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In spring 2022, an increase in metallo-ß-lactamase-producing Pseudomonas aeruginosa (MBL-Pa) infections was detected in a hospital in Upper Austria. To identify the source of infection and to stop further transmissions, an epidemiological outbreak investigation including whole-genome sequencing (WGS)-based typing was conducted. The final case definition included cases admitted to the hospital between 2020 and 2023 with an MBL-Pa in one of the three genomic clusters identified. In addition, the investigation was extended to include historical cases from 2017. Core genome multilocus sequence typing was performed to assess the genetic relatedness between the isolates. Fifty-four clinical P. aeruginosa isolates and eight P. aeruginosa isolates from the hospital environment were obtained. All but nine isolates grouped into one of three genomic clusters (ST235/blaVIM-1, ST111/blaVIM-2, or ST621/blaIMP-13), which were considered to be distinct, prolonged outbreaks involving 47 out of 52 cases. The most likely source of infection for cluster 1 (ST111/blaVIM-2) and cluster 2 (ST235/blaVIM-1) was sinks in the intensive care unit (ICU) washroom. Cluster 3 clone (ST621/blaIMP-13) could have originated in the urology ward in 2020 and then spread to the ICU years later. However, the nosocomial origin of this clone could not be proven. In March 2023, following the implementation of control measures (gowning, patient isolation, screening, and daily disinfection), no further MLB-Pa was detected, and the outbreaks were considered to be over. As ICUs play an important role in the transmission of P. aeruginosa, emphasis should be placed on genomic surveillance, infection prevention, and control in such wards. IMPORTANCE: The significance of our work lies in the successful resolution of three prolonged outbreaks of MBL-Pa infections in a hospital in Upper Austria. Through a comprehensive epidemiological investigation coupled with WGS-based typing of P. aeruginosa isolates, the study identified three distinct genomic clusters responsible for prolonged outbreaks involving 47 cases. The investigation pinpointed sinks in the ICU washroom as the likely source of infection for two of the clusters. The study demonstrates the effectiveness of control measures such as hand hygiene, gowning, patient isolation, screening, and disinfection in stopping further transmission and bringing the outbreaks to a close. This underscores the critical role of genomic surveillance and control measures, particularly in high-risk settings like ICUs, in reducing nosocomial transmission of MBL-Pa infections.
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Introduction: The emergence of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae is an urgent and alarming One Health problem. This study aimed to investigate duplications of plasmid-encoded ESBL genes and their impact on antimicrobial resistance (AMR) phenotypes in clinical and screening isolates. Methods: Multi-drug-resistant bacteria from hospitalized patients were collected during routine clinical surveillance from January 2022 to June 2023, and their antimicrobial susceptibility patterns were determined. Genotypes were extracted from long-read whole-genome sequencing data. Furthermore, plasmids and other mobile genetic elements associated with ESBL genes were characterized, and the ESBL genes were correlated to ceftazidime minimal inhibitory concentration (MIC). Results: In total, we identified four cases of plasmid-encoded ESBL gene duplications that match four genetically similar plasmids during the 18-month surveillance period: five Escherichia coli and three Klebsiella pneumoniae isolates. As the ESBL genes were part of transposable elements, the surrounding sequence regions were duplicated as well. In-depth analysis revealed insertion sequence (IS)-mediated transposition mechanisms. Isolates with duplicated ESBL genes exhibited a higher MIC for ceftazidime in comparison to isolates with a single gene copy (3-256 vs. 1.5-32 mg/L, respectively). Conclusion: ESBL gene duplications led to an increased phenotypic resistance against ceftazidime. Our data suggest that ESBL gene duplications by an IS-mediated transposition are a relevant mechanism for how AMR develops in the clinical setting and is part of the microevolution of plasmids.
