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In virtually all eukaryotes, the mitochondrial DNA (mtDNA) encodes proteins necessary for oxidative phosphorylation (OXPHOS) and RNAs required for their synthesis. The mechanisms of regulation of mtDNA copy number and expression are not completely understood but crucially ensure the correct stoichiometric assembly of OXPHOS complexes from nuclear- and mtDNA-encoded subunits. Here, we detect adenosine N6-methylation (6mA) on the mtDNA of diverse animal and plant species. This modification is regulated in C. elegans by the DNA methyltransferase DAMT-1 and demethylase ALKB-1. Misregulation of mtDNA 6mA through targeted modulation of these activities inappropriately alters mtDNA copy number and transcript levels, impairing OXPHOS function, elevating oxidative stress, and shortening lifespan. Compounding these defects, mtDNA 6mA hypomethylation promotes the cross-generational propagation of a deleterious mtDNA. Together, these results reveal that mtDNA 6mA is highly conserved among eukaryotes and regulates lifespan by influencing mtDNA copy number, expression, and heritable mutation levels in vivo.
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We describe the demographics, clinical and molecular epidemiology of extended-spectrum ß-lactamase (ESBL) Escherichia coli bloodstream infections (BSI) in Central Australia. All ESBL-producing E. coli bloodstream isolates from January 2018 to December 2020 were retrospectively identified. Demographic and clinical information was extracted by chart review. Whole-genome sequencing was performed for multi-locus sequence typing, antibiotic-resistance genes, and phylogenetic relationships. We identified 41 non-duplicate episodes of ESBL E. coli BSI. Median age was 55 years (IQR 47-63), 78% were female, 93% were Aboriginal, and half came from a remote community. Infections were predominantly urinary (68%, 28/41). In the 12 months prior, 70% (26/37) of identified patients had been hospitalised and 81% (30/37) prescribed antibiotics. Meropenem and piperacillin-tazobactam susceptibility was maintained in 100% and 95% of isolates, respectively. Co-resistance to non-ß-lactam antibiotics was 32% to gentamicin, 61% to trimethoprim/sulfamethoxazole, and 68% to ciprofloxacin. For sequenced isolates, 41% (16/35) were sequence type 131 (ST131). Mean acquired antibiotic-resistance genes for each isolate was 12.3 (SD 3.1). Four isolates carried an OXA-1 gene. Only non-ST131 isolates carried AmpC and acquired quinolone-resistance genes. There was some evidence of clustering of closely related strains, but no evidence of community or healthcare admission overlap. ESBL rates are rapidly rising in Central Australia, which is a conducive environment for antibiotic resistance development (e.g., overcrowding, socioeconomic disadvantages, high healthcare exposure and high antibiotic use). Future research is required to explore resistance-transmission dynamics in this unique setting.
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Background:Burkholderia pseudomallei, the causative agent of melioidosis, is highly genetically recombinant, resulting in significant genomic diversity. Multiple virulence factors have been associated with specific disease presentations. To date, there are limited data relating to genomic diversity and virulence factors associated with melioidosis cases in North Queensland, Australia. Aim: To describe the genetic diversity of B. pseudomallei and identify virulence factors associated with clinical risk factors and patient outcomes. Methods: Whole genome sequencing of clinical isolates was performed and analysed with clinical data obtained from a retrospective melioidosis cohort study. Results: Fifty-nine distinct sequence types (STs) were identified from the 128 clinical isolates. Six STs comprised 64/128 (50%) isolates. Novel STs accounted for 38/59 (64%) STs, with ST TSV-13 as the most prevalent (n = 7), and were less likely to possess an LPS A genotype or YLF gene cluster (p < 0.001). These isolates were most likely to be found outside the inner city (aOR: 4.0, 95% CI: 1.7-9.0, p = 0.001). ST TSV-13 was associated with increased mortality (aOR: 6.1, 95% CI: 1.2-30.9, p = 0.03). Patients with a history of alcohol excess were less likely to be infected by fhaB3 (aOR 0.2, 95% CI: 0.1-0.7, p = 0.01) or YLF (aOR: 0.4, 95% CI: 0.2-0.9, p = 0.04) positive isolates. Conclusions: There are a significant number of novel sequence types in Townsville, Australia. An emerging novel ST appears to have an association with geographic location and mortality. Ongoing investigation is required to further understand the impact of this ST on the Townsville region.
