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Gram-negative bacteria (GNB) are a major cause of neonatal sepsis in low- and middle-income countries (LMICs). Although the World Health Organization (WHO) reports that over 80% of these sepsis deaths could be prevented through improved treatment, the efficacy of the currently recommended first- and second-line treatment regimens for this condition is increasingly affected by high rates of drug resistance. Here we assess three well known antibiotics, fosfomycin, flomoxef and amikacin, in combination as potential antibiotic treatment regimens by investigating the drug resistance and genetic profiles of commonly isolated GNB causing neonatal sepsis in LMICs. The five most prevalent bacterial isolates in the NeoOBS study (NCT03721302) are Klebsiella pneumoniae, Acinetobacter baumannii, E. coli, Serratia marcescens and Enterobacter cloacae complex. Among these isolates, high levels of ESBL and carbapenemase encoding genes are detected along with resistance to ampicillin, gentamicin and cefotaxime, the current WHO recommended empiric regimens. The three new combinations show excellent in vitro activity against ESBL-producing K. pneumoniae and E. coli isolates. Our data should further inform and support the clinical evaluation of these three antibiotic combinations for the treatment of neonatal sepsis in areas with high rates of multidrug-resistant Gram-negative bacteria., (© 2024. The Author(s).)

Background: Streptococcus pneumoniae is a global cause of community-acquired pneumonia (CAP) and invasive disease in children. The CAP-IT trial (grant No. 13/88/11; https://www.capitstudy.org.uk/ ) collected nasopharyngeal swabs from children discharged from hospitals with clinically diagnosed CAP, and found no differences in pneumococci susceptibility between higher and lower antibiotic doses and shorter and longer durations of oral amoxicillin treatment. Here, we studied in-depth the genomic epidemiology of pneumococcal (vaccine) serotypes and their antibiotic resistance profiles., Methods: Three-hundred and ninety pneumococci cultured from 1132 nasopharyngeal swabs from 718 children were whole-genome sequenced (Illumina) and tested for susceptibility to penicillin and amoxicillin. Genome heterogeneity analysis was performed using long-read sequenced isolates (PacBio, n = 10) and publicly available sequences., Results: Among 390 unique pneumococcal isolates, serotypes 15B/C, 11 A, 15 A and 23B1 were most prevalent (n = 145, 37.2%). PCV13 serotypes 3, 19A, and 19F were also identified (n = 25, 6.4%). STs associated with 19A and 19F demonstrated high genome variability, in contrast to serotype 3 (n = 13, 3.3%) that remained highly stable over a 20-year period. Non-susceptibility to penicillin (n = 61, 15.6%) and amoxicillin (n = 10, 2.6%) was low among the pneumococci analysed here and was independent of treatment dosage and duration. However, all 23B1 isolates (n = 27, 6.9%) were penicillin non-susceptible. This serotype was also identified in ST177, which is historically associated with the PCV13 serotype 19F and penicillin susceptibility, indicating a potential capsule-switch event., Conclusions: Our data suggest that amoxicillin use does not drive pneumococcal serotype prevalence among children in the UK, and prompts consideration of PCVs with additional serotype coverage that are likely to further decrease CAP in this target population. Genotype 23B1 represents the convergence of a non-vaccine genotype with penicillin non-susceptibility and might provide a persistence strategy for ST types historically associated with vaccine serotypes. This highlights the need for continued genomic surveillance., (© 2024. The Author(s).)

