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The European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommends two steps for detecting beta-lactamases in Gram-negative bacteria. Screening for potential extended-spectrum beta-lactamase (ESBL), plasmid-mediated AmpC beta-lactamase, or carbapenemase production is confirmed. We aimed to validate generative pre-trained transformer (GPT)-4 and GPT-agent for pre-classification of disk diffusion to indicate potential beta-lactamases. We assigned 225 Gram-negative isolates based on phenotypic resistances against beta-lactam antibiotics and additional tests to one or more resistance mechanisms as follows: "none," "ESBL," "AmpC," or "carbapenemase." Next, we customized a GPT-agent with EUCAST guidelines and breakpoint table (v13.1). We compared routine diagnostics (reference) to those of (i) EUCAST-GPT-expert, (ii) microbiologists, and (iii) non-customized GPT-4. We determined sensitivities and specificities to flag suspect resistances. Three microbiologists showed concordance in 814/862 (94.4%) phenotypic categories and were used in median eight words (interquartile range [IQR] 4-11) for reasoning. Median sensitivity/specificity for ESBL, AmpC, and carbapenemase were 98%/99.1%, 96.8%/97.1%, and 95.5%/98.5%, respectively. Three prompts of EUCAST-GPT-expert showed concordance in 706/862 (81.9%) categories but were used in median 158 words (IQR 140-174) for reasoning. Sensitivity/specificity for ESBL, AmpC, and carbapenemase prediction were 95.4%/69.23%, 96.9%/86.3%, and 100%/98.8%, respectively. Non-customized GPT-4 could interpret 169/862 (19.6%) categories, and 137/169 (81.1%) agreed with routine diagnostics. Non-customized GPT-4 was used in median 85 words (IQR 72-105) for reasoning. Microbiologists showed higher concordance and shorter argumentations compared to GPT-agents. Humans showed higher specificities compared to GPT-agents. GPT-agent's unspecific flagging of ESBL and AmpC potentially results in additional testing, diagnostic delays, and higher costs. GPT-4 is not approved by regulatory bodies, but validation of large language models is needed., Importance: The study titled "GPT-4-based AI agents-the new expert system for detection of antimicrobial resistance mechanisms?" is critically important as it explores the integration of advanced artificial intelligence (AI) technologies, like generative pre-trained transformer (GPT)-4, into the field of laboratory medicine, specifically in the diagnostics of antimicrobial resistance (AMR). With the growing challenge of AMR, there is a pressing need for innovative solutions that can enhance diagnostic accuracy and efficiency. This research assesses the capability of AI to support the existing two-step confirmatory process recommended by the European Committee on Antimicrobial Susceptibility Testing for detecting beta-lactamases in Gram-negative bacteria. By potentially speeding up and improving the precision of initial screenings, AI could reduce the time to appropriate treatment interventions. Furthermore, this study is vital for validating the reliability and safety of AI tools in clinical settings, ensuring they meet stringent regulatory standards before they can be broadly implemented. This could herald a significant shift in how laboratory diagnostics are performed, ultimately leading to better patient outcomes.

Objectives: Carbapenem- and colistin-resistant Klebsiella pneumoniae were isolated from war victims treated in hospitals in Ukraine. The question was whether these pandrug-resistant K. pneumoniae are pathogenic and capable of causing disease in a broader context., Methods: Klebsiella pneumoniae clinical isolates (n = 37) were tested for antibiotic resistance and subjected to whole-genome sequencing (WGS). In addition, their pathogenicity was tested by serum resistance and two separate animal models., Results: Isolates belonging to the sequence types (ST) 23, 147, 307, 395, and 512 were found to harbor resistance genes against carbapenems and cephalosporins. Nine isolates carried point mutations in pmrB and phoP genes associated with colistin resistance. All bacteria were equipped with multiple virulence genes, and the colistin-resistant isolates each carried 10 different genes. Colistin-resistant K. pneumoniae were more serum-resistant, more virulent against G. mellonella larvae, and displayed an increased survival in mice compared to colistin-susceptible bacteria. The iucA, peg-344, rmpA, rmpC, and rmpD genes were associated with increased virulence in animals., Conclusions: Pandrug-resistant K. pneumoniae in Ukraine are hypervirulent and retain their pathogenicity, highlighting the need to prevent disseminated spread., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

This study was conducted to investigate decreased susceptibility (minimum inhibitory concentrations [MICs] 0.25-4 mg/L) and resistance (MICs > 4 mg/L) to aztreonam-avibactam (ATM-AVI). Contemporary non-replicate clinical isolates of carbapenemase-producing Escherichia coli (CP-EC) (n=90) and ESBL-producing E. coli (EP-EC) (n=12) were used. CP-EC belonged to 25 distinct sequence types (STs) and all EP-EC belonged to ST405. All strains were isolated from 2019 to 2022 at the Karolinska University Laboratory, Stockholm, Sweden. ATM-AVI MICs were determined using broth microdilution. The EUCAST epidemiological cut-off value of 0.125 mg/L was used to define the wild type (WT). Whole-genome sequences (Illumina) were analysed for detecting resistance determinants among WT vs. non-WT isolates. Among 102 isolates, 40 (39%) and 62 (61%) were WT and non-WT, respectively. Among non-WT isolates, resistance was noted for 20 and decreased susceptibility for 42. Resistance was observed among 14/47 New Delhi metallo-β-lactamase (NDM)-producers, 5/43 OXA-48 group producers, and 1/12 EP-EC. Decreased susceptibility was observed among 29/47 NDM, 13/43 OXA-48 group, and 3/12 EP-EC. Resistant isolates predominantly belonged to ST405, followed by STs 410, 361, 167, 617, and 1284. Penicillin-binding protein 3 (PBP3) inserts (YRIK/YRIN) were observed in 20/20 and CMY-42 in 5/20 resistant isolates. Several mutations in the ftsI (encoding PBP3) and regulatory genes of outer membrane proteins (OmpC and OmpF) and efflux pumps (AcrAB-TolC) were detected. A ≥ 2-fold reduction in MICs was observed among 20/20 vs. 7/20 isolates tested in the presence of the membrane permeabiliser, polymyxin B nanopeptide (PMBN) and efflux inhibitor, phenylalanine arginine β-naphthylamide (PAβN), respectively. In conclusion, resistance to ATM-AVI is a result of interplay of various determinants, including target alterations, deactivating enzymes, and decreased permeability., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Amoxicillin-clavulanate (AMC) is among the most frequently prescribed antibiotics globally. It has broad antibacterial activity against gram-positive, gram-negative, and anaerobic bacteria and has been used to treat infections caused by a broad range of pathogens. AMC breakpoints against Enterobacterales were initially set in the 1980s. However, since that time, increases in antibiotic resistance, advances in pharmacokinetic/pharmacodynamic analyses, and publication of additional clinical data prompted a reassessment by the Clinical and Laboratory Standards Institute (CLSI) Subcommittee on Antimicrobial Susceptibility Testing. Based on this contemporary reappraisal, the CLSI retained the Enterobacterales breakpoints but revised comments regarding dosing associated with use of the AMC breakpoints in the 2022 supplement of M100. This viewpoint provides insight into the CLSI breakpoint reevaluation process and summarizes the data and rationale used to support these revisions to the AMC Enterobacterales breakpoint., Competing Interests: Potential conflicts of interest. N. N. reports support from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (under award K23AI159396); honoraria from Astellas Pharma and Beckman Coulter; and research contracts with Merck and Shionogi Inc. E. W. reports honoraria from AbbVie Inc, Astellas Pharma, Ferring Pharmaceuticals, Melinta Therapeutics, Shionogi Inc, and Venatorx Pharmaceuticals. C. G. G. is immediate past-chair of the European Committee on Antimicrobial Susceptibility Testing. A. J. M. serves as a scientific advisor for Cepheid, DayZero Diagnostics and OpGen; and previously served as a scientific advisor for Merck, Qpex Biopharma, Venatorx Pharmaceuticals, and Melinta Therapeutics. N. M. M. reports research contracts from Cepheid, Inc and Abbott Molecular paid to his institution outside the submitted work; honoraria from the American Society for Clinical Laboratory Science; and serving as a member of the American Society for Clinical Laboratory Science Board of Directors and as a member of the Microbiology Resource Committee for the College of American Pathologists. All authors serve as unpaid volunteers for the Clinical and Laboratory Standards Institute. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Purpose: To assess Gram-positive bacterial (GPB) bloodstream infection (BSI) in neonates, covering incidence, morbidity, mortality, antimicrobial resistance patterns and biomarkers in Region Stockholm, Sweden between 2006 and 2016., Methods: A population-based retrospective epidemiological study including infants with GPB-BSI, admitted to the neonatal units at Karolinska University Hospital (KUH). Data were collected from patient records, the Swedish Neonatal Quality Register, the microbiological laboratory at KUH and the Swedish Public Health Agency., Results: We identified 357 infants with GPB-BSI, representing an incidence of 1.47/1000 live births (LB). Group B streptococcus (GBS) was the most common pathogen causing BSI in full-term infants and early-onset sepsis (EOS) (0.20/1000 LB), while coagulase-negative staphylococci (CoNS) were predominant in infants born very preterm and in late-onset sepsis (LOS) (0.79/1000 LB). There were no fatal GBS BSI cases, but 10.2% developed meningitis. The GPB case fatality rate was 9.5% and the sepsis fatality rate 2.8%. In GPB-BSI, 1/10 did not have an elevated C-reactive protein level. Staphylococcus aureus (S. aureus) BSI increased during the study period, but no methicillin or vancomycin resistant strains were found. The antimicrobial resistance (AMR) rate was highest in CoNS isolates., Conclusion: GPB-BSI was four times more common than Gram-negative BSI in neonates but resulted in lower mortality rate. GBS was the most common pathogen in full-term infants and in EOS. CoNS was the most common pathogen in LOS and infants born very preterm, and the AMR rate was high in these isolates. The increasing trend of S. aureus BSI indicates a need of further investigation., (© 2024. The Author(s).)

