RESUMEN
OBJECTIVES: Recently, reports on antimicrobial-resistant Bacteroides and Prevotella isolates have increased in the Netherlands. This urged the need for a surveillance study on the antimicrobial susceptibility profile of Bacteroides, Phocaeicola, Parabacteroides and Prevotella isolates consecutively isolated from human clinical specimens at eight different Dutch laboratories. METHODS: Each laboratory collected 20-25 Bacteroides (including Phocaeicola and Parabacteroides) and 10-15 Prevotella isolates for 3â months. At the national reference laboratory, the MICs of amoxicillin, amoxicillin/clavulanic acid, piperacillin/tazobactam, meropenem, imipenem, metronidazole, clindamycin, tetracycline and moxifloxacin were determined using agar dilution. Isolates with a high MIC of metronidazole or a carbapenem, or harbouring cfiA, were subjected to WGS. RESULTS: Bacteroides thetaiotaomicron/faecis isolates had the highest MIC90 values, whereas Bacteroides fragilis had the lowest MIC90 values for amoxicillin/clavulanic acid, piperacillin/tazobactam, meropenem, imipenem and moxifloxacin. The antimicrobial profiles of the different Prevotella species were similar, except for amoxicillin, for which the MIC50 ranged from 0.125 to 16â mg/L for Prevotella bivia and Prevotella buccae, respectively. Three isolates with high metronidazole MICs were sequenced, of which one Bacteroides thetaiotaomicron isolate harboured a plasmid-located nimE gene and a Prevotella melaninogenica isolate harboured a nimA gene chromosomally.Five Bacteroides isolates harboured a cfiA gene and three had an IS element upstream, resulting in high MICs of carbapenems. The other two isolates harboured no IS element upstream of the cfiA gene and had low MICs of carbapenems. CONCLUSIONS: Variations in resistance between species were observed. To combat emerging resistance in anaerobes, monitoring resistance and conducting surveillance are essential.
Asunto(s)
Antiinfecciosos , Metronidazol , Humanos , Meropenem , Moxifloxacino , Países Bajos , Laboratorios , Bacteroides , Antibacterianos/farmacología , Carbapenémicos , Bacteroides fragilis , Imipenem , Pruebas de Sensibilidad Microbiana , Piperacilina , Tazobactam , Prevotella/genética , Amoxicilina , Ácido ClavulánicoRESUMEN
OBJECTIVES: Five human clinical multidrug-resistant (MDR) Bacteroides fragilis isolates, including resistance to meropenem and metronidazole, were recovered at different hospitals in the Netherlands between 2014 and 2020 and sent to the anaerobic reference laboratory for full characterization. METHODS: Isolates were recovered from a variety of clinical specimens from patients with unrelated backgrounds. Long- and short-read sequencing was performed, followed by a hybrid assembly to study the presence of mobile genetic elements (MGEs) and antimicrobial resistance genes (ARGs). RESULTS: A cfxA gene was present on a transposon (Tn) similar to Tn4555 in two isolates. In two isolates a novel Tn was present with the cfxA gene. Four isolates harbored a nimE gene, located on a pBFS01_2 plasmid. One isolate contained a novel plasmid carrying a nimA gene with IS1168. The tetQ gene was present on novel conjugative transposons (CTns) belonging to the CTnDOT family. Two isolates harbored a novel plasmid with tetQ. Other ARGs in these isolates, but not on an MGE, were: cfiA, ermF, mef(EN2), and sul2. ARGs harboured differed between isolates and corresponded with the observed phenotypic resistance. CONCLUSIONS: Novel CTns, Tns, and plasmids were encountered in the five MDR B. fragilis isolates, complementing our knowledge on MDR and horizontal gene transfer in anaerobic bacteria.