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Neonatal meningitis is a devastating disease associated with high mortality and neurological sequelae. Escherichia coli is the second most common cause of neonatal meningitis in full-term infants (herein NMEC) and the most common cause of meningitis in preterm neonates. Here, we investigated the genomic relatedness of a collection of 58 NMEC isolates spanning 1974-2020 and isolated from seven different geographic regions. We show NMEC are comprised of diverse sequence types (STs), with ST95 (34.5%) and ST1193 (15.5%) the most common. No single virulence gene profile was conserved in all isolates; however, genes encoding fimbrial adhesins, iron acquisition systems, the K1 capsule, and O antigen types O18, O75, and O2 were most prevalent. Antibiotic resistance genes occurred infrequently in our collection. We also monitored the infection dynamics in three patients that suffered recrudescent invasive infection caused by the original infecting isolate despite appropriate antibiotic treatment based on antibiogram profile and resistance genotype. These patients exhibited severe gut dysbiosis. In one patient, the causative NMEC isolate was also detected in the fecal flora at the time of the second infection episode and after treatment. Thus, although antibiotics are the standard of care for NMEC treatment, our data suggest that failure to eliminate the causative NMEC that resides intestinally can lead to the existence of a refractory reservoir that may seed recrudescent infection.
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Infecciones por Escherichia coli , Meningitis , Recién Nacido , Humanos , Escherichia coli/genética , Virulencia/genética , Células ClonalesRESUMEN
PURPOSE: Early recognition and effective treatment of sepsis improves outcomes in critically ill patients. However, antibiotic exposures are frequently suboptimal in the intensive care unit (ICU) setting. We describe the feasibility of the Bayesian dosing software Individually Designed Optimum Dosing Strategies (ID-ODS™), to reduce time to effective antibiotic exposure in children and adults with sepsis in ICU. METHODS: A multi-centre prospective, non-randomised interventional trial in three adult ICUs and one paediatric ICU. In a pre-intervention Phase 1, we measured the time to target antibiotic exposure in participants. In Phase 2, antibiotic dosing recommendations were made using ID-ODS™, and time to target antibiotic concentrations were compared to patients in Phase 1 (a pre-post-design). RESULTS: 175 antibiotic courses (Phase 1 = 123, Phase 2 = 52) were analysed from 156 participants. Across all patients, there was no difference in the time to achieve target exposures (8.7 h vs 14.3 h in Phase 1 and Phase 2, respectively, p = 0.45). Sixty-one courses in 54 participants failed to achieve target exposures within 24 h of antibiotic commencement (n = 36 in Phase 1, n = 18 in Phase 2). In these participants, ID-ODS™ was associated with a reduction in time to target antibiotic exposure (96 vs 36.4 h in Phase 1 and Phase 2, respectively, p < 0.01). These patients were less likely to exhibit subtherapeutic antibiotic exposures at 96 h (hazard ratio (HR) 0.02, 95% confidence interval (CI) 0.01-0.05, p < 0.01). There was no difference observed in in-hospital mortality. CONCLUSIONS: Dosing software may reduce the time to achieve target antibiotic exposures. It should be evaluated further in trials to establish its impact on clinical outcomes.
