Higher rates of cefiderocol resistance among NDM producing Klebsiella bloodstream isolates applying EUCAST over CLSI breakpoints.

BACKGROUND: Cefiderocol is generally active against carbapenem-resistant Klebsiella spp. (CRK) with higher MICs against metallo-beta-lactamase producers. There is a variation in cefiderocol interpretive criteria determined by EUCAST and CLSI. Our objective was to test CRK isolates against cefiderocol and compare cefiderocol susceptibilities using EUCAST and CLSI interpretive criteria. METHODS: A unique collection (n = 254) of mainly OXA-48-like- or NDM-producing CRK bloodstream isolates were tested against cefiderocol with disc diffusion (Mast Diagnostics, UK). Beta-lactam resistance genes and multilocus sequence types were identified using bioinformatics analyses on complete bacterial genomes. RESULTS: Median cefiderocol inhibition zone diameter was 24 mm (interquartile range [IQR] 24-26 mm) for all isolates and 18 mm (IQR 15-21 mm) for NDM producers. We observed significant variability between cefiderocol susceptibilities using EUCAST and CLSI breakpoints, such that 26% and 2% of all isolates, and 81% and 12% of the NDM producers were resistant to cefiderocol using EUCAST and CLSI interpretive criteria, respectively. CONCLUSIONS: Cefiderocol resistance rates among NDM producers are high using EUCAST criteria. Breakpoint variability may have significant implications on patient outcomes. Until more clinical outcome data are available, we suggest using EUCAST interpretive criteria for cefiderocol susceptibility testing.

Comparison of ceftazidime-avibactam susceptibility testing methods against OXA-48-like carrying Klebsiella blood stream isolates.

Ceftazidime-avibactam exhibits good in vitro activity against carbapenem resistant Klebsiella carrying OXA-48-like enzymes. We tested two hundred unique carbapenem resistant Klebsiella blood stream isolates (71% with single OXA-48-like carbapenemases, including OXA-48, n = 62; OXA-232, n = 57; OXA-244, n = 17; OXA-181, n = 5) that were collected as part of a multicentre study against ceftazidime-avibactam using Etest (bioMérieux, Marcyl'Étoile, France), 10/4 µg disc (Thermo Fisher) and Sensititre Gram Negative EURGNCOL Plates (Lyophilized panels, Sensititre, Thermo Fisher) with the aim of comparing the performances of the Etest and disc to that of Sensititre. Ceftazidime-avibactam MIC50/90 was 2/>16 mg/L for the entire collection and was 2/4 mg/L for single OXA-48-like producers. Categorical and essential agreements between the Etest and Sensititre were 100% and 97%, respectively. Categorical agreement between the disc and Sensititre was 100%. Etest and 10/4 µg discs are suitable alternatives to Sensititre for ceftazidime-avibactam sensitivity testing for OXA-48-like producers.

Clinical Burkholderia pseudomallei isolates from north Queensland carry diverse bimABm genes that are associated with central nervous system disease and are phylogenomically distinct from other Australian strains.

BACKGROUND: Burkholderia pseudomallei is an environmental gram-negative bacterium that causes the disease melioidosis and is endemic in many countries of the Asia-Pacific region. In Australia, the mortality rate remains high at approximately 10%, despite curative antibiotic treatment being available. The bacterium is almost exclusively found in the endemic region, which spans the tropical Northern Territory and North Queensland, with clusters occasionally present in more temperate climates. Despite being endemic to North Queensland, these infections remain understudied compared to those of the Northern Territory. METHODOLOGY/PRINCIPAL FINDINGS: This study aimed to assess the prevalence of central nervous system (CNS) disease associated variant bimABm, identify circulating antimicrobial resistance mutations and genetically distinct strains from Queensland, via comparative genomics. From 76 clinical isolates, we identified the bimABm variant in 20 (26.3%) isolates and in 9 (45%) of the isolates with documented CNS infection (n = 18). Explorative analysis suggests a significant association between isolates carrying the bimABm variant and CNS disease (OR 2.8, 95% CI 1.3-6.0, P = 0.009) compared with isolates carrying the wildtype bimABp. Furthermore, 50% of isolates were identified as novel multi-locus sequence types, while the bimABm variant was more commonly identified in isolates with novel sequence types, compared to those with previously described. Additionally, mutations associated with acquired antimicrobial resistance were only identified in 14.5% of all genomes. CONCLUSIONS/SIGNIFICANCE: The findings of this research have provided clinically relevant genomic data of B. pseudomallei in Queensland and suggest that the bimABm variant may enable risk stratification for the development CNS complications and be a potential therapeutic target.