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Infecciones Bacterianas , Infecciones por Bacteroides , Humanos , Bacteroides fragilis/genética , Genes Bacterianos , Países Bajos , Secuencias Repetitivas Esparcidas , Antibacterianos/farmacología , Infecciones por Bacteroides/epidemiología , Infecciones por Bacteroides/microbiología , Pruebas de Sensibilidad MicrobianaRESUMEN
Current knowledge about the respiratory microbiome is mainly based on 16S ribosomal RNA gene sequencing. Newer sequencing approaches, such as metatranscriptomics, offer the technical ability to measure the viable microbiome response to environmental conditions such as smoking as well as to explore its functional role by investigating host-microbiome interactions. However, knowledge about its feasibility in respiratory microbiome research, especially in lung biopsies, is still very limited. RNA sequencing was performed in bronchial biopsies from clinically stable smokers (n = 5) and ex-smokers (n = 6) with COPD not using (inhaled) steroids. The Trinity assembler was used to assemble non-human reads in order to allow unbiased taxonomical and microbial transcriptional analyses. Subsequently, host-microbiome interactions were analyzed based on associations with host transcriptomic data. Ultra-low levels of microbial mass (0.009%) were identified in the RNA-seq data. Overall, no differences were identified in microbiome diversity or transcriptional profiles of microbial communities or individual microbes between COPD smokers and ex-smokers in the initial test dataset as well as a larger replication dataset. We identified an upregulated host gene set, related to the simultaneous presence of Bradyrhizobium, Roseomonas, Brevibacterium.spp., which were related to PERK-mediated unfolded protein response (UPR) and expression of the microRNA-155-5p. Our results show that metatranscriptomic profiling in bronchial biopsy samples from stable COPD patients yields ultra-low levels of microbial mass. Further, this study illustrates the potential of using transcriptional profiling of the host and microbiome to gain more insight into their interaction in the airways.
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MicroARNs , Microbiota , Enfermedad Pulmonar Obstructiva Crónica , Biopsia , Ex-Fumadores , Humanos , Microbiota/genética , Enfermedad Pulmonar Obstructiva Crónica/patología , FumadoresRESUMEN
BACKGROUND: Vancomycin-resistant enterococci (VRE), particularly Enterococcus faecium (VREfm), can cause serious nosocomial infections, and have been responsible for healthcare-associated outbreaks. Spreading of VREfm can occur both clonally and by the dissemination of mobile genetic elements. AIM: To report prospective analysis of whole-genome sequencing (WGS) data, including both core-genome multi-locus sequence typing (cgMLST) and transposon analysis, during a vanB VREfm outbreak. METHODS: Screening for vanB-positive VREfm isolates was performed by real-time polymerase chain reaction (PCR) on an overnight enriched broth and, if positive, subculture was performed. vanB-positive VREfm isolates underwent WGS. Generated data were used for molecular typing that was performed by cgMLST using SeqSphere. For transposon characterization, sequence data were mapped against the reference sequence of transposon Tn1549 using CLC Genomics Workbench, or de-novo assemblies were used for BLASTN comparisons. RESULTS: In total, 1358 real-time PCRs were performed. Two hundred and fifty-one specimens from 207 patients tested positive on PCR for vanB, of which 13 specimens obtained from six patients were identified as vanB VREfm positive on culture. These six patients harboured seven unique isolates belonging to four cluster types: CT118 (N=2), CT2483 (N=3), CT2500 (N=1) and CT2501 (N=1). Transposon analysis revealed the presence of an identical vanB-carrying transposon in the isolates cultured from all six patients that could be linked based on epidemiological data. CONCLUSION: A vanB VREfm outbreak occurred in the study hospital, including six patients with isolates belonging to four cluster types. In-depth transposon analysis revealed that dissemination of transposon Tn1549 rather than clonal spread was the cause of the outbreak.