Scope: Pseudomonas aeruginosa, a ubiquitous opportunistic pathogen considered one of the paradigms of antimicrobial resistance, is among the main causes of hospital-acquired and chronic infections associated with significant morbidity and mortality. This growing threat results from the extraordinary capacity of P. aeruginosa to develop antimicrobial resistance through chromosomal mutations, the increasing prevalence of transferable resistance determinants (such as the carbapenemases and the extended-spectrum β-lactamases), and the global expansion of epidemic lineages. The general objective of this initiative is to provide a comprehensive update of P. aeruginosa resistance mechanisms, especially for the extensively drug-resistant (XDR)/difficult-to-treat resistance (DTR) international high-risk epidemic lineages, and how the recently approved β-lactams and β-lactam/β-lactamase inhibitor combinations may affect resistance mechanisms and the definition of susceptibility profiles., Methods: To address this challenge, the European Study Group for Antimicrobial Resistance Surveillance (ESGARS) from the European Society of Clinical Microbiology and Infectious Diseases launched the 'Improving Surveillance of Antibiotic-Resistant Pseudomonas aeruginosa in Europe (ISARPAE)' initiative in 2022, supported by the Joint programming initiative on antimicrobial resistance network call and included a panel of over 40 researchers from 18 European Countries. Thus, a ESGARS-ISARPAE position paper was designed and the final version agreed after four rounds of revision and discussion by all panel members., Questions Addressed in the Position Paper: To provide an update on (a) the emerging resistance mechanisms to classical and novel anti-pseudomonal agents, with a particular focus on β-lactams, (b) the susceptibility profiles associated with the most relevant β-lactam resistance mechanisms, (c) the impact of the novel agents and resistance mechanisms on the definitions of resistance profiles, and (d) the globally expanding XDR/DTR high-risk lineages and their association with transferable resistance mechanisms., Implication: The evidence presented herein can be used for coordinated epidemiological surveillance and decision making at the European and global level., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Antimicrobials cause perturbations in the composition and diversity of the host microbiome. We aimed to compare gut microbiome perturbations caused by oral tebipenem pivoxil hydrobromide (a novel carbapenem) and by amoxicillin-clavulanic acid (an orally administered β-lactam-β-lactam inhibitor combination widely used in clinical practice)., Methods: We did a phase 1, single-centre, randomised, parallel-group, active-control trial to evaluate the effect of tebipenem pivoxil hydrobromide on the human gut microbiota. Healthy participants aged 18 years or older with no documented illnesses during recruitment were enrolled at Karolinska University Hospital (Stockholm, Sweden). Study participants were stratified by sex and block-randomised in a 1:1 ratio to treatment with either tebipenem pivoxil hydrobromide (600 mg orally every 8 h) or amoxicillin-clavulanic acid (500 mg amoxicillin and 125 mg clavulanic acid orally every 8 h). The study included 10 days of treatment (days 1-10) and four follow-up visits (days 14, 21, 90, and 180). The trial was open-label for clinical investigators and patients, but masked for microbiology investigators. Faecal samples were collected at all visits. Sequencing of 16S rDNA was used to measure the diversity metrics, and quantitative culture to quantify selected taxa. The primary outcomes were changes in the α and β diversity and log count of colony-forming units for selected taxa between samples compared with baseline (day 1), and whether any changes reverted during the follow-up period. The analyses were done in the intention-to-treat population. This study was registered with ClinicalTrials.gov (NCT04376554)., Findings: The study was conducted between Jan 23, 2020, and April 6, 2021. 49 volunteers were screened for eligibility, among whom 30 evaluable participants (14 men and 16 women) were assigned: 15 (50%) to the tebipenem pivoxil hydrobromide group and 15 (50%) to the amoxicillin-clavulanic acid group. Baseline characteristics were similar between groups. Complete follow-up was available for all participants, and all participants except one completed treatment as assigned. The diversity metrics showed significant changes from baseline during the treatment period. Significant decreases in richness were observed on days 4-10 (p≤0·0011) in the amoxicillin-clavulanic acid group and on days 4-14 (p≤0·0019) in the tebipenem pivoxil hydrobromide group. Similarly, evenness was significantly decreased during treatment in the amoxicillin-clavulanic acid group (day 4, p=0·030) and the tebipenem pivoxil hydrobromide group (days 4-10, p<0·0001) compared with baseline. Quantitative cultures showed significant decreases in Enterobacterales (days 4-7, p≤0·0030), Enterococcus spp (days 4-14, p=0·025 to p<0·0001), Bifidobacterium spp (days 2-4, p≤0·026), and Bacteroides spp (days 4-10, p≤0·030) in the tebipenem pivoxil hydrobromide group. Similarly, in amoxicillin-clavulanic acid recipients, significant changes were observed in Enterobacterales (days 4-10, p≤0·048), Bifidobacterium spp (days 2-4, p≤0·013), and Lactobacillus spp (days 2-4, p≤0·020). Samples from the follow-up period were not significantly different from those at baseline in β diversity analysis (PERMANOVA, p>0·99). By the end of the study, no significant change was observed compared with baseline in either group. There were no deaths or severe adverse events., Interpretation: The impact of tebipenem pivoxil hydrobromide on the gut microbiome was similar to that of amoxicillin-clavulanic acid. The safety of antibiotic use with regard to the microbiome should be given attention, as dysbiosis is associated with health and disease., Funding: Spero Therapeutics., Competing Interests: Declaration of interests MN, DM, PBE, and IAC were employees of Spero Therapeutics and LBG was a consultant at Spero Therapeutics during the study. No personal honoraria were paid out to any of the academic investigators. All other authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Antimicrobial stewardship (AMS) programmes are established across the world to treat infections efficiently, prioritize patient safety, and reduce the emergence of antimicrobial resistance. One of the core elements of AMS programmes is guidance to support and direct physicians in making efficient, safe and optimal decisions when prescribing antibiotics. To optimize and tailor AMS, we need a better understanding of prescribing physicians' experience with AMS guidance., Objectives: To explore the prescribing physicians' user experience, needs and targeted improvements of AMS guidance in hospital settings., Methods: Semi-structured interviews were conducted with 36 prescribing physicians/AMS guidance users from hospital settings in Canada, Germany, Israel, Latvia, Norway and Sweden as a part of the international PILGRIM trial. A socioecological model was applied as an overarching conceptual framework for the study., Results: Research participants were seeking more AMS guidance than is currently available to them. The most important aspects and targets for improvement of AMS guidance were: (i) quality of guidelines; (ii) availability of infectious diseases specialists; and (iii) suitability of AMS guidance to department context., Conclusions: Achieving prudent antibiotic use not only depends on individual and collective levels of commitment to follow AMS guidance but also on the quality, availability and suitability of the guidance itself. More substantial commitment from stakeholders is needed to allocate the required resources for delivering high-quality, available and relevant AMS guidance to make sure that the prescribers' AMS needs are met., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Objectives: Treatment options for urinary tract infections (UTIs) caused by ESBL-producing Enterobacterales are limited. Moreover, evidence to support therapeutic decisions is lacking. This study assessed current treatment strategies and patient and pathogen characteristics in relation to clinical and microbiological outcomes., Methods: Patients with UTI caused by ESBL-producing Enterobacterales were prospectively recruited by investigators at 15 infectious disease hospital departments. Data were collected on patient characteristics, treatments, clinical and microbiological cure 10-14 days after the end of treatment, and relapse within 3 months. Bacterial isolates were subjected to MIC determination and WGS., Results: In total, 235 patients (107 febrile UTI, 128 lower UTI) caused by Escherichia coli (n = 223) and Klebsiella spp. (n = 12) were included. Clinical and microbiological cure rates were 83% and 64% in febrile UTI, and 79% and 65% in lower UTI. Great variability in treatments was observed, especially in oral therapy for febrile UTI. No difference was seen in clinical outcomes with piperacillin/tazobactam (n = 28) compared with carbapenems (n = 41). Pivmecillinam was frequently used in lower UTI (n = 62), and was also associated with high clinical cure rates when used as initial therapy (10/10) or follow-up (7/8) for febrile UTI. Recurrent infection, diabetes mellitus and urogenital disease were associated (P < 0.05) with clinical failure and relapse. In E. coli, ST131 was significantly associated with relapse, and haemolysin with microbiological failure or relapse., Conclusions: Antibiotic treatments were highly variable. Patient and pathogen factors were identified as potential determinants of disease presentation and outcomes and may prove useful to guide individualized treatment and follow-up., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Extensively drug-resistant (XDR) Klebsiella pneumoniae inflict a notable burden on healthcare worldwide. Of specific concern are strains producing carbapenem-hydrolyzing enzymes, as the therapeutic options for these strains are still very limited. Specific sequence types of K. pneumoniae have been noted for their epidemic occurrence globally, but the mechanisms behind the success of specific clones remain unclear. Herein, we have characterized 20 high-risk clones (HiRCs) and 10 non-HiRCs of XDR K. pneumoniae , exploring factors connected to the epidemiological success of some clones. Isolates were subjected to core genome multilocus sequence typing analysis to determine the clonal relationships of the isolates and subsequently characterized with regard to features known to be linked to overall bacterial fitness and virulence. The genomes were analyzed in silico for capsule types, O antigens, virulence factors, antimicrobial resistance genes, prophages, and CRISPR-Cas loci. In vitro growth experiments were conducted to retrieve proxies for absolute and relative fitness for 11 HiRC and 9 non-HiRC isolates selected based on the clonal groups they belonged to, and infections in a Galleria mellonella insect model were used to evaluate the virulence of the isolates in vivo . This study did not find evidence that virulence factors, prophages, CRISPR-Cas loci, or fitness measured in vitro alone would contribute to the global epidemiological success of specific clones of carbapenemase-producing XDR K. pneumoniae . However, this study did find the HiRC group to be more virulent than the non-HiRC group when measured in vivo in a model with G. mellonella . This suggests that the virulence and epidemiological success of certain clones of K. pneumoniae cannot be explained by individual traits investigated in this study and thus warrant further experiments in the future.IMPORTANCEHerein, we explored potential explanations for the successfulness of some epidemic or high-risk clones of carbapenemase-producing Klebsiella pneumoniae . We found differences in mortality in a larva model but found no clear genomic differences in known virulence markers. Most of the research on virulence in K. pneumoniae has been focused on hypervirulent strains, but here, we try to understand differences within the group of highly resistant strains. The results from the larva virulence model could be used to design experiments in higher animals. Moreover, the data could provide further support to a differentiated infection control approach against extensively drug-resistant strains, based on their classification as high-risk clones., Competing Interests: The authors declare no conflict of interest.