Characteristics and outcomes of carbapenemase harbouring carbapenem-resistant Klebsiella spp. bloodstream infections: a multicentre prospective cohort study in an OXA-48 endemic setting.

A prospective, multicentre observational cohort study of carbapenem-resistant Klebsiella spp. (CRK) bloodstream infections was conducted in Turkey from June 2018 to June 2019. One hundred eighty-seven patients were recruited. Single OXA-48-like carbapenemases predominated (75%), followed by OXA-48-like/NDM coproducers (16%). OXA-232 constituted 31% of all OXA-48-like carbapenemases and was mainly carried on ST2096. Thirty-day mortality was 44% overall and 51% for ST2096. In the multivariate cox regression analysis, SOFA score and immunosuppression were significant predictors of 30-day mortality and ST2096 had a non-significant effect. All OXA-48-like producers remained susceptible to ceftazidime-avibactam.

Strengths and caveats of identifying resistance genes from whole genome sequencing data.

INTRODUCTION: Antimicrobial resistance (AMR) continues to present major challenges to modern healthcare. Recent advances in whole-genome sequencing (WGS) have made the rapid molecular characterization of AMR a realistic possibility for diagnostic laboratories; yet major barriers to clinical implementation exist. AREAS COVERED: We describe and compare short- and long-read sequencing platforms, typical components of bioinformatics pipelines, tools for AMR gene detection and the relative merits of read- or assembly-based approaches. The challenges of characterizing mobile genetic elements from genomic data are outlined, as well as the complexities inherent to the prediction of phenotypic resistance from WGS. Practical obstacles to implementation in diagnostic laboratories, the critical role of quality control and external quality assurance, as well as standardized reporting standards are also discussed. Future directions, such as the application of machine-learning and artificial intelligence algorithms, linked to clinically meaningful outcomes, may offer a new paradigm for the clinical application of AMR prediction. EXPERT OPINION: AMR prediction from WGS data presents an exciting opportunity to advance our capacity to comprehensively characterize infectious pathogens in a rapid manner, ultimately aiming to improve patient outcomes. Collaborative efforts between clinicians, scientists, regulatory bodies and healthcare administrators will be critical to achieve the full promise of this approach.

High prevalence of ArmA-16S rRNA methyltransferase among aminoglycoside-resistant Klebsiella pneumoniae bloodstream isolates.