Asunto(s)
Elementos Transponibles de ADN , Farmacorresistencia Bacteriana/genética , Enterococcus faecium , Infecciones por Bacterias Grampositivas , Enterococos Resistentes a la Vancomicina , Proteínas Bacterianas/genética , Brotes de Enfermedades , Enterococcus faecium/genética , Humanos , Tipificación de Secuencias Multilocus , Estudios Prospectivos , Vancomicina , Enterococos Resistentes a la Vancomicina/genética , Secuenciación Completa del GenomaRESUMEN
BACKGROUND: Carbapenemase-producing Enterobacterales (CPE) have rapidly emerged in Europe, being responsible for nosocomial outbreaks. AIM: Following an outbreak in the burn unit of Ghent University Hospital, we investigated whether CPE can spread between toilets through drain water and therefrom be transmitted to patients. METHODS: In 2017, the burn centre of our hospital experienced an outbreak of OXA-48-producing Klebsiella pneumoniae that affected five patients staying in three different rooms. Environmental samples were collected from the sink, shower, shower stretcher, hand rail of the bed, nursing carts, toilets, and drain water to explore a common source. Whole-genome sequencing and phylogenetic analysis was performed on K. pneumoniae outbreak isolates and two random K. pneumoniae isolates. FINDINGS: OXA-48-producing K. pneumoniae was detected in toilet water in four out of six rooms and drain water between two rooms. The strain persisted in two out of six rooms after two months of daily disinfection with bleach. All outbreak isolates belonged to sequence type (ST) 15 and showed isogenicity (<15 allele differences). This suggests that the strain may have spread between rooms by drain water. Unexpectedly, one random isolate obtained from a patient who became colonized while residing at the geriatric ward clustered with the outbreak isolates, suggesting the outbreak to be larger than expected. Daily application of bleach tended to be superior to acetic acid to disinfect toilet water; however, disinfection did not completely prevent the presence of carbapenemase-producing K. pneumoniae in toilet water. CONCLUSION: Toilet drain water may be a potential source of hospital room-to-room transmission of carbapenemase-producing K. pneumoniae.
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Aparatos Sanitarios/microbiología , Infección Hospitalaria/etiología , Hospitales , Infecciones por Klebsiella/transmisión , Klebsiella pneumoniae/aislamiento & purificación , Microbiología del Agua , Bélgica , Infección Hospitalaria/microbiología , Brotes de Enfermedades , Reservorios de Enfermedades/microbiología , Drenaje de Agua , Humanos , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/clasificación , Filogenia , Secuenciación Completa del Genoma , beta-Lactamasas/genéticaRESUMEN
We observed an increase in methicillin-susceptible Staphylococcus aureus (MSSA) infections at a Dutch neonatal intensive care unit. Weekly neonatal MSSA carriage surveillance and cross-sectional screenings of health care workers (HCWs) were available for outbreak tracing. Traditional clustering of MSSA isolates by spa typing and Multiple-Locus Variable number tandem repeat Analysis (MLVA) suggested that nosocomial transmission had contributed to the infections. We investigated whether whole-genome sequencing (WGS) of MSSA surveillance would provide additional evidence for transmission. MSSA isolates from neonatal infections, carriage surveillance, and HCWs were subjected to WGS and bioinformatic analysis for identification and localization of high-quality single nucleotide polymorphisms, and in-depth analysis of subsets of isolates. By measuring the genetic diversity in background surveillance, we defined transmission-level relatedness and identified isolates that had been unjustly assigned to clusters based on MLVA, while spa typing was concordant but of insufficient resolution. Detailing particular subsets of isolates provided evidence that HCWs were involved in multiple outbreaks, yet it alleviated concerns about one particular HCW. The improved resolution and accuracy of genomic outbreak analyses substantially altered the view on outbreaks, along with apposite measures. Therefore, inclusion of the circulating background population has the potential to overcome current issues in genomic outbreak inference.