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Antibacterianos , Ceftazidima , Humanos , Ceftazidima/farmacologia , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , beta-Lactamases/genética , Duplicação Gênica , Escherichia coli , Plasmídeos/genética , Enterobacteriaceae/genética , Klebsiella pneumoniae , Testes de Sensibilidade MicrobianaRESUMO
Host targeting antiviral drugs (HTA) are directed against cellular mechanisms which can be exploited by viruses. These mechanisms are essential for viral replication, because missing functions cannot be compensated by the virus. However, this assumption needs experimental proof. Here we compared the HTA Zapnometinib (ZMN), with direct acting antivirals (DAA) (Remdesivir (RDV), Molnupiravir (MPV), Nirmatrelvir (NTV), Ritonavir (RTV), Paxlovid PAX)), in terms of their potency to induce reduced drug susceptibilities in SARS-CoV-2. During serial passage of δ-B1.617.2 adaptation to all DAAs occurred, while the inhibitory capacity of ZMN was not altered. Known single nucleotide polymorphisms (SNPs) responsible for partial resistances were found for RDV, NTV and PAX. Additionally, the high mutagenic potential of MPV was confirmed and decreased drug efficacies were found for the first time. Reduced DAA efficacy did not alter the inhibitory potential of ZMN. These results show that ZMN confers a high barrier towards the development of viral resistance and has the potential to act against partially DAA-insensitive viruses.
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COVID-19 , Citidina/análogos & derivados , Hepatite C Crônica , Hidroxilaminas , Humanos , Antivirais , SARS-CoV-2 , RitonavirRESUMO
We describe the identification of a methicillin-resistant, high-level mupirocin-resistant Staphylococcus argenteus. The isolate (1801221) was characterized as t6675-ST2250-SCCmecIVc, and whole-genome sequencing revealed that the isolate possessed two plasmids. One plasmid (34,870 bp), designated p1_1801221 with rep23, harboured the mupirocin resistance (mupA) gene. The second plasmid (20,644 bp), assigned as p2_1801221 with rep5a and rep16, carried the resistance determinants for penicillin (blaZ) and cadmium (cadD). Phylogenetic analysis revealed that the isolate clustered with the European ST2250 lineage. The overall high similarity of both plasmids in S. argenteus with published DNA sequences of Staphylococcus aureus plasmids strongly suggests an interspecies transfer. The pathogenic potential, community and nosocomial spread, and acquisition of antibiotic resistance gene determinants, including the mupA gene by S. argenteus, highlight its clinical significance and the need for its correct identification.
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Staphylococcus aureus Resistente à Meticilina , Mupirocina , Resistência a Meticilina , Staphylococcus aureus Resistente à Meticilina/genética , Mupirocina/farmacologia , Filogenia , StaphylococcusRESUMO
Today, whole genome sequencing (WGS)-based typing is the gold standard approach to detect outbreaks of Shiga toxin-producing Escherichia coli (STEC) and to differentiate them from sporadic cases. Here, we describe an optimized protocol to efficiently determine the genome sequences of STEC using short read Illumina technology and provide information on helpful tools for the subsequent bioinformatic analysis.
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Biologia Computacional , Surtos de Doenças , Infecções por Escherichia coli , Genoma Bacteriano , Escherichia coli Shiga Toxigênica/genética , Sequenciamento Completo do Genoma , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/genética , HumanosRESUMO
Hybrid Shiga toxin-producing Escherichia coli (STEC) and uropathogenic E. coli (UPEC) strains of multilocus sequence type 141 (ST141) cause both urinary tract infections and diarrhea in humans and are phylogenetically positioned between STEC and UPEC strains. We used comparative genomic analysis of 85 temporally and spatially diverse ST141 E. coli strains, including 14 STEC/UPEC hybrids, collected in Germany (n = 13) and the United States (n = 1) to reconstruct their molecular evolution. Whole-genome sequencing data showed that 89% of the ST141 E. coli strains either were STEC/UPEC hybrids or contained a mixture of virulence genes from other pathotypes. Core genome analysis and ancestral reconstruction revealed that the ST141 E. coli strains clustered into two lineages that evolved from a common ancestor in the mid-19th century. The STEC/UPEC hybrid emerged â¼100 years ago by acquiring an stx prophage, which integrated into previously unknown insertion site between rcsB and rcsD, followed by the insertion of a pathogenicity island (PAI) similar to PAI II of UPEC strain 536 (PAI II536-like). The two variants of PAI II536-like were associated with tRNA genes leuX and pheU, respectively. Finally, microevolution within PAI II536-like and acquisition of the enterohemorrhagic E. coli plasmid were observed. Our data suggest that intestinal pathogenic E. coli (IPEC)/extraintestinal pathogenic E. coli (ExPEC) hybrids are widespread and that selection pressure within the ST141 E. coli population led to the emergence of the STEC/UPEC hybrid as a clinically important subgroup. We hypothesize that ST141 E. coli strains serve as a melting pot for pathogroup conversion between IPEC and ExPEC, contrasting the classical theory of pathogen emergence from nonpathogens and corroborating our recent phenomenon of heteropathogenicity among pathogenic E. coli strains.
