Biology subjects

Hasman, H.

Publications and source records attributed to Hasman, H..

7 recordsLinked to original sources

bioRxiv2024

Real-time identification of carbapenemase-producing Klebsiella pneumoniae lineages and outbreak detection using FT-IR ATR

Expansion of carbapenemase-producing Klebsiella pneumoniae (CP-Kp) is driven by nosocomial dissemination, and effective infection control depends on timely and reliable typing data. Here, we evaluated our previously developed Fourier-transform infrared spectroscopy (FT-IR) with attenuated total reflectance (ATR) workflow for real-time typing of Kp capsular (KL)-types and lineages to support infection control. FT-IR spectra were acquired from Columbia agar with 5% sheep blood cultures of all CP-Kp infection isolates (n=136) from hospitalized patients at a northern Portugal hospital (April 2022 - March 2023), and analyzed using automated machine-learning (ML) classification models. Typing results were confirmed by wzi sequencing, MLST and/or WGS. FT-IR typing on Columbia agar plates showed 73% sensitivity, 79% specificity and 74% accuracy. Our method correctly typed 94% of typeable isolates (78/83), from which 87% were comunicated in <24h. Sixty percent of non-typeable isolates were considered false negatives, but the majority (66%) was correctly predicted when re-tested in Mueller-Hinton agar, improving sensitivity (92%), specificity (76%) and accuracy (89%) of Kp typing. Three Kp lineages (ST147-KL64, ST15-KL19, ST268-KL20) represented 74% of the sample, with ST268-KL20 causing an outbreak in Neonatal Intensive Care unit, quickly recognized by FT-IR enabling immediate infection control measures. Epidemiological links between patiens were mostly found on medical, surgical and urology units, using EpiLinx software. Most isolates (98%) produced KPC-3. Our FT-IR ATR ML-based typing workflow demonstrated high performance standards in real-time and high adaptability to clonal dynamics. The unprecedent time-to-response (same day of species identification) represents an opportunity to implement timely and effective infection control measures. ImportanceThis study represents the first prospective and real-time evaluation of FT-IR spectroscopy to type multidrug resistant Klebsiella pneumoniae to support surveillance and infection control. We demonstrate a high sensitivity, specificity and accuracy of a previously developed workflow that allows precise identification of K. pneumoniae lineages. The adaptability to changes in clonal dynamics and bacterial typing in <24h offer significant advantages in both high- and low-income countries for a timely infection control and improvement of antimicrobial resistance management.

microbiology↗

bioRxiv2023

Long-term dynamics of the 'Serratia marcescens complex' in the hospital-built environment

Serratia marcescens is an opportunistic pathogen historically associated with abrupt, unpredictable, and severe outbreaks in hospital intensive care units (ICUs) and, more recently, with the spread of acquired genes encoding carbapenem resistance. However, the population biology and ecology of S. marcescens in the hospital ecosystem is still poorly understood. Here, we combine epidemiological information of 1417 Serratia isolates collected from the sinks of a large ICU ward that underwent significant demographic and operational changes (2019-2020) and 99 non-redundant outbreak/non-outbreak isolates from the same hospital (2003-2019), with genomic data of 165 isolates. We first report hospital sinks as reservoirs of heterogeneous and coexistent populations of the S. marcescens complex (SMC). A novel SMC clade congruent with the recently described Serratia nevei species is predominant, exhibiting chromosomal AmpC {beta}-lactamase with an unusual basal expression in contrast to one of the major features of S. marcescens. Persistent Serratia sink strains are identical to those involved in clonal and polyclonal outbreaks of VIM-1 and OXA-48 producers since at least 2017. The "source-sink" dynamics of SMC populations able to acquire the highly conserved plasmids such as IncL carrying blaVIM-1 or blaOXA-48 offer novel insights that might improve interventions to control outbreaks and treat Serratia infections in high-risk hospital areas.

microbiology↗

bioRxiv2021

An amphipathic and cationic antimicrobial peptide kills colistin resistant Gram-negative pathogens in vivo