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Antibacterianos , Sepsis , Adulto , Niño , Humanos , Antibacterianos/uso terapéutico , Teorema de Bayes , Enfermedad Crítica/terapia , Unidades de Cuidado Intensivo Pediátrico , Estudios Prospectivos , Sepsis/tratamiento farmacológico , Programas InformáticosRESUMEN
The past decade has seen an increase in the prevalence of sequence type (ST) 45 methicillin-resistant Staphylococcus aureus (MRSA), yet the underlying drivers for its emergence and spread remain unclear. To better understand the worldwide dissemination of ST45 S. aureus, we performed phylogenetic analyses of Australian isolates, supplemented with a global population of ST45 S. aureus genomes. Our analyses revealed a distinct lineage of multidrug-resistant ST45 MRSA harbouring qacA, predominantly found in Australia and Singapore. Bayesian inference predicted that the acquisition of qacA occurred in the late 1990s. qacA was integrated into a structurally variable region of the chromosome containing Tn552 (carrying blaZ) and Tn4001 (carrying aac(6')-aph(2")) transposable elements. Using mutagenesis and in vitro assays, we provide phenotypic evidence that qacA confers tolerance to chlorhexidine. These findings collectively suggest both antimicrobial resistance and the carriage of qacA may play a role in the successful establishment of ST45 MRSA.
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Staphylococcus aureus Resistente a Meticilina , Infecciones Estafilocócicas , Humanos , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus/genética , Teorema de Bayes , Filogenia , Infecciones Estafilocócicas/epidemiología , Proteínas de Transporte de Membrana/genética , Proteínas Bacterianas/genética , AustraliaRESUMEN
Existing tools for phylogeographic and epidemiological visualisation primarily provide a macro-geographic view of epidemic and pandemic transmission events but offer little support for detailed investigation of outbreaks in healthcare settings. Here, we present HAIviz, an interactive web-based application designed for integrating and visualising genomic epidemiological information to improve the tracking of healthcare-associated infections (HAIs). HAIviz displays and links the outbreak timeline, building map, phylogenetic tree, patient bed movements, and transmission network on a single interactive dashboard. HAIviz has been developed for bacterial outbreak investigations but can be utilised for general epidemiological investigations focused on built environments for which visualisation to customised maps is required. This paper describes and demonstrates the application of HAIviz for HAI outbreak investigations.
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Infección Hospitalaria , Genómica , Humanos , Filogenia , Brotes de Enfermedades , Infección Hospitalaria/epidemiología , PandemiasRESUMEN
The ability of Mycobacterium tuberculosis (Mtb) to persist in the host complicates and prolongs tuberculosis (TB) patient chemotherapy. Here we demonstrate that a neglected two-component system (TCS) of Mtb, TcrXY, is an autoregulated acid-sensing TCS that controls a functionally diverse 70-gene regulon required for bacterial persistence. Characterisation of two representatives of this regulon, Rv3706c and Rv3705A, implicate these genes as key determinants for the survival of Mtb in vivo by serving as important effectors to mitigate redox stress at acidic pH. We show that genetic silencing of the response regulator tcrX using CRISPR interference attenuates the persistence of Mtb during chronic mouse infection and improves treatment with the two front-line anti-TB drugs, rifampicin and isoniazid. We propose that targeting TcrXY signal transduction blocks the ability of Mtb to sense and respond to acid stress, resulting in a disordered program of persistence to render the organism vulnerable to existing TB chemotherapy.
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Genes Bacterianos , Mycobacterium tuberculosis , Animales , Humanos , Ratones , Antituberculosos/química , Genes Bacterianos/fisiología , Isoniazida , Mycobacterium tuberculosis/genética , Infección Persistente , RifampinRESUMEN
Antibiotic resistance is a significant global public health concern. Uropathogenic Escherichia coli sequence type (ST)131, a widely prevalent multidrug-resistant clone, is frequently associated with bacteraemia. This study investigates third-generation cephalosporin resistance in bloodstream infections caused by E. coli ST131. From 2013-2014 blood culture surveillance in Wales, 142 E. coli ST131 genomes were studied alongside global data. All three major ST131 clades were represented across Wales, with clade C/H30 predominant (n = 102/142, 71.8%). Consistent with global findings, Welsh strains of clade C/H30 contain ß-lactamase genes from the blaCTX-M-1 group (n = 65/102, 63.7%), which confer resistance to third-generation cephalosporins. Most Welsh clade C/H30 genomes belonged to sub-clade C2/H30Rx (58.3%). A Wales-specific sub-lineage, named GB-WLS.C2, diverged around 1996-2000. An introduction to North Wales around 2002 led to a localised cluster by 2009, depicting limited genomic diversity within North Wales. This investigation emphasises the value of genomic epidemiology, allowing the detection of genetically similar strains in local areas, enabling targeted and timely public health interventions.