Understanding the myriad pathways by which antimicrobial-resistance genes (ARGs) spread across biomes is necessary to counteract the global menace of antimicrobial resistance. We screened 17939 assembled metagenomic samples covering 21 biomes, differing in sequencing quality and depth, unevenly across 46 countries, 6 continents, and 14 years (2005-2019) for clinically crucial ARGs, mobile colistin resistance (mcr), carbapenem resistance (CR), and (extended-spectrum) beta-lactamase (ESBL and BL) genes. These ARGs were most frequent in human gut, oral and skin biomes, followed by anthropogenic (wastewater, bioreactor, compost, food), and natural biomes (freshwater, marine, sediment). Mcr-9 was the most prevalent mcr gene, spatially and temporally; bla OXA-233 and bla TEM-1 were the most prevalent CR and BL/ESBL genes, but bla GES-2 and bla TEM-116 showed the widest distribution. Redundancy analysis and Bayesian analysis showed ARG distribution was non-random and best-explained by potential host genera and biomes, followed by collection year, anthropogenic factors and collection countries. Preferential ARG occurrence, and potential transmission, between characteristically similar biomes indicate strong ecological boundaries. Our results provide a high-resolution global map of ARG distribution and importantly, identify checkpoint biomes wherein interventions aimed at disrupting ARGs dissemination are likely to be most effective in reducing dissemination and in the long term, the ARG global burden., (© 2022. The Author(s).)

Multi-drug-resistant Acinetobacter baumannii isolates are key pathogens that contribute to the global burden of antimicrobial resistance. This study aimed to investigate the phenotypic and molecular characteristics of carbapenem-resistant A. baumannii (CRAB) isolates from the EURECA clinical trial. In total, 228 CRAB clinical strains were recovered from 29 sites in 10 European countries participating in the EURECA study between May 2016 and November 2018. All strains were reconfirmed centrally for identification and antimicrobial susceptibility testing, and were then subjected to DNA isolation and whole-genome sequencing (WGS), with analysis performed using BacPipe v.1.2.6. K and O typing was performed using KAPTIVE. Overall, 226 (99.1%) strains were confirmed as CRAB isolates. The minimum inhibitory concentration (MIC 90 ) results of imipenem and meropenem were >16 mg/L. WGS showed that the isolates mainly harboured bla OXA-23 (n=153, 67.7%) or bla OXA-72 (n=70, 30.1%). Four bla OXA-72 isolates from Serbia co-harboured bla NDM-1. An IS5 transposase family element, ISAba31, was found upstream of the bla OXA-72 gene harboured on a small (~10-kb) pSE41030-EUR plasmid. The majority of isolates (n=178, 79.1%) belonged to international clone II. Strains belonging to the same sequence type but isolated in different countries or within the same country could be delineated in different clusters by core-genome multi-locus sequence typing (MLST). Whole-genome/core-genome MLST showed high diversity among the isolates, and the most common sequence type was ST2 (n=153, 67.7%). The EURECA A. baumannii strain collection represents a unique, diverse repository of carbapenem-resistant isolates that adds to the existing knowledge of A. baumannii epidemiology and resistance genes harboured by these strains., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

It is well established that antibiotic treatment selects for resistance, but the dynamics of this process during infections are poorly understood. Here we map the responses of Pseudomonas aeruginosa to treatment in high definition during a lung infection of a single ICU patient. Host immunity and antibiotic therapy with meropenem suppressed P. aeruginosa, but a second wave of infection emerged due to the growth of oprD and wbpM meropenem resistant mutants that evolved in situ. Selection then led to a loss of resistance by decreasing the prevalence of low fitness oprD mutants, increasing the frequency of high fitness mutants lacking the MexAB-OprM efflux pump, and decreasing the copy number of a multidrug resistance plasmid. Ultimately, host immunity suppressed wbpM mutants with high meropenem resistance and fitness. Our study highlights how natural selection and host immunity interact to drive both the rapid rise, and fall, of resistance during infection.

Objectives: WGS-based antimicrobial susceptibility testing (AST) is as reliable as phenotypic AST for several antimicrobial/bacterial species combinations. However, routine use of WGS-based AST is hindered by the need for bioinformatics skills and knowledge of antimicrobial resistance (AMR) determinants to operate the vast majority of tools developed to date. By leveraging on ResFinder and PointFinder, two freely accessible tools that can also assist users without bioinformatics skills, we aimed at increasing their speed and providing an easily interpretable antibiogram as output., Methods: The ResFinder code was re-written to process raw reads and use Kmer-based alignment. The existing ResFinder and PointFinder databases were revised and expanded. Additional databases were developed including a genotype-to-phenotype key associating each AMR determinant with a phenotype at the antimicrobial compound level, and species-specific panels for in silico antibiograms. ResFinder 4.0 was validated using Escherichia coli (n = 584), Salmonella spp. (n = 1081), Campylobacter jejuni (n = 239), Enterococcus faecium (n = 106), Enterococcus faecalis (n = 50) and Staphylococcus aureus (n = 163) exhibiting different AST profiles, and from different human and animal sources and geographical origins., Results: Genotype-phenotype concordance was ≥95% for 46/51 and 25/32 of the antimicrobial/species combinations evaluated for Gram-negative and Gram-positive bacteria, respectively. When genotype-phenotype concordance was <95%, discrepancies were mainly linked to criteria for interpretation of phenotypic tests and suboptimal sequence quality, and not to ResFinder 4.0 performance., Conclusions: WGS-based AST using ResFinder 4.0 provides in silico antibiograms as reliable as those obtained by phenotypic AST at least for the bacterial species/antimicrobial agents of major public health relevance considered., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