New antibiotics are needed against multidrug resistant Gram-negative pathogens that have compromised global health systems. Antimicrobial peptides are generally considered promising lead candidates for the next generation of antibiotics but have not fulfilled this expectation. Here we demonstrate activity of a cationic amphipathic undecapeptide (ChIP; Charge change Independent Peptide) against a wide panel of multidrug resistant Gram-negative pathogens. Importantly, the antimicrobial activity of ChIP is independent of the surface charge changes that confer colistin resistance through modification of Lipid A, while decreased activity of ChIP correlates with GlcN1 tri-acylation of Lipid A. In an in vivo peritonitis mouse model ChIP displays excellent activity against both colistin sensitive and resistant Escherichia coli and Acinetobacter baumannii strains. Author SummaryAntimicrobial peptides hold promise as novel treatment options for diseases caused by multidrug resistant bacteria. Here we present evidence that the ChIP peptide, comprised of 11 D-amino acids, is active against a variety of Gram-negative bacteria that ranks high on the WHO list of critically important pathogens. ChIP initially interacts with the Gram-negative outer membrane, independent of its surface charge, followed by entry into the periplasm and permeabilization of the inner membrane, leading to bacterial cell death. Detailed analyses of the outer membrane indicate that the acylation pattern of lipopolysaccharides plays an important role for ChIP activity. In a mouse infection model, ChIP display excellent activity in reducing bacterial numbers for both Escherichia coli and Acinetobacter baumannii. Importantly, ChIP is highly efficient against bacteria resistant to colistin, an antibiotic normally considered as a last resort treatment of infections caused by multidrug resistant Gram-negative bacteria.

microbiology↗

bioRxiv2020

MINTyper: A method for generating phylogenetic distance matrices with long read sequencing data

For detection of clonal outbreaks in clinical settings, we present a complete pipeline that generates a SNP-distance matrix from a set of sequencing reads. Importantly, the program is able to handle a separate mix of both short reads from the Illumina sequencing platforms and long reads from Oxford Nanopore Technologies (ONT) platforms as input. MINTyper performs automated reference identification, alignment, alignment trimming, optional methylation masking and pairwise distance calculations. With this approach, we could rapidly and accurately cluster a set of DNA sequenced isolates, with a known epidemiological relationship to confirm the clustering. Functions were built to allow for both high-accuracy methylation-aware base-called MinION reads (hac_m Q10) and fast generated lower-quality reads (fast Q8) to be used, also in combination with Illumina data. With fast Q8 reads a higher number of base pairs were excluded from the calculated distance matrix, compared to the high-accuracy methylation-aware Q10 base-calling of ONT data. Nonetheless, when using different qualities of ONT data with corresponding input parameters, the clustering of isolates were nearly identical.

bioinformatics↗

bioRxiv2019

Identification of 2CS-CHXT operon signature of chlorhexidine tolerance among Enterococcus faecium

Chlorhexidine (CHX) is a broad-spectrum antiseptic widely used in community and clinical contexts for many years, recently acquiring higher relevance in nosocomial infections control worldwide. Despite of this, CHX tolerance has been poorly understood among Enterococcus faecium, one of the leading agents causing nosocomial infections. This study provides new phenotypic and molecular data for a better identification of CHX tolerant E. faecium subpopulations in community and clinical contexts. MICCHX distribution of 106 E. faecium suggested the occurrence of tolerant subpopulations in diverse sources (human, animal, food, environment) and phylogenomic backgrounds (clades A1/A2/B), with predominance in clade A1. They carried a specific variant of 2CS-CHXT operon, here identified. It encodes a glucose and an amino-acid-polyamine-organocation family transporters, besides the DNA-binding-response-regulator ChtR with a P102H mutation previously described in only CHX tolerant clade A1 E. faecium, and the ChtS sensor. Combined data from normal MIC distribution and 2CS-CHXT operon characterization supports a tentative epidemiological cut-off (ECOFF) of 8 mg/L to CHX, useful to detect tolerant E. faecium populations in future surveillance studies. The spread of tolerant E. faecium in diverse epidemiological backgrounds alerts for a prudent use of CHX in multiple contexts.\n\nImportanceChlorhexidine is one of the substances included in the World Health Organizations List of Essential Medicines which comprises the safest and most effective medicines needed in global health systems. Although it has been widely applied as a disinfectant and antiseptic since the 1950s in healthcare (skin, hands, mouthwashes, eye drops), its use in hospitals to prevent nosocomial infections has increased worldwide in recent years. Here we provide a comprehensive study on chlorhexidine tolerance among Enterococcus faecium, one of the leading nosocomial agents worldwide, and identified a novel 2CS-CHXT operon as a signature of tolerant strains occurring in diverse phylogenomic groups. Finally, our data allowed to propose a tentative epidemiological cut-off of 8 mg/L, useful to detect tolerant E. faecium populations in future surveillance studies in community and clinical contexts.

