT-type and blaCMH-3. In conclusion, CID revealed potent activity against clinically relevant organisms of multidrug-resistant Enterobacterales and non-fermenters.

Development of an immunochromatographic lateral flow assay to rapidly detect OXA-23-, OXA-40-, OXA-58- and NDM-mediated carbapenem resistance determinants in Acinetobacter baumannii.

Introduction. Acinetobacter baumannii infections can be extremely challenging to treat owing to the worldwide prevalence of multidrug-resistant isolates, especially against carbapenems. Colonization with carbapenem-resistant A. baumannii (CRAb) requires rapid action from an infection control perspective because the organism is known for its propensity for epidemic spread. Hypothesis/Gap Statement. There is an unmet medical need to rapidly identify CRAb to enable appropriate antimicrobial treatment and to prevent transmission. Aim. Our aim was to expand the OXA-detection abilities of the rapid immunochromatographic test (ICT) OXA-23 K-SeT (Coris BioConcept) to include OXA-40- and OXA-58-like carbapenemases, which together confer carbapenem resistance to more than 94 % of CRAb isolates worldwide. Methodology. We used hybridoma technology to generate mAbs against OXA-40 and OXA-58 and selected them for productivity and specificity against recombinant and endogenous OXA-40 and OXA-58. Combinations of the resulting mAbs were analysed in ICT format for their ability to detect recombinant rOXA-40His6 or rOXA-58His6, respectively. Subsequently, selected antibody pairs were implemented into single-OXA-40 or single-OXA-58 prototypes and the final OXA-23/40/58/NDM ICT and were evaluated on clinical Acinetobacter spp. isolates with well-defined carbapenem resistance mechanisms. Results. Five anti-OXA-40 and anti-OXA-58 mAbs were selected. Competition ELISA with combinations of these antibodies revealed that the anti-OXA-40 antibodies bind to one of two binding clusters on OXA-40, while anti-OXA-58 antibodies bind to one of four binding clusters on OXA-58. Direct binding to the corresponding antigen in an ICT format has left only three antibodies against rOXA-40His6 and rOXA-58His6, respectively for the subsequent sandwich ICT selection procedure, which revealed that the anti-OXA-40 (#5) and anti-OXA-58 (#A8) mAbs in combination with the cross-reactive mAb #C8 performed best. They were implemented into single-OXA-40 and single-OXA-58 ICT prototypes and evaluated. These single ICT prototypes demonstrated 100 % specificity and sensitivity. Based on these results, an OXA-23/40/58/NDM-ICT was developed, complemented with OXA-23 and NDM-specific detection. An evaluation with selected carbapenem-resistant Acinetobacter spp. isolates (n=34) showed 100 % specificity. Conclusion. With this easy-to-use detection assay, one can save 12-48 h in diagnostics, which helps to treat patients earlier with appropriate antibiotics and allows immediate intervention to control transmission of CRAb.

Acinetobacter baumannii from Samples of Commercially Reared Turkeys: Genomic Relationships, Antimicrobial and Biocide Susceptibility.

Acinetobacter baumannii is especially known as a cause of nosocomial infections worldwide. It shows intrinsic and acquired resistances to numerous antimicrobial agents, which can render the treatment difficult. In contrast to the situation in human medicine, there are only few studies focusing on A. baumannii among livestock. In this study, we have examined 643 samples from turkeys reared for meat production, including 250 environmental and 393 diagnostic samples, for the presence of A. baumannii. In total, 99 isolates were identified, confirmed to species level via MALDI-TOF-MS and characterised with pulsed-field gel electrophoresis. Antimicrobial and biocide susceptibility was tested by broth microdilution methods. Based on the results, 26 representative isolates were selected and subjected to whole-genome sequencing (WGS). In general, A. baumannii was detected at a very low prevalence, except for a high prevalence of 79.7% in chick-box-papers (n = 118) of one-day-old turkey chicks. The distributions of the minimal inhibitory concentration values were unimodal for the four biocides and for most of the antimicrobial agents tested. WGS revealed 16 Pasteur and 18 Oxford sequence types, including new ones. Core genome MLST highlighted the diversity of most isolates. In conclusion, the isolates detected were highly diverse and still susceptible to many antimicrobial agents.