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Infecções por Escherichia coli/microbiologia , Evolução Molecular , Hibridização Genética , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Uropatogênica/genética , Genoma Bacteriano , Genômica/métodos , Humanos , Filogenia , Virulência/genética , Fatores de Virulência/genéticaRESUMO
Ceftazidime/avibactam (CZA) is a new ß-lactam/ß-lactamase inhibitor combination with promising properties as avibactam can inhibit a broad range of ß-lactamases (e.g. blaKPC, blaOXA-48). The objectives of this study were: (i) to assess CZA susceptibility rates; (ii) to compare gradient and disk diffusion tests with broth microdilution (BMD) for CZA susceptibility testing; and (iii) to study the clonal structure and antimicrobial resistance genes in multi-drug-resistant (MDR) and extensively drug-resistant (XDR) Pseudomonas aeruginosa. Isolates (n=192) from routine diagnostics (Germany, 2013-2018) were tested by BMD reference method, gradient diffusion test (Etest, bioMérieux and MIC Test Strip, Liofilchem) and disk diffusion test (MAST and Oxoid). All isolates were whole-genome sequenced to screen for metallo-ß-lactamases and to assess the clonal structure using core-genome multi-locus sequence typing. In total, 64.1% of isolates (n=123) were susceptible to CZA (minimum inhibitory concentration required to inhibit the growth of 50% of organisms 8 mg/L, minimum inhibitory concentration required to inhibit the growth of 90% of organisms >256 mg/L, range 0.5->256 mg/L). Susceptibility rates were higher in MDR (85.0%) than in XDR (49.1%) P. aeruginosa. Among commercial susceptibility testing methods, Etest showed highest accuracy in comparison to BMD (essential agreement 94.8%, categorical agreement 94.3%). CZA-resistant isolates (n=69) mainly belonged to ST235 (n=29, blaIMP-positive). In conclusion, CZA is a promising treatment option for infections caused by MDR P. aeruginosa. CZA-resistant P. aeruginosa mainly belong to the pandemic ST235 high-risk clone. Etest can be considered as an alternative to BMD.
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Antibacterianos/farmacologia , Compostos Azabicíclicos/farmacologia , Ceftazidima/farmacologia , Farmacorresistência Bacteriana Múltipla , Testes de Sensibilidade Microbiana/métodos , Pseudomonas aeruginosa/efeitos dos fármacos , Inibidores de beta-Lactamases/farmacologia , Combinação de Medicamentos , Genótipo , Alemanha , Humanos , Tipagem de Sequências Multilocus , Filogenia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/classificação , Pseudomonas aeruginosa/genética , Sequenciamento Completo do GenomaRESUMO
Clostridium difficile, recently renamed Clostridioides difficile, is the most common cause of antibiotic-associated nosocomial gastrointestinal infections worldwide. To differentiate endogenous infections and transmission events, highly discriminatory subtyping is necessary. Today, methods based on whole-genome sequencing data are increasingly used to subtype bacterial pathogens; however, frequently a standardized methodology and typing nomenclature are missing. Here we report a core genome multilocus sequence typing (cgMLST) approach developed for C. difficile Initially, we determined the breadth of the C. difficile population based on all available MLST sequence types with Bayesian inference (BAPS). The resulting BAPS partitions were used in combination with C. difficile clade information to select representative isolates that were subsequently used to define cgMLST target genes. Finally, we evaluated the novel cgMLST scheme with genomes from 3,025 isolates. BAPS grouping (n = 6 groups) together with the clade information led to a total of 11 representative isolates that were included for cgMLST definition and resulted in 2,270 cgMLST genes that were present in all isolates. Overall, 2,184 to 2,268 cgMLST targets were detected in the genome sequences of 70 outbreak-associated and reference strains, and on average 99.3% cgMLST targets (1,116 to 2,270 targets) were present in 2,954 genomes downloaded from the NCBI database, underlining the representativeness of the cgMLST scheme. Moreover, reanalyzing different cluster scenarios with cgMLST were concordant to published single nucleotide variant analyses. In conclusion, the novel cgMLST is representative for the whole C. difficile population, is highly discriminatory in outbreak situations, and provides a unique nomenclature facilitating interlaboratory exchange.