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Bacteriemia , Infecciones por Escherichia coli , Proteínas de Escherichia coli , Humanos , Escherichia coli , Infecciones por Escherichia coli/epidemiología , Gales/epidemiología , Genotipo , Proteínas de Escherichia coli/genética , Genómica , beta-Lactamasas/genética , Bacteriemia/epidemiología , Análisis por Conglomerados , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Farmacorresistencia Bacteriana Múltiple/genéticaRESUMEN
We aimed to evaluate the performance of Oxford Nanopore Technologies (ONT) sequencing from positive blood culture (BC) broths for bacterial identification and antimicrobial susceptibility prediction. Patients with suspected sepsis in four intensive care units were prospectively enrolled. Human-depleted DNA was extracted from positive BC broths and sequenced using ONT (MinION). Species abundance was estimated using Kraken2, and a cloud-based system (AREScloud) provided in silico predictive antimicrobial susceptibility testing (AST) from assembled contigs. Results were compared to conventional identification and phenotypic AST. Species-level agreement between conventional methods and AST predicted from sequencing was 94.2% (49/52), increasing to 100% in monomicrobial infections. In 262 high-quality AREScloud AST predictions across 24 samples, categorical agreement (CA) was 89.3%, with major error (ME) and very major error (VME) rates of 10.5% and 12.1%, respectively. Over 90% CA was achieved for some taxa (e.g., Staphylococcus aureus) but was suboptimal for Pseudomonas aeruginosa. In 470 AST predictions across 42 samples, with both high quality and exploratory-only predictions, overall CA, ME, and VME rates were 87.7%, 8.3%, and 28.4%. VME rates were inflated by false susceptibility calls in a small number of species/antibiotic combinations with few representative resistant isolates. Time to reporting from sequencing could be achieved within 8-16 h from BC positivity. Direct sequencing from positive BC broths is feasible and can provide accurate predictive AST for some species. ONT-based approaches may be faster but significant improvements in accuracy are required before it can be considered for clinical use.IMPORTANCESepsis and bloodstream infections carry a high risk of morbidity and mortality. Rapid identification and susceptibility prediction of causative pathogens, using Nanopore sequencing direct from blood cultures, may offer clinical benefit. We assessed this approach in comparison to conventional phenotypic methods and determined the accuracy of species identification and susceptibility prediction from genomic data. While this workflow holds promise, and performed well for some common bacterial species, improvements in sequencing accuracy and more robust predictive algorithms across a diverse range of organisms are required before this can be considered for clinical use. However, results could be achieved in timeframes that are faster than conventional phenotypic methods.
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Secuenciación de Nanoporos , Sepsis , Humanos , Cultivo de Sangre/métodos , Pruebas de Sensibilidad Microbiana , Sepsis/microbiología , Antibacterianos , Cuidados CríticosRESUMEN
BACKGROUND: The clinical and genomic epidemiology of melioidosis varies across regions. AIM: To describe the clinical and genetic diversity of B. pseudomallei across Queensland, Australia. METHODS: Whole genome sequencing of clinical isolates stored at the melioidosis reference lab from 1996-2020 was performed and analysed in conjunction with available clinical data. RESULTS: Isolates from 292 patients were analysed. Bacteraemia was present in 71% and pneumonia in 65%. The case-fatality rate was 25%. Novel sequence types (ST) accounted for 51% of all isolates. No association was identified between the variable virulence factors assessed and patient outcome. Over time, the proportion of First Nation's patients declined from 59% to 26%, and the proportion of patients aged >70 years rose from 13% to 38%. CONCLUSION: This study describes a genomically diverse and comparatively distinct collection of B. pseudomallei clinical isolates from across Queensland, Australia. An increasing incidence of melioidosis in elderly patients may be an important factor in the persistently high case-fatality in this region and warrants further investigation and directed intervention.
