Development of a novel streamlined workflow (AACRE) and database (inCREDBle) for genomic analysis of carbapenem-resistant Enterobacterales.

In response to the threat of increasing antimicrobial resistance, we must increase the amount of available high-quality genomic data gathered on antibiotic-resistant bacteria. To this end, we developed an integrated pipeline for high-throughput long-read sequencing, assembly, annotation and analysis of bacterial isolates and used it to generate a large genomic data set of carbapenemase-producing Enterobacterales (CPE) isolates collected in Spain. The set of 461 isolates were sequenced with a combination of both Illumina and Oxford Nanopore Technologies (ONT) DNA sequencing technologies in order to provide genomic context for chromosomal loci and, most importantly, structural resolution of plasmids, important determinants for transmission of antimicrobial resistance. We developed an informatics pipeline called Assembly and Annotation of Carbapenem-Resistant Enterobacteriaceae (AACRE) for the full assembly and annotation of the bacterial genomes and their complement of plasmids. To explore the resulting genomic data set, we developed a new database called inCREDBle that not only stores the genomic data, but provides unique ways to filter and compare data, enabling comparative genomic analyses at the level of chromosomes, plasmids and individual genes. We identified a new sequence type, ST5000, and discovered a genomic locus unique to ST15 that may be linked to its increased spread in the population. In addition to our major objective of generating a large regional data set, we took the opportunity to compare the effects of sample quality and sequencing methods, including R9 versus R10 nanopore chemistry, on genome assembly and annotation quality. We conclude that converting short-read and hybrid microbial sequencing and assembly workflows to the latest nanopore chemistry will further reduce processing time and cost, truly enabling the routine monitoring of resistance transmission patterns at the resolution of complete chromosomes and plasmids.

Emergence of 16S rRNA methyltransferases among carbapenemase-producing Enterobacterales in Spain studied by whole-genome sequencing.

The emergence of 16S rRNA methyltransferases (RMTs) in Gram-negative pathogens bearing other clinically relevant resistance mechanisms, such as carbapenemase-producing Enterobacterales (CPE), is becoming an alarming concern. We investigated the prevalence, antimicrobial susceptibility, resistance mechanisms, molecular epidemiology and genetic support of RMTs in CPE isolates from Spain. This study included a collection of 468 CPE isolates recovered during 2018 from 32 participating Spanish hospitals. MICs were determined using the broth microdilution method, the agar dilution method (fosfomycin) or MIC gradient strips (plazomicin). All isolates were subjected to hybrid whole-genome sequencing (WGS). Sequence types (STs), core genome phylogenetic relatedness, horizontally acquired resistance mechanisms, plasmid analysis and the genetic environment of RMTs were determined in silico from WGS data in all RMT-positive isolates. Among the 468 CPE isolates evaluated, 24 isolates (5.1%) recovered from nine different hospitals spanning five Spanish regions showed resistance to all aminoglycosides and were positive for an RMT (21 RmtF, 2 ArmA and 1 RmtC). All RMT-producers showed high-level resistance to all aminoglycosides, including plazomicin, and in most cases exhibited an extensively drug-resistant susceptibility profile. The RMT-positive isolates showed low genetic diversity and were global clones of Klebsiella pneumoniae (ST147, ST101, ST395) and Enterobacter cloacae (ST93) bearing blaOXA-48, blaNDM-1 or blaVIM-1 carbapenemase genes. RMTs were harboured in five different multidrug resistance plasmids and linked to efficient mobile genetic elements. Our findings highlight that RMTs are emerging among clinical CPE isolates from Spain and their spread should be monitored to preserve the future clinical utility of aminoglycosides and plazomicin.

An Improved Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Data Analysis Pipeline for the Identification of Carbapenemase-Producing Klebsiella pneumoniae.