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Clostridioides difficile/classificação , Genoma Bacteriano , Tipagem de Sequências Multilocus/métodos , Técnicas de Tipagem Bacteriana/normas , Teorema de Bayes , Genótipo , Humanos , Tipagem de Sequências Multilocus/normas , Filogenia , Sequenciamento Completo do GenomaRESUMO
BACKGROUND: Infections caused by MDR Pseudomonas aeruginosa are on the rise, particularly in critically ill patients. Therefore, there is a need to evaluate new antimicrobial regimens. The objectives of this study were to investigate the ceftolozane/tazobactam resistance rates of MDR and XDR P. aeruginosa, the underlying resistance genes, the clonal structure and different antimicrobial susceptibility testing (AST) methods regarding their accuracy for ceftolozane/tazobactam testing. METHODS: In total, 112 MDR and XDR P. aeruginosa (from infection and colonization) from one German tertiary care hospital were included (2013-16). AST was done using broth microdilution (BMD), gradient diffusion test strips and disc diffusion. Resistance genes were screened by PCR. A randomly selected subset of 77 isolates was subjected to WGS to assess the clonal structure. RESULTS: In total, 38 isolates (33.9%) were resistant to ceftolozane/tazobactam according to the BMD reference method. Resistance was significantly lower in MDR P. aeruginosa (4.8%) compared with XDR P. aeruginosa (50%, P < 0.0001). The underlying mechanism in carbapenemase-positive ceftolozane/tazobactam-resistant isolates (n = 38) was blaIMP (n = 25), blaVIM (n = 4) and blaGES (n = 1). The resistance mechanism of the remaining eight ceftolozane/tazobactam-resistant isolates remained unclear. Although our strain collection was diverse, resistance to ceftolozane/tazobactam was almost exclusively associated with MLST ST235. The disc diffusion method was accurate for ceftolozane/tazobactam AST (no false-susceptible results, categorical agreement = 92.9%). CONCLUSIONS: Ceftolozane/tazobactam resistance was low in MDR P. aeruginosa, but higher in XDR P. aeruginosa. The disc diffusion method showed an acceptable accuracy for ceftolozane/tazobactam AST.
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Antibacterianos/farmacologia , Cefalosporinas/farmacologia , Farmacorresistência Bacteriana Múltipla , Testes de Sensibilidade Microbiana/métodos , Ácido Penicilânico/análogos & derivados , Pseudomonas aeruginosa/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas do Citoesqueleto/genética , Alemanha , Humanos , Testes de Sensibilidade Microbiana/instrumentação , Tipagem de Sequências Multilocus , Ácido Penicilânico/farmacologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , TazobactamRESUMO
To develop novel techniques for improving blood safety, dedicated bacterial strains, which are able to persist and to proliferate in blood platelet concentrates, are needed. Here, we present draft genome sequences of the four bacterial strains approved for the first WHO repository of platelet transfusion-relevant bacterial reference strains.