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Infección Hospitalaria/epidemiología , Staphylococcus aureus Resistente a Meticilina/genética , Repeticiones de Minisatélite , Infecciones Estafilocócicas/epidemiología , Técnicas de Tipificación Bacteriana , Infección Hospitalaria/microbiología , Infección Hospitalaria/transmisión , Estudios Transversales , Brotes de Enfermedades , Humanos , Recién Nacido , Unidades de Cuidado Intensivo Neonatal , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Epidemiología Molecular , Polimorfismo de Nucleótido Simple , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/transmisión , Secuenciación Completa del GenomaRESUMEN
OBJECTIVES: The prevalence of resistance genes in two important anaerobic genera, Bacteroides and Prevotella, was assessed by applying PCR specifically directed to genes of interest. METHODS: A total of 101 Bacteroides spp. and 99 Prevotella spp. human clinical isolates were identified using MALDI-TOF MS. The presence of the resistance genes cfxA, cepA, cfiA, tetQ, ermF and nim, was assessed. Prevalence of resistance genes was compared with the phenotypic resistance against amoxicillin, clindamycin, meropenem and metronidazole. RESULTS: Even though the majority of the Bacteroides isolates (95.0%) showed resistance towards amoxicillin, only 52/101 of the isolates harboured one of the resistance genes, accounting for this resistance. Within the genus Prevotella the presence of cfxA (50/99) almost perfectly matched the amoxicillin resistance (48/99). No difference in prevalence of the ermF gene (16/101 and 9/99) and clindamycin resistance (16/101 and 10/99) was observed within Bacteroides and Prevotella, respectively. Two isolates of Prevotella were resistant to metronidazole. One harboured the nim gene. One metronidazole-susceptible isolate of Bacteroides harboured a nim gene. Within the Bacteroides and Prevotella genera, 6/101 strains and 5/99 isolates harboured three different resistance genes, respectively, among them tetQ. TetQ is often located on a conjugative transposon, increasing the chance of horizontal gene transfer between isolates. CONCLUSIONS: An unknown mechanism in Bacteroides non-fragilis isolates causes resistance to ß-lactam antibiotics. The fact that the prevalence of the tetQ gene among Prevotella is increasing and the existence of isolates harbouring three resistance genes are worrisome developments.
Asunto(s)
Bacteroides/genética , Farmacorresistencia Bacteriana/genética , Prevotella/genética , Antibacterianos/farmacología , Infecciones por Bacteroidaceae/epidemiología , Infecciones por Bacteroidaceae/microbiología , Bacteroides/efectos de los fármacos , Bacteroides/aislamiento & purificación , Genes Bacterianos/genética , Humanos , Pruebas de Sensibilidad Microbiana , Países Bajos/epidemiología , Prevalencia , Prevotella/efectos de los fármacos , Prevotella/aislamiento & purificaciónRESUMEN
Background: Vancomycin-resistant Enterococcus faecium (VREfm) has emerged as a nosocomial pathogen worldwide. The dissemination of VREfm is due to both clonal spread and spread of mobile genetic elements (MGEs) such as transposons. Objectives: We aimed to combine vanB-carrying transposon data with core-genome MLST (cgMLST) typing and epidemiological data to understand the pathways of transmission in nosocomial outbreaks. Methods: Retrospectively, 36 VREfm isolates obtained from 34 patients from seven VREfm outbreak investigations in 2014 were analysed. Isolates were sequenced on a MiSeq and a MinION instrument. De novo assembly was performed in CLC Genomics Workbench and the hybrid assemblies were obtained through Unicycler v0.4.1. Ridom SeqSphere+ was used to extract MLST and cgMLST data. Detailed analysis of each transposon and their integration points was performed using the Artemis Comparison Tool (ACT) and multiple blast analyses. Results: Four different vanB transposons were found among the isolates. cgMLST divided ST80 isolates into three cluster types (CTs); CT16, CT104 and CT106. ST117 isolates were divided into CT24, CT103 and CT105. Within VREfm isolates belonging to CT103, two different vanB transposons were found. In contrast, VREfm isolates belonging to CT104 and CT106 harboured an identical vanB transposon. Conclusions: cgMLST provides a high discriminatory power for the epidemiological analysis of VREfm. However, additional transposon analysis is needed to detect horizontal gene transfer. Combining these two methods allows investigation of both clonal spread as well as the spread of MGEs. This leads to new insights and thereby better understanding of the complex transmission routes in VREfm outbreaks.
