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1.
Artigo em Inglês | MEDLINE | ID: mdl-28373201

RESUMO

Several lines of evidence indicate that the most primitive staphylococcal species, those of the Staphylococcus sciuri group, were involved in the first stages of evolution of the staphylococcal cassette chromosome mec (SCCmec), the genetic element carrying the ß-lactam resistance gene mecA However, many steps are still missing from this evolutionary history. In particular, it is not known how mecA was incorporated into the mobile element SCC prior to dissemination among Staphylococcus aureus and other pathogenic staphylococcal species. To gain insights into the possible contribution of several species of the Staphylococcus sciuri group to the assembly of SCCmec, we sequenced the genomes of 106 isolates, comprising S. sciuri (n = 76), Staphylococcus vitulinus (n = 18), and Staphylococcus fleurettii (n = 12) from animal and human sources, and characterized the native location of mecA and the SCC insertion site by using a variety of comparative genomic approaches. Moreover, we performed a single nucleotide polymorphism (SNP) analysis of the genomes in order to understand SCCmec evolution in relation to phylogeny. We found that each of three species of the S. sciuri group contributed to the evolution of SCCmec: S. vitulinus and S. fleurettii contributed to the assembly of the mec complex, and S. sciuri most likely provided the mobile element in which mecA was later incorporated. We hypothesize that an ancestral SCCmec III cassette (an element carried by one of the most epidemic methicillin-resistant S. aureus clones) originated in S. sciuri possibly by a recombination event in a human host or a human-created environment and later was transferred to S. aureus.


Assuntos
Cromossomos Bacterianos/genética , Staphylococcus aureus Resistente à Meticilina/genética , Evolução Molecular , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Polimorfismo de Nucleotídeo Único/genética , Staphylococcus/efeitos dos fármacos , Staphylococcus/genética , Resistência beta-Lactâmica/genética
2.
Emerg Infect Dis ; 22(5): 900-2, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-27089007

RESUMO

We describe 2 fatal cases of methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 398 septicemia in persons who had no contact with livestock. Whole-genome sequencing of the isolated MRSA strains strongly suggest that both were of animal origin and that the patients had been infected through 2 independent person-to-person transmission chains.


Assuntos
Infecção Hospitalar , Hospitais , Staphylococcus aureus Resistente à Meticilina/classificação , Casas de Saúde , Sepse , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/transmissão , Idoso , Animais , Dinamarca , Fazendeiros , Evolução Fatal , Feminino , Genoma Bacteriano , Humanos , Gado , Masculino , Staphylococcus aureus Resistente à Meticilina/genética , Pessoa de Meia-Idade , Filogenia , Diálise Renal/efeitos adversos , Infecções Estafilocócicas/diagnóstico
3.
Antimicrob Agents Chemother ; 60(1): 151-60, 2016 01.
Artigo em Inglês | MEDLINE | ID: mdl-26482314

RESUMO

The origin of carbapenem-hydrolyzing metallo-ß-lactamases (MBLs) acquired by clinical bacteria is largely unknown. We investigated the frequency, host range, diversity, and functionality of MBLs in the soil microbiota. Twenty-five soil samples of different types and geographical origins were analyzed by antimicrobial selective culture, followed by phenotypic testing and expression of MBL-encoding genes in Escherichia coli, and whole-genome sequencing of MBL-producing strains was performed. Carbapenemase activity was detected in 29 bacterial isolates from 13 soil samples, leading to identification of seven new MBLs in presumptive Pedobacter roseus (PEDO-1), Pedobacter borealis (PEDO-2), Pedobacter kyungheensis (PEDO-3), Chryseobacterium piscium (CPS-1), Epilithonimonas tenax (ESP-1), Massilia oculi (MSI-1), and Sphingomonas sp. (SPG-1). Carbapenemase production was likely an intrinsic feature in Chryseobacterium and Epilithonimonas, as it occurred in reference strains of different species within these genera. The amino acid identity to MBLs described in clinical bacteria ranged between 40 and 69%. Remarkable features of the new MBLs included prophage integration of the encoding gene (PEDO-1), an unusual amino acid residue at a key position for MBL structure and catalysis (CPS-1), and overlap with a putative OXA ß-lactamase (MSI-1). Heterologous expression of PEDO-1, CPS-1, and ESP-1in E. coli significantly increased the MICs of ampicillin, ceftazidime, cefpodoxime, cefoxitin, and meropenem. Our study shows that MBL producers are widespread in soil and include four genera that were previously not known to produce MBLs. The MBLs produced by these bacteria are distantly related to MBLs identified in clinical samples but constitute resistance determinants of clinical relevance if acquired by pathogenic bacteria.