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Burkholderia pseudomallei , Melioidosis , Humanos , Anciano , Melioidosis/epidemiología , Queensland/epidemiología , Burkholderia pseudomallei/genética , Australia/epidemiología , GenómicaRESUMEN
BACKGROUND: Cluster and transmission analysis utilising pairwise SNP distance are increasingly used in genomic epidemiological studies. However, current methods are often challenging to install and use, and lack interactive functionalities for easy data exploration. RESULTS: GraphSNP is an interactive visualisation tool running in a web browser that allows users to rapidly generate pairwise SNP distance networks, investigate SNP distance distributions, identify clusters of related organisms, and reconstruct transmission routes. The functionality of GraphSNP is demonstrated using examples from recent multi-drug resistant bacterial outbreaks in healthcare settings. CONCLUSIONS: GraphSNP is freely available at https://github.com/nalarbp/graphsnp . An online version of GraphSNP, including demonstration datasets, input templates, and quick start guide is available for use at https://graphsnp.fordelab.com .
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Genómica , Programas Informáticos , Genómica/métodos , Navegador Web , Genoma , Brotes de EnfermedadesRESUMEN
To investigate an outbreak of vancomycin-resistant Enterococcus faecium (VREfm) sequence type 78 (ST78) in a large tertiary Australian hospital. A collection of 63 VREfm ST78 isolates, identified during a routine genomic surveillance program, were subjected to genomic epidemiological analysis based on whole-genome sequencing (WGS) data. The population structure was reconstructed using phylogenetic analysis, and a collection of publicly available VREfm ST78 genomes were used to provide global context. Core genome single nucleotide polymorphism (SNP) distances and available clinical metadata were used to characterize outbreak clusters and reconstruct transmission events. In silico genotyping confirmed that all study isolates were vanB-type VREfm carrying virulence characteristics of the hospital-associated E. faecium. Phylogenetic analysis identified two distinct phylogenetic clades, only one of which was responsible for a hospital outbreak. Four outbreak subtypes could be defined with examples of recent transmissions. Inference on transmission trees suggested complex transmission routes with unknown environmental reservoirs mediating the outbreak. WGS-based cluster analysis with publicly available genomes identified closely related Australian ST78 and ST203 isolates, highlighting the capacity for WGS to resolve complex clonal relationships between the VREfm lineages. Whole genome-based analysis has provided a high-resolution description of an outbreak of vanB-type VREfm ST78 in a Queensland hospital. Combined routine genomic surveillance and epidemiological analysis have facilitated better understanding of the local epidemiology of this endemic strain, providing valuable insight for better targeted control of VREfm. IMPORTANCE Vancomycin-resistant Enterococcus faecium (VREfm) is a leading cause of health care-associated infections (HAIs) globally. In Australia, the spread of hospital-adapted VREfm is largely driven by a single clonal group (clonal complex [CC]), CC17, to which the lineage ST78 belongs. While implementing a genomic surveillance program in Queensland, we observed increased incidence of ST78 colonizations and infections among patients. Here, we demonstrate the use of real-time genomic surveillance as a tool to support and enhance infection control (IC) practices. Our results show that real-time whole-genome sequencing (WGS) can efficiently disrupt outbreaks by identifying transmission routes that in turn can be targeted using resource-limited interventions. Additionally, we demonstrate that by placing local outbreaks in a global context, high-risk clones can be identified and targeted prior to them becoming established within clinical environments. Finally, the persistence of these organism within the hospital highlights the need for routine genomic surveillance as a management tool to control VRE transmission.