Asunto(s)
Brotes de Enfermedades , Enterococcus faecium/genética , Transferencia de Gen Horizontal , Infecciones por Bacterias Grampositivas/transmisión , Secuencias Repetitivas Esparcidas , Enterococos Resistentes a la Vancomicina/genética , Técnicas de Tipificación Bacteriana , Infección Hospitalaria/epidemiología , Infección Hospitalaria/microbiología , Elementos Transponibles de ADN , Enterococcus faecium/clasificación , Enterococcus faecium/efectos de los fármacos , Genoma Bacteriano , Genotipo , Humanos , Tipificación de Secuencias Multilocus , Filogenia , Estudios Retrospectivos , Análisis de Secuencia de ADNRESUMEN
Objectives: In this study we assess the antibiotic resistance genes in three metronidazole-resistant Prevotella bivia clinical isolates. Methods: Strains were whole-genome sequenced. De novo assembly was performed and genes were annotated in RAST. Manual adjustments were made, when required, to the annotation and length of the genes. Results: In all three strains a novel nim gene, nimK, was encountered located on a mobile genetic element (MGE). The nimK gene was associated with an IS1380 family transposase. On the same MGE, genes encoding an efflux small MDR (SMR) transporter were present and were associated with a crp/fnr regulator. Conclusions: This is the first description of the presence of a novel nim gene in metronidazole-resistant P. bivia clinical isolates. This gene is co-located with an efflux SMR transporter on an MGE, which has been named Tn6456 (MG827401). The identification of these resistance genes on an MGE is worrisome, since this indicates the horizontal gene transfer of antibiotic and/or biocide resistance from one strain to the other.
Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana/genética , Secuencias Repetitivas Esparcidas , Proteínas de Transporte de Membrana/genética , Metronidazol/farmacología , Prevotella/efectos de los fármacos , Prevotella/genética , Infecciones por Bacteroidaceae/microbiología , ADN Bacteriano/genética , Genes Bacterianos , Genoma Bacteriano , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Secuenciación Completa del GenomaRESUMEN
OBJECTIVES: The increasing use of fosfomycin requires reliable susceptibility testing in clinical practice. The reference standard, agar dilution (AD), is rarely used in routine settings. The fosfomycin Etest (BioMérieux) is frequently used, although reading MICs can be hampered by the interpretation of the growth of macrocolonies in the inhibition zone. We investigated the interobserver (IO), interlaboratory (IL), and interobserver-interlaboratory (IOIL) agreement of the fosfomycin Etest and evaluated the agreement with AD. METHODS: Etests were performed for 57 extended spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae of four bacterial species (Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca and Enterobacter cloacae) in two laboratories. Photographs of fosfomycin Etests were interpreted by four observers following manufacturer's instructions. RESULTS: Essential agreement (EA) and categorical agreement (CA) between Etest and AD were 57% and 89% (κ-value 0.68), respectively, with an underestimation of Etest interpretations compared with AD of 0.26 (95% confidence interval [CI] 0.03-0.48) 2-fold dilutions. Between Etest observations, IO-EA and -CA were reached in 82% and 94% of comparisons; IL-EA and -CA in 38% and 85% of comparisons; and IOIL-EA and -CA in 40% and 85% of comparisons, respectively. Agreement of the Etest with AD and between Etests was better for E. coli than for other species. Ignoring all macrocolonies and haze during Etest interpretation improved the agreement with AD (CA κ-value 0.80) and between Etests (CA κ-value from 0.68 to 0.81). CONCLUSIONS: In this study on 57 ESBL-producing Enterobacteriaceae, IOIL agreement was low with an EA of 40% and a CA of 85%, affected most by IL agreement and to a lesser extent by IO agreement.