The increasing emergence of carbapenemase-producing Klebsiella pneumoniae (CPK) isolates is a global health alarm. Rapid methods that require minimum sample preparation and rapid data analysis are urgently required. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been used by clinical laboratories for identification of antibiotic-resistant bacteria; however, discrepancies have arisen regarding biological and technical issues. The aim of this study was to standardize an operating procedure and data analysis for identification of CPK by MALDI-TOF MS. To evaluate this approach, a series of 162 K. pneumoniae isolates (112 CPK and 50 non-CPK) were processed in the MALDI BioTyper system (Bruker Daltonik, Germany) following a standard operating procedure. The study was conducted in two stages; the first is denominated the "reproducibility stage" and the second "CPK identification." The first stage was designed to evaluate the biological and technical variation associated with the entire analysis of CPK and the second stage to assess the final accuracy of MALDI-TOF MS for the identification of CPK. Therefore, we present an improved MALDI-TOF MS data analysis pipeline using neural network analysis implemented in Clover MS Data Analysis Software (Clover Biosoft, Spain) that is designed to reduce variability, guarantee interlaboratory reproducibility, and maximize the information selected from the bacterial proteome. Using the random forest (RF) algorithm, 100% of CPK isolates were correctly identified when all the peaks in the spectra were selected as input features and total ion current (TIC) normalization was applied. Thus, we have demonstrated that real-time direct tracking of CPK is possible using MALDI-TOF MS.

Occurrence of the p019 Gene in the blaKPC-Harboring Plasmids: Adverse Clinical Impact for Direct Tracking of KPC-Producing Klebsiella pneumoniae by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been used for the direct detection of KPC-producing isolates by analysis of the 11,109 Da mass peak representing the P019 protein. In this study, we evaluate the presence of the 11,109 Da mass peak in a collection of 435 unduplicated Klebsiella pneumoniae clinical isolates. The prevalence of the P019 peak in the blaKPC K. pneumoniae isolates was 49.2% (32/65). The 11,109 Da mass peak was not observed in any of the other carbapenemase (319) or noncarbapenemase producers (116). Computational analysis of the presence of the p019 gene was performed in the aforementioned carbapenemase-producing K. pneumoniae isolates fully characterized by whole-genome sequencing (WGS) and in a further collection of 1,649 K. pneumoniae genomes included in EuSCAPE. Herein, we have demonstrated that the p019 gene is not exclusively linked to the pKpQil plasmid but that it is present in the following plasmids: IncFIB(K)/IncFII(K)/ColRNAI, IncFIB(pQil), IncFIB(pQil)/ColRNAI, IncFIB(pQil)/IncFII(K), IncFIB(K)/IncFII(K), and IncX3. In addition, we have proven the independent movement of the Tn4401 and the ISKpn31, of which the p019 gene is a component. The absence of the p019 gene was obvious in Col440I, Col(pHAD28), IncFIB(K)/IncX3/IncFII(K), and IncFIB(K)/IncFII(K) plasmids. In addition, we also observed another plasmid in which neither Tn4401 nor ISKpn31 was found, IncP6. In the EuSCAPE, the occurrence of p019 varied from 0% to 100% among the different geographical locations. The adverse clinical impact of the diminished prevalence of the p019 gene within the plasmid encoding KPC-producing Klebsiella pneumoniae puts forward the need for reconsideration when applying this technique in a clinical setting.

Activity of imipenem/relebactam against a Spanish nationwide collection of carbapenemase-producing Enterobacterales.