A 60-year-old woman received meropenem/colistin treatment for bilateral pneumonia caused by a ST15 carbapenemase producing Klebsiella pneumoniae. The patient recovered but re-infection with the same (ST15), but now colistin-resistant K. pneumoniae, occurred. The molecular mechanism of the emerged colistin resistance was identified as mgrB gene modification by insertion element (IS) IS903B., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The initial report of the mcr-1 (mobile colistin resistance) gene has led to many reports of mcr-1 variants and other mcr genes from different bacterial species originating from human, animal and environmental samples in different geographical locations. Resistance gene nomenclature is complex and unfortunately problems such as different names being used for the same gene/protein or the same name being used for different genes/proteins are not uncommon. Registries exist for some families, such as bla (β-lactamase) genes, but there is as yet no agreed nomenclature scheme for mcr genes. The National Center for Biotechnology Information (NCBI) recently took over assigning bla allele numbers from the longstanding Lahey β-lactamase website and has agreed to do the same for mcr genes. Here, we propose a nomenclature scheme that we hope will be acceptable to researchers in this area and that will reduce future confusion.

Susceptibility testing for colistin remains challenging primarily due to its inherent properties. We evaluated colistin stability in agar and reproducibility of colistin MICs obtained by agar dilution, broth macro- and micro-dilution and MIC gradient strips on 3-7 iterations of each method using clinical Klebsiella pneumoniae (susceptible-CS, and resistant-CR, n = 2 each), mcr-harboring Escherichia coli (n = 2), and reference strains E. coli ATCC25922 and Pseudomonas aeruginosa ATCC27853. MICs for reference strains were not in the given range using Etest and broth microdilution (ATCC25922, 0.125 and 4 μg/ml, respectively). MICs of CR-1 and CR-2, and of the mcr-harboring E. coli showed high concordance between agar and broth dilution varying up to one 2-fold dilution. However, remarkable variations were observed on broth dilution with CS-1 and CS-2 (MIC range 0.25-32 and 0.5-64 μg/ml, respectively); whereas for agar dilution the MIC for both CS strains was 0.5 μg/ml in all the runs. MICs obtained by MIC gradient strips were lower than those obtained by dilution methods (1-2 dilutions for CS and mcr strains, and up to five dilutions for CR strains). To confirm uniform distribution of colistin in agar, a single strain was spotted in five different regions of the same plate. All spots showed concordant growth with maximum one dilution difference. No effect on MIC was found due to storage of colistin-containing agar plates for 7 days at 4 °C. In our hands, agar dilution was superior in terms of reproducibility and robustness, compared to broth dilution methods, for colistin MIC determination.

In two pairs of clinical colistin-susceptible/colistin-resistant (Cst(s)/Cst(r)) Acinetobacter baumannii strains, the Cst(r) strains showed significantly decreased biofilm formation in static and dynamic assays (P < 0.001) and lower relative fitness (P < 0.05) compared with those of the Cst(s) counterparts. The whole-genome sequencing comparison of strain pairs identified a mutation converting a stop codon to lysine (*241K) in LpsB (involved in lipopolysaccharide [LPS] synthesis) in one Cst(r) strain and a frameshift mutation in CarO and the loss of a 47,969-bp element containing multiple genes associated with biofilm production in the other., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)