Klebsiella pneumonia in Sudan: Multidrug Resistance, Polyclonal Dissemination, and Virulence.

The emergence and global expansion of hyper-virulent and multidrug resistant (MDR) Klebsiella pneumoniae is an increasing healthcare threat worldwide. The epidemiology of MDR K. pneumoniae is under-characterized in many parts of the world, particularly Africa. In this study, K. pneumoniae isolates from hospitals in Khartoum, Sudan, have been whole-genome sequenced to investigate their molecular epidemiology, virulence, and resistome profiles. Eighty-six K. pneumoniae were recovered from patients in five hospitals in Khartoum between 2016 and 2020. Antimicrobial susceptibility was performed by disk-diffusion and broth microdilution. All isolates underwent whole genome sequencing using Illumina MiSeq; cgMLST was determined using Ridom SeqSphere+, and 7-loci MLST virulence genes and resistomes were identified. MDR was observed at 80%, with 35 isolates (41%) confirmed carbapenem-resistant. Thirty-seven sequence types were identified, and 14 transmission clusters (TC). Five of these TCs involved more than one hospital. Ybt9 was the most common virulence gene detected, in addition to some isolates harbouring iuc and rmp1. There is a diverse population of K. pneumoniae in Khartoum hospitals, harbouring multiple resistance genes, including genes coding for ESBLs, carbapenemases, and aminoglycoside-modifying enzymes, across multiple ST's. The majority of isolates were singletons and transmissions were rare.

First identification of multidrug-resistant Acinetobacter bereziniae isolates harboring bla NDM-1 from hospitals in South China.

This study is a first report on the identification of multidrug-resistant (MDR) Acinetobacter bereziniae among non-baumannii acinetobacters that had previously escaped automated laboratory detection, and characterize their clinical courses of infection at two tertiary-care hospitals in Shenzhen city, China (2015-2017). Herein, definitive identification by PCR was performed with universal and species-specific primers targeting 16S rDNA and rpoB genes, respectively, followed by Sanger sequencing and blast analysis. Antimicrobial susceptibility of A. bereziniae isolates was assessed accordingly. Three of the five identified A. bereziniae isolates exhibited carbapenem-resistance and were subjected to a multiplex PCR assay to detect drug-resistance genes. Sequences of the rpoB amplicon were aligned with curated sequences from global databases for phylogenetic analysis on evolutionary relations. Five clinical isolates of A. bereziniae were thereby re-identified, whose infections were primarily nosocomial. Automated identification and susceptibility testing systems (Phoenix-100 and VITEK 2) proved insufficient for discriminating A. bereziniae from other acinetobacters such as Acinetobacter baumannii and Acinetobacter guillouiae. Among these isolates, three exhibited carbapenem-resistant phenotypes indistinguishable from that of carbapenem-resistant A. baumannii. The carbapenem-resistant A. bereziniae isolates were subsequently confirmed to carry a bla NDM-1 (New Delhi metallo-ß-lactamase-1) gene downstream of ISAba125. Phylogenetic analysis revealed that A. bereziniae isolates evolved slowly but independently in local habitats. A. bereziniae isolates are difficult to distinguish by traditional automated detection systems. PCR-based identification via amplification and sequencing of selected house-keeping genes provides sufficient resolution for discriminating the isolates.

Darobactins Exhibiting Superior Antibiotic Activity by Cryo-EM Structure Guided Biosynthetic Engineering.

Over recent decades, the pipeline of antibiotics acting against Gram-negative bacteria is running dry, as most discovered candidate antibiotics suffer from insufficient potency, pharmacokinetic properties, or toxicity. The darobactins, a promising new small peptide class of drug candidates, bind to novel antibiotic target BamA, an outer membrane protein. Previously, we reported that biosynthetic engineering in a heterologous host generated novel darobactins with enhanced antibacterial activity. Here we utilize an optimized purification method and present cryo-EM structures of the Bam complex with darobactin 9 (D9), which served as a blueprint for the biotechnological generation of twenty new darobactins including halogenated analogs. The newly engineered darobactin 22 binds more tightly to BamA and outperforms the favorable activity profile of D9 against clinically relevant pathogens such as carbapenem-resistant Acinetobacter baumannii up to 32-fold, without observing toxic effects.