microbiology↗

bioRxiv2019

Complete genome assembly of clinical multidrug resistant Bacteroides fragilis isolates enables comprehensive identification of antimicrobial resistance genes and plasmids

Bacteroides fragilis constitutes a significant part of the normal human gut microbiota and can also act as an opportunistic pathogen. Antimicrobial resistance and the prevalence of antimicrobial resistance genes are increasing, and prediction of antimicrobial susceptibility based on sequence information could support targeted antimicrobial therapy in a clinical setting. Complete identification of insertion sequence (IS) elements carrying promoter sequences upstream of resistance genes is necessary for prediction of antimicrobial resistance. However, de novo assemblies from short reads alone are often fractured due to repeat regions and the presence multiple copies of identical IS elements. Identification of plasmids in clinical isolates can aid in the surveillance of the dissemination of antimicrobial resistance and comprehensive sequence databases support microbiome and metagenomic studies. Here we test several short-read, hybrid and long-lead assembly pipelines by assembling the type strain B. fragilis CCUG4856T (=ATCC25285=NCTC9343) with Illumina short reads and long reads generated by Oxford Nanopore Technologies (ONT) MinION sequencing. Hybrid assembly with Unicycler, using quality filtered Illumina reads and Filtlong filtered and Canu corrected ONT reads produced the assembly of highest quality. This approach was then applied to six clinical multidrug resistant B. fragilis isolates and, with minimal manual finishing of chromosomal assemblies of three isolates, complete, circular assemblies of all isolates were produced. Eleven circular, putative plasmids were identified in the six assemblies of which only three corresponded to a known cultured Bacteroides plasmid. Complete IS elements could be identified upstream of antimicrobial resistance genes, however there was not complete correlation between the absence of IS elements and antimicrobial susceptibility. As our knowledge on factors that increase expression of resistance genes in the absence of IS elements is limited, further research is needed prior to implementing antimicrobial resistance prediction for B. fragilis from whole genome sequencing.\n\nREPOSITORIESSequence files (MinION reads de-multiplexed with Deepbinner and basecalled with Albacore in fast5 format and Illumina MiSeq reads in fastq format) and final genome assemblies have been deposited to NCBI/ENA/DDBJ under Bioproject accessions PRJNA525024, PRJNA244942, PRJNA244943, PRJNA244944, PRJNA253771, PRJNA254401, and PRJNA254455\n\nIMPACT STATEMENTBacterial whole genome sequencing is increasingly used in public health, clinical, and research laboratories for typing, identification of virulence factors, phylogenomics, outbreak investigation and identification of antimicrobial resistance genes. In some settings, diagnostic microbiome amplicon sequencing or metagenomic sequencing directly from clinical samples is already implemented and informs treatment decisions. The prospect of prediction of antimicrobial susceptibility based on resistome identification holds promises for shortening time from sample to report and informing treatment decisions. Databases with comprehensive reference sequences of high quality are a necessity for these purposes. Bacteroides fragilis is an important part of the human commensal gut microbiota and is also the most commonly isolated anaerobic bacterium from non-faecal clinical samples but few complete genome assemblies are available through public databases. The fragmented assemblies from short read de novo assembly often negate the identification of insertion sequences upstream of antimicrobial resistance gens, which is necessary for prediction of antimicrobial resistance from whole genome sequencing. Here we test multiple assembly pipelines with short read Illumina data and long read data from Oxford Nanopore Technologies MinION sequencing to select an optimal pipeline for complete genome assembly of B. fragilis. However, B. fragilis is a highly plastic genome with multiple inversive repeat regions, and complete genome assembly of six clinical multidrug resistant isolates still required minor manual finishing for half the isolates. Complete identification of known insertion sequences and resistance genes was possible from the complete genome. In addition, the current catalogue of Bacteroides plasmid sequences is augmented by eight new plasmid sequences that do not have corresponding, complete entries in the NCBI database. This work almost doubles the number of publicly available complete, finished chromosomal and plasmid B. fragilis sequences paving the way for further studies on antimicrobial resistance prediction and increased quality of microbiome and metagenomic studies.\n\nDATA SUMMARYO_LISequence read files (Oxford Nanopore (ONT) fast5 files and Illumina fastq files) as well as the final genome assemblies have been deposited to NCBI/ENA/DDBJ under Bioproject accessions PRJNA525024, PRJNA244942, PRJNA244943, PRJNA244944, PRJNA253771, PRJNA254401, and PRJNA254455.\nC_LIO_LIFastq format of demultiplexed ONT reads trimmed of adapters and barcode sequences are available at doi.org/10.5281/zenodo.2677927\nC_LIO_LIGenome assemblies from the assembly pipeline validation are available at doi: doi.org/10.5281/zenodo.2648546.\nC_LIO_LIGenome assemblies corresponding to each stage of the process of the assembly are available at doi.org/10.5281/zenodo.2661704.\nC_LIO_LIFull commands and scripts used are available from GitHub: https://github.com/thsyd/bfassembly as well as a static version at doi.org/10.5281/zenodo.2683511\nC_LI