The aim of this study was to investigate antimicrobial susceptibilities and genomic characteristics of mupirocin-resistant MRSA isolates in Stockholm, Sweden. In total, 44 non-duplicate mupirocin-resistant MRSA isolates detected in Stockholm during 2010-2022 were investigated. Antimicrobial susceptibility testing was performed using broth microdilution method and further tested for high-level mupirocin-resistance (MuH) and rifampicin by Etest®. All isolates were subjected to whole genome sequencing. 41 isolates presented MuH with MICs ≥1024 mg/L whilst three isolates displayed low-level mupirocin resistance (MuL). mupA-gene was detected in all MuH isolates. Point mutations in ileS gene leading to N213D and V588F were identified in the three MuL isolates. Mutation in rpoB (H481N) was detected in a rifampicin-resistant isolate. Among the isolates, 15 multi-locus sequence types (MLST) were identified, with the four most common sequence types (ST22, ST72, ST8, and ST125) accounting for 66% of the isolates. Mupirocin-resistant MRSA in Stockholm was uncommon, with a percentage of <0.5% among MRSA cases during 2010-2022. In the present study, most mupirocin-resistant isolates were MuH and mupA-positive, predominantly linked to ST22 or ST72 isolates. MuL-resistance was associated with a point mutation in the IleS protein. A multidrug-resistant ST1-MRSA-IV strain was resistant to both mupirocin and rifampicin., (© 2023 The Authors. APMIS published by John Wiley & Sons Ltd on behalf of Scandinavian Societies for Pathology, Medical Microbiology and Immunology.)

Background: Therapeutic drug monitoring (TDM) has been suggested to optimize antimicrobial target attainment, typically using 100%T>MIC, in β-lactam treatment in the ICU. The MIC parameter used in this equation is mostly the worst case scenario MIC (MICWCS)-the highest MIC the empirical treatment should cover. However, the impact of the MIC parameter used in pharmacokinetic/pharmacodynamic calculations has been poorly investigated., Objectives: To assess the influence of target attainment rates for two different MIC parameters using actual MICs of the causative pathogens as the primary reference., Methods: In a Swedish multicentre study of target attainment for 138 ICU patients treated with β-lactams, the causative pathogen was isolated and subjected to reference MIC testing. Whenever the strain belonged to the WT distribution, we assigned it to the category MICECOFF (epidemiological cut-off value). In the calculations we compared the MICECOFF and the MICWCS., Results: The proportion of patients with target attainment failure for all antibiotics using 100%T>MIC was 45% (95% CI, 37%-53%) for MICWCS and 23% (95% CI, 16%-31%) for MICECOFF. When the target 50%T>4×MIC was used, corresponding attainment failures were 57% (95% CI, 49%-66%) and 25% (95% CI, 17%-32%) for MICWCS and MICECOFF, respectively., Conclusions: MICWCS can overestimate target attainment failure. The use of MICWCS could be one reason for the difficulties in establishing a relationship between target failure and mortality in other studies. Based on findings herein, the MICECOFF, which is based on the MIC of the causative pathogen, should be considered a more suitable alternative. When no pathogen is detected, the MICECOFF of likely pathogens according to infection type should be used., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Automated testing of antimicrobial susceptibility is common in clinical microbiology laboratories but their ability to detect low-level resistance has been questioned. This Nordic multicentre study aimed to evaluate the performance of commercially available automated AST systems. A phenotypically well-characterised collection of gram-negative bacilli (Escherichia coli (n = 7), Klebsiella pneumoniae (n = 6) and Pseudomonas aeruginosa (n = 7)) with and without resistance mechanisms was examined by Danish (n = 1), Finnish (n = 6), Norwegian (n = 16) and Swedish (n = 5) laboratories. Minimum inhibitory concentrations (MICs) were determined for 12 antimicrobials with automated systems and compared with MICs obtained with gold standard broth microdilution. The automated systems used were VITEK 2 (n = 23), Phoenix (n = 4), MicroScan (n = 1), and ARIS (n = 1). Very major errors were identified for six antimicrobials; cefotaxime (6.9%), meropenem (0.4%), ciprofloxacin (0.7%), ertapenem (4.3%), amikacin (3.4%) and colistin (6.4%). Categorical agreement of MIC for the automated systems compared to broth microdilution ranged from 83% for imipenem to 100% for ampicillin and trimethoprim-sulfamethoxazole. The analysis revealed several important antimicrobials where resistance was underestimated, potentially with significant consequences in patient treatment. The results cast doubt on the use of automated AST in the management of patients with serious infections and suggests that more work is needed to define their limitations., (© 2023 The Authors. APMIS published by John Wiley & Sons Ltd on behalf of Scandinavian Societies for Pathology, Medical Microbiology and Immunology.)

Nucleoside analogues have been in clinical use since 1960s and they are still used as the first therapeutic option for several cancers and viral infections, due to their high therapeutic efficacy. However, their wide clinical acceptance has been limited due to their high toxicity and severe side effects to patients. Herein, we report on a nanocarrier system that delivers nucleosides analogues in a target-specific manner, making nucleoside-based therapeutics safer and with the possibility to be used in other human conditions. This system, named, Therapeutic OligonUCleotides Activated by Nucleases" (TOUCAN) combines: i) the recognition power of oligonucleotides as substrates, ii) the use of nucleases as enzymatic biomarkers and iii) the clinical efficacy of nucleoside analogues, in a single approach. As a proof-of-concept, we report on a TOUCAN that is activated by a specific nuclease produced by bacteria and releases a therapeutic nucleoside, floxuridine. We demonstrate, for the first time, that, by incorporating a therapeutic nucleoside analogue into oligonucleotide probes, we can specifically inhibit bacterial growth in cultures. In this study, Staphylococcus aureus was selected as the targeted bacteria and the TOUCAN strategy successfully inhibited its growth with minimal inhibitory concentration (MIC) values ranging from 0.62 to 40 mg/L across all tested strains. Moreover, our results indicate that the intravenous administration of TOUCANs at a dose of 20 mg/kg over a 24-h period is a highly effective method for treating bacterial infections in a mouse model of pyomyositis. Importantly, no signs of toxicity were observed in our in vitro and in vivo studies. This work can significantly impact the current management of bacterial infections, laying the grounds for the development of a different class of antibiotics. Furthermore, it can provide a safer delivery platform for clinical nucleoside therapeutics in any human conditions, such as cancer and viral infection, where specific nuclease activity has been reported., Competing Interests: Declaration of Competing Interest BAB, LIH, FJH are inventors in patent that describes the use of TOUCAN technology andfounders of Theralia AB, a spin-off company from LiU that develops TOUCANs for clinical applications. The other authors declare no competing financial interests., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: P. aeruginosa bacteremia is a common and severe infection carrying high mortality in older adults. We aimed to evaluate outcomes of P. aeruginosa bacteremia among old adults (≥ 80 years)., Methods: We included the 464/2394 (19%) older adults from a retrospective multinational (9 countries, 25 centers) cohort study of individuals hospitalized with P. aeruginosa bacteremia. Bivariate and multivariable logistic regression models were used to evaluate risk factors for 30-day mortality among older adults., Results: Among 464 adults aged ≥ 80 years, the mean age was 84.61 (SD 3.98) years, and 274 (59%) were men. Compared to younger patients, ≥ 80 years adults had lower Charlson score; were less likely to have nosocomial acquisition; and more likely to have urinary source. Thirty-day mortality was 30%, versus 27% among patients 65-79 years (n = 894) and 25% among patients < 65 years (n = 1036). Multivariate analysis for predictors of mortality among patients ≥ 80 years, demonstrated higher SOFA score (odds ratio [OR] 1.36, 95% confidence interval [CI] 1.23-1.51, p < 0.001), corticosteroid therapy (OR 3.15, 95% CI: 1.24-8.01, p = 0.016) and hospital acquired P. aeruginosa bacteremia (OR 2.30, 95% CI: 1.33-3.98, p = 0.003) as predictors. Appropriate empirical therapy within 24 h, type of definitive anti-pseudomonal drug, and type of regimen (monotherapy or combination) were not associated with 30-day mortality., Conclusions: In older adults with P. aeruginosa bacteremia, background conditions, place of acquisition, and disease severity are associated with mortality, rather than the antimicrobial regimen. In this regard, preventive efforts and early diagnosis before organ failure develops might be beneficial for improving outcomes., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany.)