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Infección Hospitalaria , Enterococcus faecium , Infecciones por Bacterias Grampositivas , Enterococos Resistentes a la Vancomicina , Humanos , Vancomicina , Enterococcus faecium/genética , Queensland/epidemiología , Centros de Atención Terciaria , Filogenia , Australia/epidemiología , Enterococos Resistentes a la Vancomicina/genética , Genómica , Brotes de Enfermedades , Infección Hospitalaria/epidemiología , Infecciones por Bacterias Grampositivas/epidemiologíaRESUMEN
Elizabethkingia meningoseptica is an uncommonly encountered multidrug-resistant gram-negative bacterium that causes infections primarily among vulnerable hosts. A true opportunistic pathogen, its ability to cause severe sepsis and complicated infection in selected patients has been noted. Very limited preclinical and clinical data exist with regard to suitable therapeutic options. In this study, we present the case of prolonged bloodstream and central nervous system infection due to E. meningoseptica treated with dose-optimized combination antibiotic therapy, with evidence of microbiological (including development of adaptive resistance mechanisms) and clinical failure.
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Chryseobacterium , Infecciones por Flavobacteriaceae , Sepsis , Humanos , Antibacterianos/farmacología , Infecciones por Flavobacteriaceae/tratamiento farmacológico , Infecciones por Flavobacteriaceae/microbiología , Pruebas de Sensibilidad Microbiana , Sepsis/tratamiento farmacológico , Sepsis/microbiología , Insuficiencia del TratamientoRESUMEN
BACKGROUND: Prospective whole-genome sequencing (WGS)-based surveillance may be the optimal approach to rapidly identify transmission of multi-drug resistant (MDR) bacteria in the healthcare setting. METHODS: We prospectively collected methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), carbapenem-resistant Acinetobacter baumannii (CRAB), extended-spectrum beta-lactamase (ESBL-E), and carbapenemase-producing Enterobacterales (CPE) isolated from blood cultures, sterile sites, or screening specimens across three large tertiary referral hospitals (2 adult, 1 paediatric) in Brisbane, Australia. WGS was used to determine in silico multi-locus sequence typing (MLST) and resistance gene profiling via a bespoke genomic analysis pipeline. Putative transmission events were identified by comparison of core genome single nucleotide polymorphisms (SNPs). Relevant clinical meta-data were combined with genomic analyses via customised automation, collated into hospital-specific reports regularly distributed to infection control teams. RESULTS: Over 4 years (April 2017 to July 2021) 2660 isolates were sequenced. This included MDR gram-negative bacilli (n = 293 CPE, n = 1309 ESBL), MRSA (n = 620), and VRE (n = 433). A total of 379 clinical reports were issued. Core genome SNP data identified that 33% of isolates formed 76 distinct clusters. Of the 76 clusters, 43 were contained to the 3 target hospitals, suggesting ongoing transmission within the clinical environment. The remaining 33 clusters represented possible inter-hospital transmission events or strains circulating in the community. In 1 hospital, proven negligible transmission of non-multi-resistant MRSA enabled changes to infection control policy. CONCLUSIONS: Implementation of routine WGS for MDR pathogens in clinical laboratories is feasible and can enable targeted infection prevention and control interventions.