microbiology↗

bioRxiv2018

Emergence of enteroaggregative Escherichia coli within the ST131 lineage as a cause of extraintestinal infections

Escherichia coli sequence type 131 (ST131) is a major cause of urinary and bloodstream infections and its association with extended-spectrum {beta}-lactamases (ESBL) significantly complicates treatment. Most notorious is its rapidly expanding H30-Rx clade (named for containing allele 30 of the type-1 fimbrial adhesin gene fimH and extensive antimicrobial resistance), which appears to have emerged in the United States due in part due to the acquisition of the ESBL-encoding blaCTX-M-15 gene and resistance to fluoroquinolones. However, non-H30 ST131 lineages with acquired CTX-M-type resistance genes also are emerging. Based on whole-genome analyses, we describe here the presence of an (fimH) H27 E. coli ST131 lineage that currently is causing an outbreak of community-acquired bacteremia and recurrent urinary tract infections (UTIs) in Denmark. This lineage has acquired both a virulence plasmid (pAA) that defines the enteroaggregative E. coli (EAEC) diarrheagenic pathotype and multiple genes associated with extraintestinal E. coli (ExPEC) that combined has made this particular ST131 lineage highly successful at colonizing its human host and cause recurrent UTI. Moreover, using a historic World Health Organization E. coli collection and publically available genome sequences, we identify a global H27 EAEC ST131 lineage dating back as far as 1998. Most H27 EAEC ST131 isolates harbor pAA or pAA-like plasmids, which analysis strongly imply was caused by a single ancestral acquisition. These findings illustrate the profound plasticity of this important pathogenic E. coli H27 lineage in general, and the genetic acquisitions of EAEC-specific virulence traits that likely confer an enhanced ability to cause intestinal colonization.\n\nImportanceThe E. coli ST131 lineage is a notorious extraintestinal pathogen. A signature characteristic of ST131 is its ability to asymptomatically colonize the gastrointestinal tract and then opportunistically cause extraintestinal infections, such as cystitis, pyelonephritis and urosepsis. In this study, we report a novel ST131 sublineage that has acquired the enteroaggregative diarrheagenic phenotype, spread across multiple continents and has been associated with multiple outbreaks of community-acquired bloodstream infections in Denmark. The strains ability to both cause diarrhea and colonize the human gastrointestinal tract may facilitate its dissemination and establishment in the community, whereas the strains clonal nature may facilitate targeted control strategies, such as vaccination.

microbiology↗