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Today, next-generation whole-genome sequencing (WGS) is increasingly used to determine the genetic relationships of bacteria on a nearly whole-genome level for infection control purposes and molecular surveillance. Here, we conducted a multicenter ring trial comprising five laboratories to determine the reproducibility and accuracy of WGS-based typing. The participating laboratories sequenced 20 blind-coded Staphylococcus aureus DNA samples using 250-bp paired-end chemistry for library preparation in a single sequencing run on an Illumina MiSeq sequencer. The run acceptance criteria were sequencing outputs >5.6 Gb and Q30 read quality scores of >75%. Subsequently, spa typing, multilocus sequence typing (MLST), ribosomal MLST, and core genome MLST (cgMLST) were performed by the participants. Moreover, discrepancies in cgMLST target sequences in comparisons with the included and also published sequence of the quality control strain ATCC 25923 were resolved using Sanger sequencing. All five laboratories fulfilled the run acceptance criteria in a single sequencing run without any repetition. Of the 400 total possible typing results, 394 of the reported spa types, sequence types (STs), ribosomal STs (rSTs), and cgMLST cluster types were correct and identical among all laboratories; only six typing results were missing. An analysis of cgMLST allelic profiles corroborated this high reproducibility; only 3 of 183,927 (0.0016%) cgMLST allele calls were wrong. Sanger sequencing confirmed all 12 discrepancies of the ring trial results in comparison with the published sequence of ATCC 25923. In summary, this ring trial demonstrated the high reproducibility and accuracy of current next-generation sequencing-based bacterial typing for molecular surveillance when done with nearly completely locked-down methods.
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Técnicas de Genotipagem/métodos , Tipagem Molecular/métodos , Staphylococcus aureus/classificação , Staphylococcus aureus/genética , Genoma Bacteriano , Humanos , Epidemiologia Molecular/métodos , Reprodutibilidade dos Testes , Análise de Sequência de DNARESUMO
The colonization of afro-tropical wildlife with Staphylococcus aureus and the derived clade Staphylococcus schweitzeri remains largely unknown. A reservoir in bats could be of importance since bats and humans share overlapping habitats. In addition, bats are food sources in some African regions and can be the cause of zoonotic diseases. Here, we present a cross-sectional survey employing pharyngeal swabs of captured and released bats (n=133) in a forest area of Gabon. We detected low colonization rates of S. aureus (4-6%) and S. schweitzeri (4%) in two out of four species of fruit bats, namely Rousettus aegyptiacus and Micropteropus pusillus, but not in insectivorous bats. Multilocus sequence typing showed that S. aureus from Gabonese bats (ST2984, ST3259, ST3301, ST3302) were distinct from major African human associated clones (ST15, ST121, ST152). S. schweitzeri from bats (ST1697, ST1700) clustered with S. schweitzeri from other species (bats, monkeys) from Nigeria and Côte d'Ivoire. In conclusion, colonization rates of bats with S. aureus and S. schweitzeri were low in our study. Phylogenetic analysis supports an intense geographical dispersal of S. schweitzeri among different mammalian wildlife hosts.
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Quirópteros/microbiologia , Reservatórios de Doenças/microbiologia , Staphylococcus aureus , Animais , Estudos Transversais , Gabão , Faringe/microbiologia , Filogenia , Infecções Estafilocócicas , Staphylococcus aureus/classificação , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , ZoonosesRESUMO
The increasing prevalence of multidrug-resistant (MDR) bacteria is a serious global challenge. Here, we studied prospectively whether bacterial whole-genome sequencing (WGS) for real-time MDR surveillance is technical feasible, returns actionable results, and is cost-beneficial. WGS was applied to all MDR isolates of four species (methicillin-resistant Staphylococcus aureus [MRSA], vancomycin-resistant Enterococcus faecium, MDR Escherichia coli, and MDR Pseudomonas aeruginosa) at the University Hospital Muenster, Muenster, Germany, a tertiary care hospital with 1,450 beds, during two 6-month intervals. Turnaround times (TAT) were measured, and total costs for sequencing per isolate were calculated. After cancelling prior policies of preemptive isolation of patients harboring certain Gram-negative MDR bacteria in risk areas, the second interval was conducted. During interval I, 645 bacterial isolates were sequenced. From culture, TATs ranged from 4.4 to 5.3 days, and costs were 202.49 per isolate. During interval II, 550 bacterial isolates were sequenced. Hospital-wide transmission rates of the two most common species (MRSA and MDR E. coli) were low during interval I (5.8% and 2.3%, respectively) and interval II (4.3% and 5.0%, respectively). Cancellation of isolation of patients infected with non-pan-resistant MDR E. coli in risk wards did not increase transmission. Comparing sequencing costs with avoided costs mostly due to fewer blocked beds during interval II, we saved in excess of 200,000. Real-time microbial WGS in our institution was feasible, produced precise actionable results, helped us to monitor transmission rates that remained low following a modification in isolation procedures, and ultimately saved costs.