BACKGROUND: Imipenem/relebactam is a novel carbapenem/ß-lactamase inhibitor combination, developed to act against carbapenemase-producing Enterobacterales (CPE). OBJECTIVES: To assess the in vitro activity of imipenem/relebactam against a Spanish nationwide collection of CPE by testing the susceptibility of these isolates to 16 widely used antimicrobials and to determine the underlying ß-lactam resistance mechanisms involved and the molecular epidemiology of carbapenemases in Spain. MATERIALS AND METHODS: Clinical CPE isolates (n = 401) collected for 2 months from 24 hospitals in Spain were tested. MIC50, MIC90 and susceptibility/resistance rates were interpreted in accordance with the EUCAST guidelines. ß-Lactam resistance mechanisms and molecular epidemiology were characterized by WGS. RESULTS: For all isolates, high rates of susceptibility to colistin (86.5%; MIC50/90 = 0.12/8 mg/L), imipenem/relebactam (85.8%; MIC50/90 = 0.5/4 mg/L) and ceftazidime/avibactam (83.8%, MIC50/90 = 1/≥256 mg/L) were observed. The subgroups of isolates producing OXA-48-like (n = 305, 75.1%) and KPC-like enzymes (n = 44, 10.8%) were highly susceptible to ceftazidime/avibactam (97.7%, MIC50/90 = 1/2 mg/L) and imipenem/relebactam (100.0%, MIC50/90 = ≤0.25/1 mg/L), respectively.The most widely disseminated high-risk clones of carbapenemase-producing Klebsiella pneumoniae across Spain were found to be ST11, ST147, ST392 and ST15 (mostly associated with OXA-48) and ST258/512 (in all cases producing KPC). CONCLUSIONS: Imipenem/relebactam, colistin and ceftazidime/avibactam were the most active antimicrobials against all CPEs. Imipenem/relebactam is a valuable addition to the antimicrobial arsenal used in the fight against CPE, particularly against KPC-producing isolates, which in all cases were susceptible to this combination.

267 Spanish Exomes Reveal Population-Specific Differences in Disease-Related Genetic Variation.

Recent results from large-scale genomic projects suggest that allele frequencies, which are highly relevant for medical purposes, differ considerably across different populations. The need for a detailed catalog of local variability motivated the whole-exome sequencing of 267 unrelated individuals, representative of the healthy Spanish population. Like in other studies, a considerable number of rare variants were found (almost one-third of the described variants). There were also relevant differences in allelic frequencies in polymorphic variants, including ∼10,000 polymorphisms private to the Spanish population. The allelic frequencies of variants conferring susceptibility to complex diseases (including cancer, schizophrenia, Alzheimer disease, type 2 diabetes, and other pathologies) were overall similar to those of other populations. However, the trend is the opposite for variants linked to Mendelian and rare diseases (including several retinal degenerative dystrophies and cardiomyopathies) that show marked frequency differences between populations. Interestingly, a correspondence between differences in allelic frequencies and disease prevalence was found, highlighting the relevance of frequency differences in disease risk. These differences are also observed in variants that disrupt known drug binding sites, suggesting an important role for local variability in population-specific drug resistances or adverse effects. We have made the Spanish population variant server web page that contains population frequency information for the complete list of 170,888 variant positions we found publicly available (http://spv.babelomics.org/), We show that it if fundamental to determine population-specific variant frequencies to distinguish real disease associations from population-specific polymorphisms.

High-resolution hepatitis C virus subtyping using NS5B deep sequencing and phylogeny, an alternative to current methods.

Hepatitis C virus (HCV) is classified into seven major genotypes and 67 subtypes. Recent studies have shown that in HCV genotype 1-infected patients, response rates to regimens containing direct-acting antivirals (DAAs) are subtype dependent. Currently available genotyping methods have limited subtyping accuracy. We have evaluated the performance of a deep-sequencing-based HCV subtyping assay, developed for the 454/GS-Junior platform, in comparison with those of two commercial assays (Versant HCV genotype 2.0 and Abbott Real-time HCV Genotype II) and using direct NS5B sequencing as a gold standard (direct sequencing), in 114 clinical specimens previously tested by first-generation hybridization assay (82 genotype 1 and 32 with uninterpretable results). Phylogenetic analysis of deep-sequencing reads matched subtype 1 calling by population Sanger sequencing (69% 1b, 31% 1a) in 81 specimens and identified a mixed-subtype infection (1b/3a/1a) in one sample. Similarly, among the 32 previously indeterminate specimens, identical genotype and subtype results were obtained by direct and deep sequencing in all but four samples with dual infection. In contrast, both Versant HCV Genotype 2.0 and Abbott Real-time HCV Genotype II failed subtype 1 calling in 13 (16%) samples each and were unable to identify the HCV genotype and/or subtype in more than half of the non-genotype 1 samples. We concluded that deep sequencing is more efficient for HCV subtyping than currently available methods and allows qualitative identification of mixed infections and may be more helpful with respect to informing treatment strategies with new DAA-containing regimens across all HCV subtypes.