Objective: For non-tuberculous mycobacteria (NTM), minimum inhibitory concentration (MIC) distributions of wild-type isolates have not been systematically evaluated despite their importance for establishing antimicrobial susceptibility testing (AST) breakpoints., Methods: We gathered MIC distributions for drugs used against the Mycobacterium avium complex (MAC) and Mycobacterium abscessus (MAB) obtained by commercial broth microdilution (SLOMYCOI and RAPMYCOI) from 12 laboratories. Epidemiological cut-off values (ECOFFs) and tentative ECOFFs (TECOFFs) were determined by EUCAST methodology including quality control (QC) strains., Results: The clarithromycin ECOFF was 16 mg/L for M. avium (n = 1271) whereas TECOFFs were 8 mg/L for M. intracellulare (n = 415) and 1 mg/L for MAB (n = 1014) confirmed by analysing MAB subspecies without inducible macrolide resistance (n = 235). For amikacin, the ECOFFs were 64 mg/L for MAC and MAB. For moxifloxacin, the WT spanned >8 mg/L for both MAC and MAB. For linezolid, the ECOFF and TECOFF were 64 mg/L for M. avium and M. intracellulare, respectively. Current CLSI breakpoints for amikacin (16 mg/L), moxifloxacin (1 mg/L) and linezolid (8 mg/L) divided the corresponding WT distributions. For QC M. avium and M. peregrinum, ≥95% of MIC values were well within recommended QC ranges., Conclusion: As a first step towards clinical breakpoints for NTM, (T)ECOFFs were defined for several antimicrobials against MAC and MAB. Broad wild-type MIC distributions indicate a need for further method refinement which is now under development within the EUCAST subcommittee for anti-mycobacterial drug susceptibility testing. In addition, we showed that several CLSI NTM breakpoints are not consistent in relation to the (T)ECOFFs., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objectives: Antimicrobial susceptibility testing (AST) of anaerobic bacteria has until recently been done by MIC methods. We have carried out a multi-centre evaluation of the newly validated EUCAST disk diffusion method for AST of Bacteroides spp., Methods: A panel of 30 Bacteroides strains was assembled based on reference agar dilution MICs, resistance gene detection and quantification of cfiA carbapenemase gene expression. Nordic clinical microbiology laboratories (n = 45) performed disk diffusion on Fastidious Anaerobe Agar with 5% mechanically defibrinated horse blood (FAA-HB) for piperacillin-tazobactam, meropenem and metronidazole., Results: A total of 43/45 (95.6%) laboratories carried out disk diffusion per protocol. Intraclass correlation coefficients were 0.87 (0.80-0.93) for piperacillin-tazobactam, 0.95 (0.91-0.97) for meropenem and 0.89 (0.83-0.94) for metronidazole. For metronidazole, one media lot yielded smaller zones and higher variability than another. Piperacillin-tazobactam and meropenem zone diameters correlated negatively with cfiA expression. A meropenem zone diameter of <28 mm in B. fragilis indicated presence of cfiA. Piperacillin-tazobactam had the most false susceptible results. Categorical errors for this antimicrobial were particularly prevalent in cfiA-positive strains, and piperacillin-tazobactam had the highest number of comments describing zone reading difficulties., Conclusions: Inter-laboratory agreement by disk diffusion was good or very good. The main challenges were media-related variability for metronidazole and categorical disagreement with the reference method for piperacillin-tazobactam in some cfiA-positive strains. An area of technical uncertainty specific for such strains may be warranted., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: The aim was to compare rapid antigen detection test (RADT) and throat culture for group A streptococci (GAS) among patients recently treated with penicillin V for GAS pharyngotonsillitis., Design and Setting: The study was a secondary analysis within a randomized controlled trial comparing 5 versus 10 days of penicillin V for GAS pharyngotonsillitis. Patients were recruited at 17 primary health care centres in Sweden., Subjects: We included 316 patients ≥ 6 years of age, having 3-4 Centor criteria, a positive RADT and a positive throat culture for GAS at inclusion, and also having a RADT and throat culture for GAS taken at a follow-up visit within 21 days., Main Outcome Measures: RADT and conventional throat culture for GAS., Results: This prospective study showed 91% agreement between RADT and culture at follow-up within 21 days. Only 3/316 participants had negative RADT with a positive throat culture for GAS at follow-up, and 27/316 patients with positive RADT had a negative culture for GAS. Log rank test did not reveal any difference in the decline over time of positive tests between RADT and throat culture ( p  = 0.24). Agreement between RADT and throat culture for GAS at the follow-up was not associated with treatment duration, number of days from inclusion until follow-up, throat symptoms at follow-up, gender, or age., Conclusion: RADT and culture for GAS agreed to a high extent also after recent penicillin V treatment. RADT for GAS means a low risk for missing the presence of GAS.KEY POINTSTesting for group A streptococci (GAS) before antibiotic treatment can reduce antibiotic prescription for pharyngotonsillitis. It has been proposed that rapid antigen detection tests (RADT) for group A streptococci after recent penicillin V treatment may be falsely positive due to possible persisting antigens from non-viable bacteria.The decline of the presence of GAS was similar between RADT and conventional throat culture in patients who had recently completed penicillin V treatment for GAS pharyngotonsillitisRADT for GAS is useful in identifying the presence of GAS after recent penicillin V treatment.

Background: Identification of pathogens is crucial to efficiently treat and prevent bacterial infections. However, existing diagnostic techniques are slow or have a too low resolution for well-informed clinical decisions., Methods: In this study, we have developed an optical DNA mapping-based method for strain-level bacterial typing and simultaneous plasmid characterisation. For the typing, different taxonomical resolutions were examined and cultivated pure Escherichia coli and Klebsiella pneumoniae samples were used for parameter optimization. Finally, the method was applied to mixed bacterial samples and uncultured urine samples from patients with urinary tract infections., Results: We demonstrate that optical DNA mapping of single DNA molecules can identify Escherichia coli and Klebsiella pneumoniae at the strain level directly from patient samples. At a taxonomic resolution corresponding to E. coli sequence type 131 and K. pneumoniae clonal complex 258 forming distinct groups, the average true positive prediction rates are 94% and 89%, respectively. The single-molecule aspect of the method enables us to identify multiple E. coli strains in polymicrobial samples. Furthermore, by targeting plasmid-borne antibiotic resistance genes with Cas9 restriction, we simultaneously identify the strain or subtype and characterize the corresponding plasmids., Conclusion: The optical DNA mapping method is accurate and directly applicable to polymicrobial and clinical samples without cultivation. Hence, it has the potential to rapidly provide comprehensive diagnostics information, thereby optimizing early antibiotic treatment and opening up for future precision medicine management., (© 2023. The Author(s).)