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Infecção Hospitalar/transmissão , Enterococcus faecium/genética , Escherichia coli/genética , Genoma Bacteriano/genética , Controle de Infecções/métodos , Staphylococcus aureus Resistente à Meticilina/genética , Pseudomonas aeruginosa/genética , Enterococos Resistentes à Vancomicina/genética , Antibacterianos/farmacologia , Cefotaxima/farmacologia , Ciprofloxacina/farmacologia , Infecção Hospitalar/microbiologia , Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/isolamento & purificação , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/transmissão , Infecções por Bactérias Gram-Positivas/transmissão , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Testes de Sensibilidade Microbiana , Piperacilina/farmacologia , Estudos Prospectivos , Infecções por Pseudomonas/transmissão , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/isolamento & purificação , Análise de Sequência de DNA , Infecções Estafilocócicas/transmissão , Enterococos Resistentes à Vancomicina/efeitos dos fármacos , Enterococos Resistentes à Vancomicina/isolamento & purificaçãoRESUMO
We characterized two epidemiologically similar Acinetobacter baumannii clusters from two separate intensive care units (ICU) using core genome multilocus sequence typing. Clonal spread was confirmed in ICU-1 (12 of 14 isolates shared genotypes); in ICU-2, all genotypes (13 isolates) were diverse, thus excluding transmissions and enabling adequate infection control measures.
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Infecções por Acinetobacter/epidemiologia , Acinetobacter baumannii/classificação , Acinetobacter baumannii/genética , Infecção Hospitalar/epidemiologia , Genoma Bacteriano , Epidemiologia Molecular/métodos , Tipagem de Sequências Multilocus/métodos , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/isolamento & purificação , Análise por Conglomerados , Infecção Hospitalar/microbiologia , Humanos , Unidades de Terapia IntensivaRESUMO
Enterococcus faecium, a common inhabitant of the human gut, has emerged in the last 2 decades as an important multidrug-resistant nosocomial pathogen. Since the start of the 21st century, multilocus sequence typing (MLST) has been used to study the molecular epidemiology of E. faecium. However, due to the use of a small number of genes, the resolution of MLST is limited. Whole-genome sequencing (WGS) now allows for high-resolution tracing of outbreaks, but current WGS-based approaches lack standardization, rendering them less suitable for interlaboratory prospective surveillance. To overcome this limitation, we developed a core genome MLST (cgMLST) scheme for E. faecium. cgMLST transfers genome-wide single nucleotide polymorphism(SNP) diversity into a standardized and portable allele numbering system that is far less computationally intensive than SNP-based analysis of WGS data. The E. faecium cgMLST scheme was built using 40 genome sequences that represented the diversity of the species. The scheme consists of 1,423 cgMLST target genes. To test the performance of the scheme, we performed WGS analysis of 103 outbreak isolates from five different hospitals in the Netherlands, Denmark, and Germany. The cgMLST scheme performed well in distinguishing between epidemiologically related and unrelated isolates, even between those that had the same sequence type (ST), which denotes the higher discriminatory power of this cgMLST scheme over that of conventional MLST. We also show that in terms of resolution, the performance of the E. faecium cgMLST scheme is equivalent to that of an SNP-based approach. In conclusion, the cgMLST scheme developed in this study facilitates rapid, standardized, and high-resolution tracing of E. faecium outbreaks.