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Infección Hospitalaria , Staphylococcus aureus Resistente a Meticilina , Adulto , Humanos , Niño , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Tipificación de Secuencias Multilocus , Infección Hospitalaria/epidemiología , Staphylococcus aureus Resistente a Meticilina/genética , Centros de Atención TerciariaRESUMEN
The application of direct metagenomic sequencing from positive blood culture broth may solve the challenges of sequencing from low-bacterial-load blood samples in patients with sepsis. Forty prospectively collected blood culture broth samples growing Gram-negative bacteria were extracted using commercially available kits to achieve high-quality DNA. Species identification via metagenomic sequencing and susceptibility prediction via a machine-learning algorithm (AREScloud) were compared to conventional methods and other rapid diagnostic platforms (Accelerate Pheno and blood culture identification [BCID] panel). A two-kit method (using MolYsis Basic and Qiagen DNeasy UltraClean kits) resulted in optimal extractions. Taxonomic profiling by direct metagenomic sequencing matched conventional identification in 38/40 (95%) samples. In two polymicrobial samples, a second organism was missed by sequencing. Prediction models were able to accurately infer susceptibility profiles for 6 common pathogens against 17 antibiotics, with an overall categorical agreement (CA) of 95% (increasing to >95% for 5/6 of the most common pathogens, if Klebsiella oxytoca was excluded). The performance of whole-genome sequencing (WGS)-antimicrobial susceptibility testing (AST) was suboptimal for uncommon pathogens (e.g., Elizabethkingia) and some ß-lactamase inhibitor antibiotics (e.g., ticarcillin-clavulanate). The time to pathogen identification was the fastest with BCID (1 h from blood culture positivity). Accelerate Pheno provided a susceptibility result in approximately 8 h. Illumina-based direct sequencing methods provided results in time frames similar to those of conventional culture-based methods. Direct metagenomic sequencing from blood cultures for pathogen detection and susceptibility prediction is feasible. Additional work is required to optimize algorithms for uncommon species and complex resistance genotypes as well as to streamline methods to provide more rapid results.
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Cultivo de Sangre , Ácidos Nucleicos , Cultivo de Sangre/métodos , Pruebas de Sensibilidad Microbiana , Antibacterianos/farmacología , FenotipoRESUMEN
OBJECTIVES: To develop instrument-free point-of-care methods using recombinase polymerase amplification (RPA) technology coupled with a simple lateral flow detection system to detect Neisseria gonorrhoeae and susceptibility to ciprofloxacin. METHODS: For identification of gonococcal infection, an RPA-based method was developed targeting the gonococcal porA pseudogene (NG-porA-RPA). For ciprofloxacin susceptibility, predictive WT sequences at codons 91 and 95 of the gonococcal gyrA DNase gene were targeted. Given the known complexities of SNP detection using RPA (e.g. the ability to accommodate mismatches) we trialled several different assays incorporating various additional non-template mismatches in the oligonucleotide sequences to reduce affinity for the mutant (resistant) gyrA sequences. Assays were evaluated using a bank of N. gonorrhoeae (nâ=â10) and non-gonococcal (nâ=â5) isolates and a panel of N. gonorrhoeae nucleic acid amplification test (NAAT)-positive clinical sample extracts (nâ=â40). RESULTS: The NG-porA-RPA assay was specific to N. gonorrhoeae and provided a positive percentage agreement (PPA) of 87.5% (35/40) compared with a commercial N. gonorrhoeae NAAT when applied to the 40 clinical sample extracts. For gyrA, the non-template bases successfully reduced banding intensity for double-mutant strains (mutations at both 91 and 95), but not for rarer single-mutant (91 only) strains. The most promising gyrA assay, NG-gyrA-RPA08, correctly detected 83% (25/30) of infections from NAAT-positive clinical samples confirmed to have WT gyrA sequences based on Sanger sequencing. CONCLUSIONS: These proof-of-concept data show that RPA technology has considerable promise for detecting N. gonorrhoeae and associated antibiotic susceptibility and would offer a diagnostic-based stewardship strategy identified as urgently needed by the WHO.