Introduction. Aminoglycosides are used for the treatment of carbapenemase-producing Klebsiella pneumoniae (CPK) infections. 16S rRNA methyltransferases (RMTs) confer resistance to all aminoglycosides and are often cocarried with NDM.Hypothesis/Gap Statement. There is a dart of studies looking at the aminoglycoside resistance mechanisms for invasive CPK isolates, particularly in OXA-48 endemic settings.Aim. We aimed to determine the prevalence of RMTs and their association with beta lactamases and MLSTs amongst aminoglycoside-resistant CPK bloodstream isolates in an OXA-48 endemic setting.Methodology. CPK isolates (n=181), collected as part of a multicentre cohort study, were tested for amikacin, gentamicin and tobramycin susceptibility using custom-made sensititre plates (GN2XF, Thermo Fisher Scientific). All isolates were previously subjected to whole-genome sequencing. Carbapenemases, RMTs, MLSTs and plasmid incompatibility groups were detected on the assembled genomes.Results. Of the 181 isolates, 109(60 %) were resistant to all three aminoglycosides, and 96 of 109(88 %) aminoglycoside-resistant isolates carried an RMT (85 ArmA, 10 RmtC, 4 RmtF1; three isolates cocarried ArmA and RmtC). Main clonal types associated with ArmA were ST2096 (49/85, 58 %) and ST14 (24/85, 28 %), harbouring mainly OXA-232 and OXA-48 +NDM, respectively. RmtC was cocarried with NDM (5/10) on ST395, and NDM +OXA-48 or NDM +KPC (4/10) on ST14, ST15 and ST16. All RMT producers also carried CTX-M-15, and the majority cocarried SHV-106, TEM-150 and multiple other antibiotic resistance genes. The majority of the isolates harboured a combination of IncFIB, IncH and IncL/M type plasmids. Non-NDM producing isolates remained susceptible to ceftazidime-avibactam.Conclusion. Aminoglycoside resistance amongst CPK bloodstream isolates is extremely common and mainly driven by clonal spread of ArmA carried on ST2096 and ST14, associated with OXA-232 and OXA48 +NDM carriage, respectively.

Burkholderia pseudomallei Clinical Isolates Are Highly Susceptible In Vitro to Cefiderocol, a Siderophore Cephalosporin.

Cefiderocol is a cephalosporin designed to treat multidrug-resistant Gram-negative infections. By forming a chelated complex with ferric iron, cefiderocol is transported into the periplasmic space via bacterial iron transport systems and primarily binds to penicillin-binding protein 3 (PBP3) to inhibit peptidoglycan synthesis. This mode of action results in cefiderocol having greater in vitro activity against many Gram-negative bacilli than currently used carbapenems, ß-lactam/ß-lactamase inhibitor combinations, and cephalosporins. Thus, we investigated the in vitro activity of cefiderocol against a total of 246 clinical isolates of Burkholderia pseudomallei from Queensland, Australia. The collection was composed primarily of bloodstream (56.1%), skin and soft tissue (16.3%), and respiratory (15.9%) isolates. MICs of cefiderocol ranged from ≤0.03 to 16 mg/liter, whereas the MIC90 was 0.125 mg/liter. Based upon CLSI clinical breakpoints for cefiderocol against Pseudomonas aeruginosa, Acinetobacter baumannii, and Stenotrophomonas maltophilia, three isolates (1.2%) would be classified as nonsusceptible (MIC > 4 mg/liter). Using EUCAST non-species-specific (pharmacokinetic/pharmacodynamic [PK/PD]) clinical breakpoints or those set for Pseudomonas aeruginosa, four isolates (1.6%) would be resistant (MIC > 2 mg/liter). Further testing for coresistance to meropenem, ceftazidime, trimethoprim-sulfamethoxazole, amoxicillin-clavulanate, and doxycycline was performed on the four isolates with elevated cefiderocol MICs (>2 mg/liter); all isolates exhibited resistance to amoxicillin-clavulanic acid, while three isolates also displayed resistance to at least one other antimicrobial. Cefiderocol was found to be highly active in vitro against B. pseudomallei primary clinical isolates. This compound shows great potential for the treatment of melioidosis in countries of endemicity and should be explored further.

Genotype-free demultiplexing of pooled single-cell RNA-seq.

A variety of methods have been developed to demultiplex pooled samples in a single cell RNA sequencing (scRNA-seq) experiment which either require hashtag barcodes or sample genotypes prior to pooling. We introduce scSplit which utilizes genetic differences inferred from scRNA-seq data alone to demultiplex pooled samples. scSplit also enables mapping clusters to original samples. Using simulated, merged, and pooled multi-individual datasets, we show that scSplit prediction is highly concordant with demuxlet predictions and is highly consistent with the known truth in cell-hashing dataset. scSplit is ideally suited to samples without external genotype information and is available at: https://github.com/jon-xu/scSplit.