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Enterococcus faecium/classificação , Enterococcus faecium/genética , Tipagem de Sequências Multilocus/métodos , Biologia Computacional/métodos , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Dinamarca/epidemiologia , Surtos de Doenças , Enterococcus faecium/isolamento & purificação , Genoma Bacteriano , Alemanha/epidemiologia , Infecções por Bactérias Gram-Positivas/epidemiologia , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Epidemiologia Molecular/métodos , Países Baixos/epidemiologia , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Whole-genome sequencing (WGS) has emerged today as an ultimate typing tool to characterize Listeria monocytogenes outbreaks. However, data analysis and interlaboratory comparability of WGS data are still challenging for most public health laboratories. Therefore, we have developed and evaluated a new L. monocytogenes typing scheme based on genome-wide gene-by-gene comparisons (core genome multilocus the sequence typing [cgMLST]) to allow for a unique typing nomenclature. Initially, we determined the breadth of the L. monocytogenes population based on MLST data with a Bayesian approach. Based on the genome sequence data of representative isolates for the whole population, cgMLST target genes were defined and reappraised with 67 L. monocytogenes isolates from two outbreaks and serotype reference strains. The Bayesian population analysis generated five L. monocytogenes groups. Using all available NCBI RefSeq genomes (n = 36) and six additionally sequenced strains, all genetic groups were covered. Pairwise comparisons of these 42 genome sequences resulted in 1,701 cgMLST targets present in all 42 genomes with 100% overlap and ≥90% sequence similarity. Overall, ≥99.1% of the cgMLST targets were present in 67 outbreak and serotype reference strains, underlining the representativeness of the cgMLST scheme. Moreover, cgMLST enabled clustering of outbreak isolates with ≤10 alleles difference and unambiguous separation from unrelated outgroup isolates. In conclusion, the novel cgMLST scheme not only improves outbreak investigations but also enables, due to the availability of the automatically curated cgMLST nomenclature, interlaboratory exchange of data that are crucial, especially for rapid responses during transsectorial outbreaks.
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Listeria monocytogenes/classificação , Listeria monocytogenes/genética , Listeriose/microbiologia , Listeriose/veterinária , Tipagem de Sequências Multilocus/métodos , Animais , Microbiologia de Alimentos , Variação Genética , Genoma Bacteriano , Genótipo , Humanos , Listeriose/epidemiologia , Epidemiologia MolecularRESUMO
Enterohemorrhagic Escherichia coli (EHEC) O26:H11/Hâ» is the predominant non-O157 EHEC serotype among patients with diarrhea, bloody diarrhea, and hemolytic uremic syndrome (HUS) worldwide. To elucidate their phylogeny and association between their phylogenetic background and clinical outcome of the infection, we investigated 120 EHEC O26:H11/Hâ» strains isolated between 1965 and 2012 from asymptomatic carriers and patients with diarrhea or HUS. Whole-genome shotgun sequencing (WGS) was applied to ten representative EHEC O26 isolates to determine single nucleotide polymorphism (SNP) localizations within a predefined set of core genes. A multiplex SNP assay, comprising a randomly distributed subset of 48 SNPs, was established to detect SNPs in 110 additional EHEC O26 strains. Within approximately 1 Mb of core genes, WGS resulted in 476 high-quality bi-allelic SNP localizations. Forty-eight of these were subsequently investigated in 110 EHEC O26 and four different SNP clonal complexes (SNP-CC) were identified. SNP-CC2 was significantly associated with the development of HUS. Within the subsequently established evolutionary model of EHEC O26, we dated the emergence of human EHEC O26 to approximately 19,700 years ago and demonstrated a recent evolution within humans into the 4 SNP-CCs over the past 1,650 years. WGS and subsequent SNP typing enabled us to gain new insights into the evolution of EHEC O26 suggesting a common theme in this EHEC group with analogies to EHEC O157. In addition, the SNP-CC analysis may help to assess a risk in infected individuals for the progression to HUS and to implement more specific infection control measures.