Improved prediction of salvage antiretroviral therapy outcomes using ultrasensitive HIV-1 drug resistance testing.

BACKGROUND: The clinical relevance of ultrasensitive human immunodeficiency virus type 1 (HIV-1) genotypic resistance testing in antiretroviral treatment (ART)-experienced individuals remains unknown. METHODS: This was a retrospective, multicentre, cohort study in ART-experienced, HIV-1-infected adults who initiated salvage ART including, at least 1 ritonavir-boosted protease inhibitor, raltegravir or etravirine. Presalvage ART Sanger and 454 sequencing of plasma HIV-1 were used to generate separate genotypic sensitivity scores (GSS) using the HIVdb, ANRS, and REGA algorithms. Virological failure (VF) was defined as 2 consecutive HIV-1 RNA levels ≥200 copies/mL at least 12 weeks after salvage ART initiation, whereas subjects remained on the same ART. The ability of Sanger and 454-GSS to predict VF was assessed by receiver operating characteristic (ROC) curves and survival analyses. RESULTS: The study included 132 evaluable subjects; 28 (21%) developed VF. Using HIVdb, 454 predicted VF better than Sanger sequencing in the ROC curve analysis (area under the curve: 0.69 vs 0.60, Delong test P = .029). Time to VF was shorter for subjects with 454-GSS < 3 vs 454-GSS ≥ 3 (Log-rank P = .003) but not significantly different between Sanger-GSS < 3 and ≥3. Factors independently associated with increased risk of VF in multivariate Cox regression were a 454-GSS < 3 (HR = 4.6, 95 CI, [1.5, 14.0], P = .007), and the number of previous antiretrovirals received (HR = 1.2 per additional drug, 95 CI, [1.1, 1.3], P = .001). Equivalent findings were obtained with the ANRS and REGA algorithms. CONCLUSIONS: Ultrasensitive HIV-1 genotyping improves GSS-based predictions of virological outcomes of salvage ART relative to Sanger sequencing. This may improve the clinical management of ART-experienced subjects living with HIV-1. CLINICAL TRIALS REGISTRATION: NCT01346878.

Genomic analysis of the emergence and evolution of multidrug resistance during a Klebsiella pneumoniae outbreak including carbapenem and colistin resistance.

OBJECTIVES: To characterize at the genomic level the evolution of multiresistance during an outbreak of Klebsiella pneumoniae in a burns intensive care unit. The outbreak involved a DHA-1 ß-lactamase-producing strain that later acquired carbapenem and fosfomycin resistance, and in one case colistin resistance. METHODS: The genomes of two isolates were sequenced and compared with a previously sequenced genome. The role of hypermutability was investigated by measuring the mutation frequencies of the isolates and comparison with a collection of control strains. RESULTS: Sequence comparison identified four single-nucleotide variants and two transposon insertions. Analysis of the variants in the whole collection related carbapenem and fosfomycin resistance to a nonsense mutation in the ompK36 porin gene and colistin resistance to an IS1 insertion in the mgrB gene. The plasmid carrying the blaDHA-1 gene was unstable in the absence of antibiotics, and analysis of isolates that had lost the plasmid showed that the porin mutation alone was not sufficient to generate carbapenem resistance. The mutation frequencies were similar among all the strains analysed. CONCLUSIONS: Carbapenem resistance required production of the DHA-1 ß-lactamase and decreased permeability, but fosfomycin resistance depended only on permeability. Resistance to colistin might be related to an alteration in the regulation of the phoPQ system. Hypermutation is not related to the selection of porin mutants. Plasmid instability might be due to the high number of mobile elements and suggests a major role for antibiotic selection pressure in the emergence and evolution of this outbreak.