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Antibacterianos/farmacología , Pruebas Antimicrobianas de Difusión por Disco , Enterobacter cloacae/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Fosfomicina/farmacología , Klebsiella/efectos de los fármacos , Humanos , Variaciones Dependientes del Observador , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Bloodstream infections are a major cause of death with increasing incidence and severity. Blood cultures are still the reference standard for microbiological diagnosis, but are rather slow. Molecular methods can be used as add-on complementary assays. They can be useful to speed up microbial identification and to predict antimicrobial susceptibility, applied to direct blood samples or positive blood cultures. AIM: To review recent developments in molecular-based diagnostic platforms used for the identification of bloodstream infections, with a focus on assays performed directly on blood samples and positive blood cultures. SOURCES: Peer reviewed articles, conference abstracts, and manufacturers' websites. CONTENT: We give an update on recent developments of molecular methods in diagnosing BSIs. We first describe the currently available molecular methods to be used for positive blood cultures including: a) in situ hybridization-based methods; b) DNA-microarray-based hybridization technology; c) nucleic acid amplification-based methods; and d) combined methods. Subsequently, molecular methods applied directly to whole blood samples are discussed, including the use of nucleic acid amplification-based methods, T2 magnetic resonance-based methods, and metagenomics for diagnosing BSIs. IMPLICATIONS: Advances in molecular-based methods complementary to conventional blood culture diagnostics and antimicrobial stewardship programmes may optimize infection management by allowing rapid identification of pathogens and relevant antimicrobial resistance genes. Rapid diagnosis of the causing microorganism and relevant resistance determinants is important for early administration and modification of appropriate antimicrobial therapy. Ultimately, this may lead to improved quality and cost-effectiveness of health care, as well as reduced antimicrobial resistance selection.
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Bacteriemia/diagnóstico , Técnicas Bacteriológicas/métodos , Técnicas de Diagnóstico Molecular/métodos , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Cultivo de Sangre , HumanosRESUMEN
Bacteria often live together in complex communities. Insight into these microbial ecosystems is essential to make it possible to intervene when these ecosystems lead to disease. Bacteria do not only respond to their host, but they also affect each other, which may have far-reaching consequences for the course of the disease. In this article we describe that clinical isolates in a polymicrobial infection can be seen as ecosystems. These ecosystems often have properties that separate isolates do not have; they may, for example, be more virulent or more resistant to antibiotics. We therefore emphasise that the whole is greater than the sum of its parts, even for infections.
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Antibacterianos/farmacología , Bacterias/patogenicidad , Fenómenos Fisiológicos Bacterianos/efectos de los fármacos , Coinfección/microbiología , Interacciones Microbiota-Huesped/fisiología , Humanos , Interacciones Microbianas/fisiologíaRESUMEN
Shiga toxins (Stx) are the main virulence factor of a pathogroup of Escherichia coli strains that cause severe human diseases. These toxins are encoded in prophages (Stx prophages), and generally their expression depends on prophage induction. Several studies have reported high diversity among both Stx prophages and Stx. In particular, the toxin subtype Stx2a is associated with high virulence and HUS. Here, we report the genome of ArgO145, an inducible Stx2a prophage identified in a bovine O145:H- strain which produced high levels of Shiga toxin and Stx phage particles. The ArgO145 genome shared lambda phage organization, with recombination, regulation, replication, lysis, and head and tail structural gene regions, although some lambda genes encoding regulatory proteins could not be identified. Remarkably, some Stx2a phages of strains isolated from patients in other countries showed high similarity to ArgO145.