Background: The intestinal microbiota functions as a reservoir of antibiotic resistance., Objectives: To evaluate penicillin V (phenoxymethylpenicillin) effects on the faecal microbiota with focus on beta-lactam resistance., Methods: We included 31 primary care patients with group A streptococcal pharyngotonsillitis treated with penicillin V for 5 (800 mg × 4) or 10 days (1000 mg × 3). Twenty-nine patients contributed with three faecal swab samples each. The faecal specimens were collected at the start of penicillin V treatment, after the last dose and at follow-up 7-9 days after completed treatment. Samples were inoculated semiquantitatively on selective screening agar plates to study beta-lactam resistance, species shifts among Enterobacterales and enterococci, and colonization with Candida spp. and Clostridioides difficile . Representative colonies were identified using MALDI-TOF. Results were analysed by non-parametric statistical methods., Results: An increase in the proportion of patients colonized with ampicillin-resistant Enterobacterales, from 52% to 86% ( P  = 0.007), and Enterobacterales with decreased susceptibility to third-generation cephalosporins, from 32% to 52% ( P  = 0.034), was observed between the first and second samples. This increase was no longer significant at follow-up. New colonization with ampicillin-resistant Enterobacterales species and non-Enterobacterales Gram-negative species was observed, and persisted at follow-up., Conclusions: Following treatment with penicillin V, we observed decreased susceptibility to ampicillin and third-generation cephalosporins, and prolonged colonization with non- Escherichia coli Gram-negative species. These findings challenge the perception that penicillin V has limited ecological effect on the intestinal microbiota, and emphasizes the importance of avoiding even narrow-spectrum antimicrobials when possible., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Multiomics technologies improve the biological understanding of health status in people living with HIV on antiretroviral therapy (PWH). Still, a systematic and in-depth characterization of metabolic risk profile during successful long-term treatment is lacking. Here, we used multi-omics (plasma lipidomic, metabolomic, and fecal 16 S microbiome) data-driven stratification and characterization to identify the metabolic at-risk profile within PWH. Through network analysis and similarity network fusion (SNF), we identified three groups of PWH (SNF-1-3): healthy (HC)-like (SNF-1), mild at-risk (SNF-3), and severe at-risk (SNF-2). The PWH in the SNF-2 (45%) had a severe at-risk metabolic profile with increased visceral adipose tissue, BMI, higher incidence of metabolic syndrome (MetS), and increased di- and triglycerides despite having higher CD4 + T-cell counts than the other two clusters. However, the HC-like and the severe at-risk group had a similar metabolic profile differing from HIV-negative controls (HNC), with dysregulation of amino acid metabolism. At the microbiome profile, the HC-like group had a lower α-diversity, a lower proportion of men having sex with men (MSM) and was enriched in Bacteroides. In contrast, in at-risk groups, there was an increase in Prevotella , with a high proportion of MSM, which could potentially lead to higher systemic inflammation and increased cardiometabolic risk profile. The multi-omics integrative analysis also revealed a complex microbial interplay of the microbiome-associated metabolites in PWH. Those severely at-risk clusters may benefit from personalized medicine and lifestyle intervention to improve their dysregulated metabolic traits, aiming to achieve healthier aging., Competing Interests: FM, MG, RB, AK, BV, JH, JH, TB, DM, CG, AM, MT, SN, UN No competing interests declared, (© 2023, Mikaeloff et al.)

Objectives: Colistin is a last-resort antibiotic, but there has been a rapid increase in colistin resistance, threatening its use in the treatment of infections with carbapenem-resistant Enterobacterales (CRE). Plasmid-mediated colistin resistance, in particular the mcr-1 gene, has been identified and WGS is the go-to method in identifying plasmids carrying mcr-1 genes. The goal of this study is to demonstrate the use of optical DNA mapping (ODM), a fast, efficient and amplification-free technique, to characterize plasmids carrying mcr-1 ., Methods: ODM is a single-molecule technique, which we have demonstrated can be used for identifying plasmids harbouring antibiotic resistance genes. We here applied the technique to plasmids isolated from 12 clinical Enterobacterales isolates from patients at a major hospital in Thailand and verified our results using Nanopore long-read sequencing., Results: We successfully identified plasmids encoding the mcr-1 gene and, for the first time, demonstrated the ability of ODM to identify resistance gene sites in small (∼30 kb) plasmids. We further identified bla CTX-M genes in different plasmids than the ones encoding mcr-1 in three of the isolates studied. Finally, we propose a cut-and-stretch assay, based on similar principles, but performed using surface-functionalized cover slips for DNA immobilization and an inexpensive microscope with basic functionalities, to identify the mcr-1 gene in a plasmid sample., Conclusions: Both ODM and the cut-and-stretch assay developed could be very useful in identifying plasmids encoding antibiotic resistance in hospitals and healthcare facilities. The cut-and-stretch assay is particularly useful in low- and middle-income countries, where existing techniques are limited., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Objectives: Carbapenemase-producing Enterobacterales (CPE) are high priority targets of global antimicrobial surveillance. Herein, we determined the colonization rate of CPE on admission to intensive care units in Vientiane, Lao PDR in August-September 2019., Methods: Data regarding clinical conditions, infection control, and antibiotic usage were collected during admission. Rectal swab samples (n = 137) collected during admission were inoculated to selective chromogenic agars, followed by confirmatory tests for extended-spectrum beta-lactamases and carbapenemases. All CPE isolates were sequenced on Illumina (HiSeq2500), reads assembled using SPAdes 3.13, and the draft genomes used to query a database (https://www.genomicepidemiology.org) for resistome, plasmid replicons, and sequence types (ST). Optical DNA mapping (ODM) was used to characterize plasmids and to determine location of resistance genes. Minimum spanning tree was generated using the Bacterial Isolate Genome Sequence database (BIGSdb) and annotated using iTOL., Result: From 47 Enterobacterales isolated on selective agars, K. pneumoniae (25/47) and E. coli (12/47) were the most prevalent species, followed by K aerogenes (2/47), K. variicola (1/47), and K. oxytoca (1/47). The overall prevalence of ESBLs was 51.0%; E. coli 83.3% (10/12) and Klebsiella spp. 41.3% (12/29). Twenty percent of the K. pneumoniae (5/25) isolates were carbapenem-resistant, and 4/5 contained the blaNDM-1 gene. All blaNDM-1 isolates belonged to ST147 and were indistinguishable with cgMLST. ODM showed that the blaNDM-1 gene was located on identical plasmids in all isolates., Conclusion: The prevalence of ESBL-producing Enterobacterales was high, while carbapenemases were less common. However, the detection of clonal dissemination of blaNDM-1-producing K. pneumoniae isolates in one of the intensive care units calls for vigilance. Stringent infection prevention and antimicrobial stewardship strategies are highly important measures., Competing Interests: No Authors have competing interest.

Objectives: To evaluate the activity of meropenem-amikacin and meropenem-colistin combinations with checkerboard broth microdilution (CKBM) compared with isothermal microcalorimetry (ITMC) assays against a multi-centric collection of multi-drug-resistant Gram-negative clinical isolates; and to compare the fractional inhibitory concentration (FIC) index and time to results of CKBM and ITMC., Methods: A collection of 333 multi-drug-resistant Gram-negative clinical isolates showing reduced susceptibility to meropenem (121 Klebsiella pneumoniae, 14 Escherichia coli, 130 Pseudomonas aeruginosa and 68 Acinetobacter baumannii) isolated from different centres (Florence, Madrid, Rotterdam and Stockholm) was included in the study. The antimicrobial activity of meropenem-amikacin and meropenem-colistin combinations was evaluated with CKBM and ITMC. FIC index results were interpreted as synergistic/additive and indifferent for values ≤0.5/0.51, respectively. Whole-genome sequencing data of a subset of strains were used to evaluate their clonality., Results: In total, 254 and 286 strains were tested with meropenem-colistin and meropenem-amikacin combinations with ITMC and CKBM, respectively. Synergistic/additive effects were observed for 46 strains (20 K. pneumoniae, four E. coli, 22 P. aeruginosa) and 20 strains (three K. pneumoniae, 11 P. aeruginosa and six A. baumannii) with meropenem-amikacin and meropenem-colistin combinations, respectively, with CKBM. ITMC showed good concordance with CKBM, with 89.5% and 92.2% of cases interpreted within the same FIC index category for meropenem-amikacin and meropenem-colistin combinations, respectively. Most of the synergistic/additive effects were detected within 6 h by ITMC., Conclusions: ITMC showed very good concordance with CKBM against a large collection of multi-drug-resistant Gram-negative clinical isolates, and could be implemented for the rapid evaluation of in-vitro activity of antimicrobial combinations., (Copyright © 2022 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)