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Escherichia coli Êntero-Hemorrágica/genética , Evolução Molecular , Polimorfismo de Nucleotídeo Único/genética , Escherichia coli Êntero-Hemorrágica/patogenicidade , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Síndrome Hemolítico-Urêmica/genética , Síndrome Hemolítico-Urêmica/microbiologia , Humanos , FilogeniaRESUMO
BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) O26 causes diarrhea and hemolytic uremic syndrome (HUS). Strains harboring the stx1a gene prevail, but strains with stx2a as the sole Shiga toxin-encoding gene are now emerging. The traits and virulence of the latter set of strains are unknown. We correlated stx genotypes of 272 EHEC O26 strains isolated in 7 European countries between 1996 and 2012 with disease phenotypes. We determined phylogeny, clonal structure, and plasmid gene profiles of the isolates and portray geographic and temporal distribution of the different subgroups. METHODS: The stx genotypes and plasmid genes were identified using polymerase chain reaction, phylogeny was assigned using multilocus sequence typing, and clonal relatedness was established using pulsed-field gel electrophoresis. RESULTS: Of the 272 EHEC O26 isolates, 107 (39.3%), 139 (51.1%), and 26 (9.6%) possessed stx1a, stx2a, or both genes, respectively. Strains harboring stx2a only were significantly associated with HUS (odds ratio, 14.2; 95% confidence interval, 7.9-25.6; P < .001) compared to other stx genotypes. The stx2a-harboring strains consist of 2 phylogenetically distinct groups defined by sequence type (ST) 21 and ST29. The ST29 strains are highly conserved and correspond by plasmid genes to the new virulent clone of EHEC O26 that emerged in Germany in the 1990s. This new clone occurred in 6 of the 7 countries and represented approximately 50% of all stx2a-harboring EHEC O26 strains isolated between 1996 and 2012. CONCLUSIONS: A new highly virulent clone of EHEC O26 has emerged in Europe. Its reservoirs and sources warrant identification.
Assuntos
Escherichia coli Êntero-Hemorrágica/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Adolescente , Adulto , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Distribuição de Qui-Quadrado , Criança , Pré-Escolar , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Escherichia coli Êntero-Hemorrágica/genética , Escherichia coli Êntero-Hemorrágica/patogenicidade , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/epidemiologia , Europa (Continente)/epidemiologia , Feminino , Humanos , Lactente , Masculino , Testes de Sensibilidade Microbiana , Fenótipo , Filogenia , Plasmídeos/genética , Reação em Cadeia da Polimerase , Resultado do Tratamento , Adulto JovemRESUMO
A GC-MS method for the quantification of bacterial lipopolysaccharides (LPS, endotoxin) is presented. After hydrolytic cleavage of 3-hydroxy fatty acids (3-OH FAs) from the lipid A region of LPS, derivatisation of both the hydroxyl and the carboxyl group was performed in one step with a mixture of methyl-bis(trifluoracetamide)(MBTFA) and N-methyl-N-(tert-butyldimethylsilyl)trifluoracetamide (MTBSTFA). Using GC-MS in the EI mode with selected ion monitoring (SIM) for analysis, baseline separation of 3-OH FAs (and of possibly interfering 2-OH FAs) was achieved. The sensitivity of the method (LOD 7-50 pg/injection for the different 3-OH FAs investigated) allows for the efficient quantification of LPS in occupational and environmental samples. Degradation of 3-OH FAs as well as of their derivatives during sample preparation and GC-MS separation as a possible source of errors in analytical methods based on 3-OH FA determination is reported for the first time. Thermal elimination of water from the underivatised 3-OH FAs and of trifluoroacetic acid from the derivatives was identified as the cause of degradation. The resulting alpha,beta-unsaturated compounds showing the same mass spectra as the 3-OH FA derivatives were detected as more or less prominent satellite peaks. By using alkaline instead of acidic hydrolysis and cool on-column instead of split/splitless injection, elimination was reduced to an acceptable level.