Asunto(s)
Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/veterinaria , Profagos/genética , Escherichia coli Shiga-Toxigénica/patogenicidad , Escherichia coli Shiga-Toxigénica/virología , Animales , Bovinos , HumanosRESUMEN
Porphyromonas gingivalis is one of the major oral pathogens implicated in the widespread inflammatory disorder periodontitis. Moreover, in recent years, P. gingivalis has been associated with the autoimmune disease rheumatoid arthritis. The peptidylarginine deiminase enzyme of P. gingivalis (PPAD) is a major virulence factor that catalyzes the citrullination of both bacterial and host proteins, potentially contributing to production of anticitrullinated protein antibodies. Considering that these antibodies are very specific for rheumatoid arthritis, PPAD appears to be a link between P. gingivalis, periodontitis, and the autoimmune disorder rheumatoid arthritis. PPAD was thus far considered unique among prokaryotes, with P. gingivalis being the only bacterium known to produce and secrete it. To challenge this hypothesis, we investigated the possible secretion of PPAD by 11 previously collected Porphyromonas isolates from a dog, 2 sheep, 3 cats, 4 monkeys, and a jaguar with periodontitis. Our analyses uncovered the presence of secreted PPAD homologues in 8 isolates that were identified as Porphyromonas gulae (from a dog, monkeys, and cats) and Porphyromonas loveana (from sheep). In all 3 PPAD-producing Porphyromonas species, the dominant form of the secreted PPAD was associated with outer membrane vesicles, while a minor fraction was soluble. Our results prove for the first time that the citrullinating PPAD exoenzyme is not unique to only 1 prokaryotic species. Instead, we show that PPAD is produced by at least 2 other oral pathogens.
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Porphyromonas/enzimología , Desiminasas de la Arginina Proteica/metabolismo , Animales , Western Blotting , Gatos , Perros , Electroforesis en Gel de Poliacrilamida , Haplorrinos , Panthera , Periodontitis/enzimología , Periodontitis/microbiología , Periodontitis/veterinaria , Filogenia , Porphyromonas/genética , Porphyromonas gingivalis/enzimología , Desiminasas de la Arginina Proteica/genética , Desiminasas de la Arginina Proteica/aislamiento & purificación , Análisis de Secuencia de ADN , OvinosRESUMEN
BACKGROUND: Next generation sequencing (NGS) is increasingly being used in clinical microbiology. Like every new technology adopted in microbiology, the integration of NGS into clinical and routine workflows must be carefully managed. AIM: To review the practical aspects of implementing bacterial whole genome sequencing (WGS) in routine diagnostic laboratories. SOURCES: Review of the literature and expert opinion. CONTENT: In this review, we discuss when and how to integrate whole genome sequencing (WGS) in the routine workflow of the clinical laboratory. In addition, as the microbiology laboratories have to adhere to various national and international regulations and criteria for their accreditation, we deliberate on quality control issues for using WGS in microbiology, including the importance of proficiency testing. Furthermore, the current and future place of this technology in the diagnostic hierarchy of microbiology is described as well as the necessity of maintaining backwards compatibility with already established methods. Finally, we speculate on the question of whether WGS can entirely replace routine microbiology in the future and the tension between the fact that most sequencers are designed to process multiple samples in parallel whereas for optimal diagnosis a one-by-one processing of the samples is preferred. Special reference is made to the cost and turnaround time of WGS in diagnostic laboratories. IMPLICATIONS: Further development is required to improve the workflow for WGS, in particular to shorten the turnaround time, reduce costs, and streamline downstream data analyses. Only when these processes reach maturity will reliance on WGS for routine patient management and infection control management become feasible, enabling the transformation of clinical microbiology into a genome-based and personalized diagnostic field.
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Enfermedades Transmisibles/diagnóstico , Pruebas Diagnósticas de Rutina/métodos , Técnicas Microbiológicas/métodos , Secuenciación Completa del Genoma/métodos , Flujo de Trabajo , HumanosRESUMEN
Health care of severe burn patients is highly specialized and may require international patient transfer. Burn patients have an increased risk of developing infections. Patients that have been hospitalized in countries where carbapenemase-producing microorganisms (CPMO) are endemic may develop infections that are difficult to treat. In addition, there is a risk on outbreaks with CPMOs in burn centers. This study underlines that burn patients may extensively be colonized with CPMOs, and it provides best practice recommendations regarding clinical microbiology and infection control. We evaluated CPMO-carriage and wound colonization in a burn patient initially treated in Romania, and transported to the Netherlands. The sequence types and acquired beta-lactamase genes of highly-resistant microorganisms were derived from next generation sequencing data. Next, we searched literature for reports on CPMOs in burn patients. Five different carbapenemase-producing isolates were cultured: two unrelated OXA-48-producing Klebsiella pneumoniae isolates, OXA-23-producing Acinetobacter baumanii, OXA-48-producing Enterobacter cloacae, and NDM-1-producing Providencia stuartii. Also, multi-drug resistant Pseudomonas aeruginosa isolates were detected. Among the sampling sites, there was high variety in CPMOs. We found 46 reports on CPMOs in burn patients. We listed the epidemiology of CPMOs by country of initial treatment, and summarized recommendations for care of these patients based on these reports and our study.