Background: Infantile central nervous system infections (CNSIs) can be life-threatening and cause severe sequelae. However, the causative microorganism remains unknown in >40% of patients with aseptic infections. This study aimed to analyze the metagenome for detection of pathogens and the transcriptome for host immune responses during infection in a single cerebrospinal fluid (CSF) sample using 2 different next-generation sequencing (NGS) platforms, Nanopore and Illumina., Methods: Twenty-eight CNSIs patients (<12 months) were enrolled, and 49 clinical samples (28 CSF and 21 blood) were collected. The DNA extracted from all 49 samples was sequenced using the Illumina sequencer for the detection of pathogens. Extracted RNA was obtained in sufficient quantities from 23 CSF samples and subjected to sequencing on both Nanopore and Illumina platforms. Human-derived reads subtracted during pathogen detection were used for host transcriptomic analysis from both Nanopore and Illumina sequencing., Results: RNA metagenomic sequencing using both sequencing platforms revealed putative viral pathogens in 10 cases. DNA sequencing using the Illumina sequencer detected 2 pathogens. The results of Nanopore and Illumina RNA sequencing were consistent; however, the mapping coverage and depth to the detected pathogen genome of Nanopore RNA sequencing were greater than those of Illumina. Host transcriptomic analysis of Nanopore sequencing revealed highly expressed genes related to the antiviral roles of innate immunity from pathogen-identified cases., Conclusions: The use of Nanopore RNA sequencing for metagenomic diagnostics of CSF samples should help to elucidate both pathogens and host immune responses of CNSI and could shed light on the pathogenesis of these infections., Competing Interests: Potential conflicts of interest. All authors: no reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Introduction: Pseudomonas aeruginosa ( P. aeruginosa ) and Acinetobacter baumannii ( A. baumannii ) can cause difficult-to-treat infections. We characterized molecular epidemiology of ceftazidime-resistant P. aeruginosa and carbapenem-resistant A. baumannii at a tertiary hospital in Ethiopia., Materials and Methods: Non-fermenting gram-negative bacilli ( n = 80) isolated from admitted patients were subjected for species identification by MALDI-TOF. Pseudomonas species resistant to ceftazidime or meropenem, and Acinetobacter species resistant to meropenem, or imipenem were selected for whole genome sequencing. DNA extracted with EZ1 Advanced XL instrument (Qiagen, Hilden, Germany) was sequenced on Illumina (HiSeq2500) using libraries prepared by NEXTRA-kits (Illumina). Raw reads were assembled using SPAdes 3.13.0, and assembled genomes were used to query databases for resistome profile and sequence types., Result: Among Pseudomonas species isolated, 31.7% (13/41), and 7.3% (3/41) were non-susceptible to ceftazidime, and meropenem, respectively. Carbapenem-resistance was 56.4% (22/39) among Acinetobacter species. Moreover, 92% (12/13) of Pseudomonas species non-susceptible to ceftazidime and/or meropenem, and 89.4% (17/19) of Acinetobacter species encoded multiple resistance genes for at least three classes of antimicrobials. The prevalent β - lactamase genes were bla OXA-486 (53.8%, 7/13), bla CTX-M-15 (23.0%, 3/13) among Pseudomonas , and bla GES-11 (57.8%, 11/19) among Acinetobacter . The bla OXA-51 -like β - lactamase, bla OXA-69 (63.1%, 12/19) was the most prevalent carbapenemase gene among Acinetobacter isolates. Single isolates from both P. aeruginosa , and A. baumannii were detected with the bla NDM-1 . Sequence type (ST)1 A. baumannii and ST274 P. aeruginosa were the prevalent sequence types. A cgMLST analysis of the ST1 A. baumannii isolates showed that they were closely related and belonged to the international clonal complex one (ICC1). Similarly, ST274 P. aeruginosa isolates were clonally related., Conclusion: The prevalence of MDR isolates of Pseudomonas and Acinetobacter spp. was high. A. baumannii isolates were clonally spreading in the admission wards at the hospital. Emergence of bla NDM-1 in the intensive care, and surgical wards of the hospital is a severe threat that requires urgent intervention., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sewunet, Asrat, Woldeamanuel, Aseffa and Giske.)

Introduction: There is no consensus regarding optimal duration of antibiotic therapy for Pseudomonas aeruginosa bacteremia. We aimed to evaluate the impact of short antibiotic course., Methods: We present a retrospective multicenter study including patients with P. aeruginosa bacteremia during 2009-2015. We evaluated outcomes of patients treated with short (6-10 days) versus long (11-15 days) antibiotic courses. The primary outcome was a composite of 30-day mortality or bacteremia recurrence and/or persistence. Univariate and inverse probability treatment-weighted (IPTW) adjusted multivariate analysis for the primary outcome was performed. To avoid immortal time bias, the landmark method was used., Results: We included 657 patients; 273 received a short antibiotic course and 384 a long course. There was no significant difference in baseline characteristics of patients. The composite primary outcome occurred in 61/384 patients in the long-treatment group (16%) versus 32/273 in the short-treatment group (12%) (p = 0.131). Mortality accounted for 41/384 (11%) versus 25/273 (9%) of cases, respectively. Length of hospital stay was significantly shorter in the short group [median 13 days, interquartile range (IQR) 9-21 days, versus median 15 days, IQR 11-26 days, p = 0.002]. Ten patients in the long group discontinued antibiotic therapy owing to adverse events, compared with none in the short group. On univariate and multivariate analyses, duration of therapy was not associated with the primary outcome., Conclusions: In this retrospective study, 6-10 days of antibiotic course for P. aeruginosa bacteremia were as effective as longer courses in terms of survival and recurrence. Shorter therapy was associated with reduced length of stay and less drug discontinuation., (© 2022. The Author(s).)

Brucellosis, mainly caused by Brucella (B.) melitensis , is associated with a risk of chronification and relapses. Antimicrobial susceptibility testing (AST) standards for B. melitensis are not available, and the agent is not yet listed in the EUCAST breakpoint tables. CLSI recommendations for B. melitensis exist, but they do not fulfill the requirements of the ISO 20776 standard regarding the culture medium and the incubation conditions. Under the third EU Health Programme, laboratories specializing in the diagnostics of highly pathogenic bacteria in their respective countries formed a working group within a Joint Action aiming to develop a suitable method for the AST of B. melitensis . Under the supervision of EUCAST representatives, this working group adapted the CLSI M45 document to the ISO 20776 standard after testing and validation. These adaptations included the comparison of various culture media, culture conditions and AST methods. A Standard Operation Procedure was derived and an interlaboratory validation was performed in order to evaluate the method. The results showed pros and cons for both of the two methods but also indicate that it is not necessary to abandon Mueller-Hinton without additives for the AST of B. melitensis .

We investigated the concordance between the Unyvero Hospitalized Pneumonia (HPN) application and quantitative culture for detection of bacterial pathogens from serial lower respiratory tract (LRT) specimens collected from the same subject. Comparison of results from HPN application and culture was evaluated using 69 LRT samples from 27 subjects, using two evaluation approaches. False positive detections by the HPN application was 29% (20/69) in Evaluation I vs 10% (7/68) in Evaluation II. Additional pathogens detected by the HPN application could be confirmed in many instances by culture positivity for the same organism from previous or subsequent samples from the same subject., (© 2022. The Author(s).)

Antimicrobial resistance (AMR) is a fast-growing threat to global health. The genes conferring AMR to bacteria are often located on plasmids, circular extrachromosomal DNA molecules that can be transferred between bacterial strains and species. Therefore, effective methods to characterize bacterial plasmids and detect the presence of resistance genes can assist in managing AMR, for example, during outbreaks in hospitals. However, existing methods for plasmid analysis either provide limited information or are expensive and challenging to implement in low-resource settings. Herein, we present a simple assay based on CRISPR/Cas9 excision and DNA combing to detect antimicrobial resistance genes on bacterial plasmids. Cas9 recognizes the gene of interest and makes a double-stranded DNA cut, causing the circular plasmid to linearize. The change in plasmid configuration from circular to linear, and hence the presence of the AMR gene, is detected by stretching the plasmids on a glass surface and visualizing by fluorescence microscopy. This single-molecule imaging based assay is inexpensive, fast, and in addition to detecting the presence of AMR genes, it provides detailed information on the number and size of plasmids in the sample. We demonstrate the detection of several β-lactamase-encoding genes on plasmids isolated from clinical samples. Furthermore, we demonstrate that the assay can be performed using standard microbiology and clinical laboratory equipment, making it suitable for low-resource settings., (© 2022. The Author(s).)