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Gonorrea , Neisseria gonorrhoeae , Humanos , Neisseria gonorrhoeae/genética , Ciprofloxacina/farmacología , Pruebas de Sensibilidad Microbiana , Farmacorresistencia Bacteriana , Antibacterianos/farmacología , Gonorrea/diagnósticoRESUMEN
Extra-intestinal pathogenic Escherichia coli (ExPEC) belong to a critical priority group of antibiotic resistant pathogens. ExPEC establish gut reservoirs that seed infection of the urinary tract and bloodstream, but the mechanisms of gut colonisation remain to be properly understood. Ucl fimbriae are attachment organelles that facilitate ExPEC adherence. Here, we investigated cellular receptors for Ucl fimbriae and Ucl expression to define molecular mechanisms of Ucl-mediated ExPEC colonisation of the gut. We demonstrate differential expression of Ucl fimbriae in ExPEC sequence types associated with disseminated infection. Genome editing of strains from two common sequence types, F11 (ST127) and UTI89 (ST95), identified a single nucleotide polymorphism in the ucl promoter that changes fimbriae expression via activation by the global stress-response regulator OxyR, leading to altered gut colonisation. Structure-function analysis of the Ucl fimbriae tip-adhesin (UclD) identified high-affinity glycan receptor targets, with highest affinity for sialyllacto-N-fucopentose VI, a structure likely to be expressed on the gut epithelium. Comparison of the UclD adhesin to the homologous UcaD tip-adhesin from Proteus mirabilis revealed that although they possess a similar tertiary structure, apart from lacto-N-fucopentose VI that bound to both adhesins at low-micromolar affinity, they recognize different fucose- and glucose-containing oligosaccharides. Competitive surface plasmon resonance analysis together with co-structural investigation of UcaD in complex with monosaccharides revealed a broad-specificity glycan binding pocket shared between UcaD and UclD that could accommodate these interactions. Overall, our study describes a mechanism of adaptation that augments establishment of an ExPEC gut reservoir to seed disseminated infections, providing a pathway for the development of targeted anti-adhesion therapeutics.
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Infecciones por Escherichia coli , Escherichia coli Patógena Extraintestinal , Adhesinas Bacterianas/metabolismo , Adhesinas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Infecciones por Escherichia coli/metabolismo , Escherichia coli Patógena Extraintestinal/genética , Escherichia coli Patógena Extraintestinal/metabolismo , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Humanos , Enfermedades Intestinales , Polisacáridos/metabolismoRESUMEN
Extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli are a global public-health concern. We evaluated the pharmacodynamic activity of piperacillin/tazobactam (TZP) dosing regimens against ESBL-producing versus non-ESBL-producing E. coli. Five E. coli clinical isolates were obtained from Bangladesh. Broth microdilution and whole-genome sequencing (WGS) were performed on the five studied isolates. Three TZP-susceptible ESBL-producing and two non-ESBL-producing E. coli were exposed to TZP regimens of 4.5 g every 6 h (q6h) and every 8 h (q8h) as a 30-min infusion in a dynamic hollow-fibre infection model over 7 days. The extent of bacterial killing was â¼4-5 log10 CFU/mL against ESBL-producing and non-ESBL-producing E. coli with TZP q6h and q8h regimens over the first 8 h. Bacterial killing was similar between two of three ESBL-producing (CTAP#168 and CTAP#169) and two non-ESBL-producing (CTAP#179 and CTAP#180) E. coli clinical isolates over the course of the experiment. ESBL-producing CTAP#173 E. coli was poorly killed (â¼1 log) compared with two non-ESBL-producing E. coli over 168 h. WGS revealed that ESBL-producing E. coli isolates co-harboured multiple antimicrobial resistance genes such as blaCTX-M-15, blaEC, blaOXA-1, blaTEM-1 and aac(6')-Ib-cr5. Overall, TZP q6h and q8h dosing regimens attained >3 log bacterial kill against all ESBL-producing or non-ESBL-producing E. coli within 24 h and maintained and prevented the emergence of resistance through the end of the experiment. In conclusion, TZP standard regimens resulted in similar bacterial killing and prevented the emergence of resistance against CTX-M-15-type ESBL-producing and non-ESBL-producing E. coli clinical isolates.