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Acinetobacter baumannii/aislamiento & purificación , Antibacterianos/uso terapéutico , Proteínas Bacterianas/metabolismo , Quemaduras/microbiología , Enterobacter cloacae/aislamiento & purificación , Klebsiella pneumoniae/aislamiento & purificación , Providencia/aislamiento & purificación , Pseudomonas aeruginosa/aislamiento & purificación , beta-Lactamasas/metabolismo , Acinetobacter baumannii/efectos de los fármacos , Colistina/uso terapéutico , Desastres , Enterobacter cloacae/efectos de los fármacos , Humanos , Kanamicina/uso terapéutico , Klebsiella pneumoniae/efectos de los fármacos , Linezolid/uso terapéutico , Pruebas de Sensibilidad Microbiana , Países Bajos , Ácido Penicilánico/análogos & derivados , Ácido Penicilánico/uso terapéutico , Piperacilina/uso terapéutico , Combinación Piperacilina y Tazobactam , Providencia/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Rumanía , Sulfadiazina de Plata/uso terapéuticoAsunto(s)
Brotes de Enfermedades , Infecciones por Klebsiella/epidemiología , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/aislamiento & purificación , beta-Lactamasas/genética , Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple , Genoma Bacteriano , Grecia/epidemiología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/enzimología , Estudios Retrospectivos , beta-Lactamasas/biosíntesisRESUMEN
Shiga toxin-producing Escherichia coli (STEC) is a significant cause of gastrointestinal infection and the haemolytic-uremic syndrome (HUS). STEC outbreaks are commonly associated with food but animal contact is increasingly being implicated in its transmission. We report an outbreak of STEC affecting young infants at a nursery in a rural community (three HUS cases, one definite case, one probable case, three possible cases and five carriers, based on the combination of clinical, epidemiological and laboratory data) identified using culture-based and molecular techniques. The investigation identified repeated animal contact (animal farming and petting) as a likely source of STEC introduction followed by horizontal transmission. Whole genome sequencing (WGS) was used for real-time investigation of the incident and revealed a unique strain of STEC O26:H11 carrying stx2a and intimin. Following a public health intervention, no additional cases have occurred. This is the first STEC outbreak reported from Israel. WGS proved as a useful tool for rapid laboratory characterization and typing of the outbreak strain and informed the public health response at an early stage of this unusual outbreak.
Asunto(s)
Brotes de Enfermedades , Infecciones por Escherichia coli/epidemiología , Síndrome Hemolítico-Urémico/epidemiología , Escherichia coli Shiga-Toxigénica/fisiología , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Genómica , Síndrome Hemolítico-Urémico/microbiología , Humanos , Lactante , Israel/epidemiología , Casas Cuna , Filogenia , Salud Pública , Análisis de Secuencia de ADN , Escherichia coli Shiga-Toxigénica/genéticaRESUMEN
We report a case of severe infection with liver abscess and endophthalmitis caused by a hypervirulent Klebsiella pneumoniae strain in an immunocompetent German male patient without travel history to Asia. Phenotypic and molecular characterization showed high similarity to the reference genome NTUH-K2044 isolated in Asia. The isolate was assigned as ST2398 (clonal complex 66). The findings underline global spread of hypervirulent Klebsiella pneumoniae strains to Europe.