Objectives: The burden of antimicrobial resistance and spread of epidemic clones are rarely reported from low-income countries. We aimed to investigate the genome-based epidemiology of extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) at a tertiary hospital in Jimma, Ethiopia., Methods: Bacteria were isolated from clinical specimens at Jimma Medical Center and subjected to species identification (MALDI-TOF), antimicrobial susceptibility testing (disk diffusion) and whole-genome sequencing (Illumina, HiSeq2500). Genomic data analysis was performed using EnteroBase and Center for Genomic Epidemiology bioinformatics pipelines. A maximum likelihood tree was generated using FastTree/2.1.8 based on single nucleotide polymorphisms (SNPs) in shared genomic regions to identify transmission clusters., Results: Escherichia coli isolates (n = 261) were collected from 1087 single non-duplicate clinical specimens over a 5-month period in 2016. The prevalence of ESBL-EC was 54.8% (143/261), 96% of which were resistant to multiple antibiotic classes. The bla CTX-M-15 ESBL gene was present in 88.4.% of isolates (122/138). Genes conferring resistance to aminoglycosides and ciprofloxacin [aac(6')-Ib-cr, 62.3% (86/138)], phenicols [catB3, 56.5% (78/138)], sulfonamides [sul1, 68.1% (94/138), trimethoprim [dfrA17, 58.0% (80/138)] and macrolides [mph(A), 67.4% (93/138) were detected. The most prevalent sequence types were ST410 (23%), ST648 (17%), ST131 (10%) and ST167 (7%). Isolates of the same sequence type collected from different units of the hospital were highly similar in the SNP analysis., Conclusion: A high prevalence of ESBLs and dissemination of bla CTX-M-15 through multiple high-risk E. coli clones was detected. Nosocomial spread of multidrug-resistant ESBL-EC within the hospital puts vulnerable patients at risk of difficult-to-treat infections., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Carbapenem-resistant Enterobacteriaceae (CRE) have been thoroughly studied as the pathogens associated with hospital acquired infections. However, data on Serratia marcescens are not enough. S. marcescens is now becoming a propensity for its highly antimicrobial-resistant clinical infections., Methods: Four carbapenem-resistant S. marcescens (CR-SM) isolates were obtained from hospitalized patients through routine microbiological experiments. We assembled the isolates genomes using whole genome sequencing (WGS) and compared their resistome and virulome patterns., Results: The average length and CG content of chromosomes was 5.33 Mbp and 59.8%, respectively. The number of coding sequences (CDSs) ranged from 4,959 to 4,989. All strains had one single putative conjugative plasmid with IncL incompatibility (Inc) group. The strains harbored bla CTX-M-15 , bla TEM-1 and bla SHV-134 . All plamsids were positive for bla OXA-48 . No bla NDM-1 , bla KPC , bla VIM and bla IMP were identified. The bla SRT-2 and aac(6')-Ic genes were chromosomally-encoded. Class 1 integron was detected in strains P8, P11 and P14. The Escher&#95;RCS47 and Salmon&#95;SJ46 prophages played major role in plasmid-mediated carraige of extended spectrum β-lactamases (ESBLs). The CR-SM strains were equipt with typical virulence factors of oppotunistic pathogens including biofilm formation, adhesins, secretory systems and siderophores. The strains did not have ability to produce prodigiosin but were positive for chitinase and EstA., Conclusion: The presence of conjugative plasmids harboring major β-lactamases within prophage and class 1 integron structures highlights the role of different mobile genetic elements (MGEs) in distribution of AMR factors and more specifically carbapenemases. More molecular studies are required to determine the status of carbapenem resistance in clinical starins. However, appropriate strategies to control the global dissemination of CR-SM are urgent., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Objectives: Accurate and rapid microbiological diagnostics are crucial to tailor treatment and improve outcomes in patients with severe infections. This study aimed to assess blood culture diagnostics in the Nordic countries and to compare them with those of a previous survey conducted in Sweden in 2013., Methods: An online questionnaire was designed and distributed to the Nordic clinical microbiology laboratories (CMLs) (n = 76) in January 2018., Results: The response rate was 64% (49/76). Around-the-clock incubation of blood cultures (BCs) was supported in 82% of the CMLs (40/49), although in six of these access to the incubators around the clock was not given to all of the cabinets in the catchment area, and 41% of the sites (20/49) did not assist with satellite incubators. Almost half (49%, 24/49) of the CMLs offered opening hours for ≥10 h during weekdays, more commonly in CMLs with an annual output ≥30 000 BCs. Still, positive BCs were left unprocessed for 60-70% of the day due to restrictive opening hours. Treatment advice was given by 23% of CMLs (11/48) in ≥75% of the phone contacts. Rapid analyses (species identification and susceptibility testing with short incubation), performed on aliquots from positive cultures, were implemented in 18% of CMLs (9/49). Compared to 2013, species identification from subcultured colonies (<6 h) had become more common., Conclusions: CMLs have taken action to improve aspects of BC diagnostics, implementing satellite incubators, rapid species identification and susceptibility testing. However, the limited opening hours and availability of clinical microbiologists are confining the advantages of these changes., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objectives: Ventilator-associated lower respiratory tract infections (VA-LRTIs) are associated with prolonged length of stay and increased mortality. We aimed to investigate the occurrence of bacterial VA-LRTI among mechanically ventilated COVID-19 patients and compare these findings to non-COVID-19 cohorts throughout the first and second wave of the pandemic., Design: Retrospective cohort study., Setting: Karolinska University Hospital, Stockholm, Sweden., Patients: All patients greater than or equal to 18 years treated with mechanical ventilation between January 1, 2011, and December 31, 2020., Interventions: None., Measurements and Main Results: The cohort consisted of 20,223 ICU episodes (479 COVID-19), with a VA-LRTI incidence proportion of 30% (129/426) in COVID-19 and 18% (1,081/5,907) in non-COVID-19 among patients ventilated greater than or equal to 48 hours. The median length of ventilator treatment for COVID-19 patients was 10 days (interquartile range, 5-18 d), which was significantly longer than for all other investigated specific diagnoses. The VA-LRTI incidence rate per 1,000 ventilator days at risk was 31 (95% CI, 26-37) for COVID-19 and 34 (95% CI, 32-36) for non-COVID-19. With COVID-19 as reference, adjusted subdistribution hazard ratios for VA-LRTI was 0.29-0.50 (95% CI, < 1) for influenza, bacterial pneumonia, acute respiratory distress syndrome, and severe sepsis, but 1.38 (95% CI, 1.15-1.65) for specific noninfectious diagnoses. Compared with COVID-19 in the first wave of the pandemic, COVID-19 in the second wave had adjusted subdistribution hazard ratio of 1.85 (95% CI, 1.14-2.99). In early VA-LRTI Staphylococcus aureus was more common and Streptococcus pneumoniae, Haemophilus influenzae, and Escherichia coli less common in COVID-19 patients, while Serratia species was more often identified in late VA-LRTI., Conclusions: COVID-19 is associated with exceptionally long durations of mechanical ventilation treatment and high VA-LRTI occurrence proportions. The incidence rate of VA-LRTI was compared with the pooled non-COVID-19 cohort, however, not increased in COVID-19. Significant differences in the incidence of VA-LRTI occurred between the first and second wave of the COVID-19 pandemic., Competing Interests: Dr. Hedberg received funding from the Swedish Innovation Agency (Vinnova), Region Stockholm, and Emil och Wera Cornell Stiftelse; he was supported by Karolinska Institutet (combined clinical studies and PhD training program). Dr. Ternhag’s institution received funding from the Swedish Innovation Agency (Vinnova). The remaining authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2022 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved.)

Background: Carbapenemase-producing multidrug-resistant (MDR) Acinetobacter baumannii is a global health care problem. MDR A. baumannii has emerged as an important nosocomial pathogen, costing many lives worldwide including Bangladesh., Aim: To investigate the detailed molecular epidemiology of carbapenem-resistant A. baumannii (CRAB) both from patients and the hospital environment, to shed light on genetic characteristics and transmission dynamics., Methods: A set of 49 clinical A. baumannii strains collected during early 2015 was received from the clinical microbiology laboratory of Dhaka Medical College Hospital (DMCH) in Bangladesh. Additionaly, 100 environmental samples were also collected from the hospital surfaces of Dhaka Medical College Hospital and analyzed for carbapenamase-producing A. baumannii . CRAB were identified by culture on selective plates, biochemical testing and MALDI-TOF. All isolates were characterized by susceptibility testing, realtime-PCRs, conventional PCR, MLST and sequencing., Findings: Clinical A. baumannii were resistant to ciprofloxacin (100%), imipenem (91.8%), meropenem (91.8%), gentamicin (91.8%), amikacin (87.7%), and trimethoprim-sulfamethoxazole (61.2%). The majority (59%) of the isolates were MDR. All environmental A. baumannii (n=10) were resistant to imipenem, meropenem, gentamicin, amikacin, and ciprofloxacin. Strains carried the following antibiotic resistant genes; bla OXA-23, bla OXA-58, bla PER-7, qnrB1 , qnrC1, aac(6')1b-cr and armA . A total of 36 different clones were identified by rep-PCR and common clonal clusters were found both in patients and hospital environments. MLST analysis revealed different sequence types (ST2, ST10, ST149, ST575, ST1063 and ST1065). In clinical and environmental settings. A. baumannii ST2 dominated in both clinical and environmental settings. Both clinical and environmental A. baumannii strains with known STs carried several biofilm-related genes; bap, csuE, and pgaB ., Conclusion: Widespread dissemination of MDR A. baumannii in the DMC hospital of Bangladesh is a serious problem., (© 2022 The Authors.)

The upper respiratory tract (URT) microbiome can contribute to the acquisition and severity of respiratory viral infections. The described associations between URT microbiota and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are limited at microbiota genus level and by the lack of functional interpretation. Our study, therefore, characterized the URT bacterial microbiome at species level and their encoded pathways in patients with COVID-19 and correlated these to clinical outcomes. Whole metagenome sequencing was performed on nasopharyngeal samples from hospitalized patients with critical COVID-19 ( n = 37) and SARS-CoV-2-negative individuals ( n = 20). Decreased bacterial diversity, a reduction in commensal bacteria, and high abundance of pathogenic bacteria were observed in patients compared to negative controls. Several bacterial species and metabolic pathways were associated with better respiratory status and lower inflammation. Strong correlations were found between species biomarkers and metabolic pathways associated with better clinical outcome, especially Moraxella lincolnii and pathways of vitamin K 2 biosynthesis. Our study demonstrates correlations between the URT microbiome and COVID-19 patient outcomes; further studies are warranted to validate these findings and to explore the causal roles of the identified microbiome biomarkers in COVID-19 pathogenesis.

Objectives: Clinicians worldwide struggle to identify the bacterial aetiology of bone and joint infections. Failure to unequivocally identify the pathogen is linked to poor clinical outcomes. We explored the added value of analysing multiple samples per patient with 16S ribosomal DNA (16S rDNA) sequencing in diagnosing postoperative bone and joint infections. All patients had received antimicrobials prior to sampling, and false-negative cultures could be suspected. Bone biopsies obtained from patients with postoperative bone and joint infections for cultures were also subjected to 16S rDNA sequencing., Results: In 5/28 infectious episodes, sequencing identified the causative organism of the infection when cultures failed. In 8/28 episodes, the methods led to different results, potentially leading to different antimicrobial choices. The analysis of multiple samples per patient helped rule out potential contaminating pathogens. We conclude that 16S rDNA sequencing has diagnostic value for patients receiving antibiotic treatment. We regard the method as a complement to culturing when the cultures are negative. Multiple samples per patient should be analysed to determine the clinical significance of positive findings., (© 2022. The Author(s).)

The European Committee on Antimicrobial Susceptibility Testing (EUCAST) is an international susceptibility testing committee, organized by the European Society for Clinical Microbiology and Infectious Diseases (ESCMID) and functioning as the breakpoint advisory committee of the European Medicines Agency (EMA). The original remit of EUCAST was to harmonize European clinical breakpoints, but very soon, the activities expanded beyond the borders of Europe and included newly licensed agents in Europe. Among the milestones were the aggregating of large numbers of MIC distributions, creating software to display these distributions, the EUCAST concept of identifying epidemiological cutoff values (ECOFF), and the development of a EUCAST disk diffusion method. The EUCAST Development Laboratory has played a critical role in the development of antimicrobial susceptibility testing (AST) methodology, including development work for novel antimicrobial agents and for rapid AST directly from blood culture bottles. EUCAST has several standing subcommittees, including for AST in fungi (AFST) and mycobacteria (AMST) and for microorganisms of veterinary interest (VetCAST), and ad hoc subcommittees on subjects such as anaerobic bacteria, MIC and zone diameter distributions and epidemiological cutoff values, the relationship between phenotypic and genotypic resistance, and expert rules and methods for the detection of resistance mechanisms. All EUCAST decisions are subjected to the EUCAST public consultation process, the only exception being breakpoints of novel antimicrobial agents where confidentiality agreements during the licensing process prevent public participation. EUCAST has recently revised the definitions of clinical susceptibility interpretive categories S, I, and R, acknowledging the intimate relationship between drug exposure and susceptibility reporting.

Objectives: The Enterobacter cloacae complex (ECC) are causatives of hospital-acquired infections. The antimicrobial resistance (AMR) and virulence profiling of ECC promotes our knowledge for their elimination in clinical settings., Methods: We assembled the whole genome of four clinical carbapenem-resistant ECCs and characterized their AMR and virulence profiles using whole genome sequencing., Results: The chromosomes length scaled from minimum 3 949 952 bp (for P2) to maximum 4 976 575 bp (for P3). Strains P1 and P2 belonged to sequence type (ST)182. P3 and P4 belonged to ST477 and ST134, respectively. The bla CTX-M-15 gene was detected in P1 plasmid. P1 and P4 harboured the bla TEM-1 and bla OXA-1 genes. bla NDM-1 was found in P1, P3, and P4. No bla OXA-48 , bla KPC , bla VIM , or bla IMP were identified. The plasmids were nontransferable and had IncFIB, IncFII, Col, and IncC incompatibility (Inc) groups . Class 1 integron was detected in all strains. Virulence genes related to biofilms, adhesins, siderophores (aerobactin, enterobactin, and salmochelin), intrinsic antimicrobial efflux pumps, secretory systems type I to VI, environmental and antibiotic stress response , outer membrane proteins, and heavy metal (copper, tellurite, arsenic, and zinc) resistance were found. The number of positive virulence factors was higher for P1 compared with other strains., Conclusion: The accumulation of AMR genes in Enterobacter spp. and their high endurance in hostile environments is a serious health problem. More genomic investigations are required to determine their AMR and virulence genetic reservoirs at the global level., Competing Interests: Competing interests Authors declare that they had no commercial, personal, or political conflict of interests., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Use of third-generation cephalosporins, such as cefotaxime, is associated with an increased risk of selection for antimicrobial resistance, so alternative antibiotics need to be considered. The aim of the present study was to evaluate intestinal colonisation with third-generation cephalosporin-resistant pathogens following use of temocillin-an alternative antibiotic to cefotaxime that is potentially less prone to disturbing the intestinal microbiota-in empirical treatment of febrile urinary tract infection (UTI)., Methods: We did a randomised, multicentre, superiority, open-label phase 4 trial in patients who had been admitted to inpatient care in 12 Swedish hospitals with suspected or diagnosed febrile UTI (complicated or uncomplicated). To meet inclusion criteria, a patient was required to have at least one sign or symptom of pyelonephritis (ie, flank pain; costovertebral angle tenderness; and changes to urinary frequency or urgency or dysuria), a fever of 38·0°C or higher, and a positive urine dipstick (for nitrites, white blood cells, or both). Participants were also required to have an indication for intravenous antibiotic treatment. Participants were randomly assigned (1:1) to receive either 2 g temocillin or 1-2 g cefotaxime, by local investigators opening consecutive sealed randomisation envelopes that were generated centrally in advance. Both drugs were administered intravenously every 8 h. The trial was open label for investigators and patients, but those doing the microbiological analyses were masked to the groups. Participants were treated with antibiotics for 7-10 days (or up to 14 days if they had bacteraemia), at least 3 days of which were on the study drug; at day 4 and later, participants who were showing improvement could be given an oral antibiotic (ciprofloxacin, ceftibuten, cefixime, or co-trimoxazole). Patients not showing improvement were regarded as having treatment failures. Rectal swabs were collected at three timepoints: at baseline (before the first dose), after the last dose of study drug, and 7-10 days after treatment stopped. The composite primary outcome was colonisation with Enterobacterales with reduced susceptibility to third-generation cephalosporins, or colonisation with toxin-producing Clostridioides difficile, or both, to evaluate disturbance of the intestinal microbiota. The study is registered in the EU Clinical Trials Register (EudraCT 2015-003898-15)., Findings: Between May 20, 2016, and July 31, 2019, 207 patients were screened for eligibility, of whom 55 patients were excluded. 152 participants were randomly assigned to groups: 77 (51%) patients received temocillin, 75 (49%) patients received cefotaxime. The composite primary endpoint was met by 18 (26%) of 68 participants receiving temocillin versus 30 (48%) of 62 patients receiving cefotaxime (risk difference -22% [95% CI -42% to -3%]), showing superiority of temocillin versus cefotaxime (ie, less disturbance of the intestinal microbiota). 43 adverse events were reported in 40 (52%) of 77 patients in the temocillin group, versus 46 adverse events in 34 (45%) of 75 patients in the cefotaxime group. Most events were of mild to moderate severity. 21 (27%) patients in the temocillin and 17 (23%) patients in the cefotaxime group had an adverse event that was considered to be associated with the study drug., Interpretation: Temocillin was found to be less selective than cefotaxime of Enterobacterales with reduced susceptibility to third-generation cephalosporins, and it could therefore be a favourable alternative in the empirical treatment of febrile UTI. Use of this antibiotic could reduce hospital transmission and health-care-associated infections by these pathogens., Funding: Public Health Agency of Sweden., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Objectives: The carbapenemase-encoding gene bla NDM-1 has been reported in Vietnam during the last 10 years, and bla NDM -producing Enterobacteriaceae are now silently and rapidly spreading. A key factor behind dissemination of bla NDM-1 is plasmids, mobile genetic elements that commonly carry antibiotic resistance genes and spread via conjugation. The diversity of bla NDM-1 -encoding plasmids from neonates at a large Vietnamese hospital was characterized in this study., Methods: 18 fecal Klebsiella pneumoniae and Klebsiella quasipneumoniae isolates collected from 16 neonates at a large pediatric hospital in Vietnam were studied using optical DNA mapping (ODM) and next-generation sequencing (NGS). Plasmids carrying the bla NDM-1 gene were identified by combining ODM with Cas9 restriction. The plasmids in the isolates were compared to investigate whether the same plasmid was present in different patients., Results: Although the same plasmid was found in some isolates, ODM confirmed that there were at least 10 different plasmids encoding bla NDM-1 among the 18 isolates, thus indicating wide plasmid diversity. The ODM results concur with the NGS data. Interestingly, some isolates had two distinct plasmids encoding bla NDM-1 that could be readily identified with ODM. The coexistence of different plasmids carrying the same bla NDM-1 gene in a single isolate has rarely been reported, probably because of limitations in plasmid characterization techniques., Conclusions: The plasmids encoding the bla NDM-1 gene in this study cohort were diverse and may represent a similar picture in Vietnamese society. The study highlights important aspects of the usefulness of ODM for plasmid analysis., Competing Interests: Competing Interests None to declare., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)