Assuntos
Chryseobacterium/enzimologia , Pedobacter/enzimologia , Microbiologia do Solo , Sphingomonas/enzimologia , Resistência beta-Lactâmica/genética , beta-Lactamases/genética , Sequência de Aminoácidos , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Chryseobacterium/efeitos dos fármacos , Chryseobacterium/genética , Chryseobacterium/isolamento & purificação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Europa (Continente) , Expressão Gênica , Hidrólise , Dados de Sequência Molecular , Pedobacter/efeitos dos fármacos , Pedobacter/genética , Pedobacter/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sphingomonas/efeitos dos fármacos , Sphingomonas/genética , Sphingomonas/isolamento & purificação , beta-Lactamases/metabolismo
4.
Appl Environ Microbiol ; 82(15): 4705-14, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27235431

RESUMO

UNLABELLED: CMY-2 is the most common plasmid-mediated AmpC ß-lactamase in Escherichia coli isolates of human and animal origin. The aim of this study was to elucidate the epidemiology of CMY-2-producing E. coli in Denmark. Strain and plasmid relatedness was studied in 93 CMY-2-producing clinical and commensal E. coli isolates collected from 2006 to 2012 from humans, retail poultry meat, broilers, and dogs. Multilocus sequence typing (MLST), antimicrobial susceptibility testing, and conjugation were performed in conjunction with plasmid replicon typing, plasmid multilocus sequence typing (pMLST), restriction fragment length polymorphism (RFLP), and sequencing of selected blaCMY-2-harboring plasmids. MLST revealed high strain diversity, with few E. coli lineages occurring in multiple host species and sample types. blaCMY-2 was detected on plasmids in 83 (89%) isolates. Most (75%) of the plasmids were conjugative and did not (96%) cotransfer resistance to antimicrobials other than cephalosporins. The main replicon types identified were IncI1-Iγ (55%) and IncK (39%). Isolates from different host species mainly carried distinct plasmid subtypes. Seven of the 18 human isolates harbored IncI1-Iγ/sequence type 2 (ST2), IncI1-Iγ/ST12, or IncK plasmids highly similar to those found among animal isolates, even though highly related human and animal plasmids differed by nonsynonymous single nucleotide polymorphisms (SNPs) or insertion sequence elements. This study clearly demonstrates that the epidemiology of CMY-2 can be understood only by thorough plasmid characterization. To date, the spread of this ß-lactam resistance determinant in Denmark is mainly associated with IncK and IncI1-Iγ plasmids that are generally distributed according to host-specific patterns. These baseline data will be useful to assess the consequences of the increasing human exposure to CMY-2-producing E. coli via animal sources. IMPORTANCE: CMY-2 is the most common plasmid-mediated AmpC ß-lactamase in Escherichia coli This ß-lactamase is poorly inhibited by clavulanic acid and confers resistance to cephamycins, third-generation cephalosporins, and aztreonam. Furthermore, resistance to carbapenems has been reported in E. coli as a result of production of plasmid-encoded CMY-2 ß-lactamase in combination with decreased outer membrane permeability. The gene encoding CMY-2 generally resides on transferable plasmids belonging to different incompatibility groups. The prevalence of CMY-2-mediated cephalosporin resistance in E. coli varies significantly depending on the geographical region and host. This study demonstrates that the epidemiology of CMY-2 can be understood only by thorough plasmid characterization. To date, the spread of this ß-lactam resistance determinant in Denmark is mainly associated with IncK and IncI1-Iγ plasmids, which are generally distributed according to host-specific patterns. These data will be useful to assess the consequences of the increasing human exposure to CMY-2-producing E. coli via animal sources.


Assuntos
Doenças do Cão/microbiologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Escherichia coli/fisiologia , Carne/microbiologia , Plasmídeos/genética , Doenças das Aves Domésticas/microbiologia , beta-Lactamases/genética , Animais , Galinhas , Dinamarca , Cães , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Variação Genética , Especificidade de Hospedeiro , Humanos , Filogenia , Plasmídeos/metabolismo , beta-Lactamases/metabolismo
5.
BMC Microbiol ; 16: 63, 2016 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-27067536

RESUMO

BACKGROUND: Escherichia coli is the most common cause of urinary tract infection (UTI). The pathogenic isolates are becoming increasingly resistant to antibiotics; with a worldwide dissemination of resistant sequence types (ST). We characterized three different uropathogenic E. coli populations, from non-hospitalized patients to describe the genetic kinship between resistant and susceptible isolates. We studied the populations by use of multi-locus sequence typing (MLST) and abbreviated-multi locus variable number of tandem repeat analysis (a-MLVA). Urine samples submitted for testing, by general practitioners, were identified at Dept. of Clinical Microbiology at Hvidovre Hospital, Denmark, from Oct. 2011 to July 2012. We included 94 fully susceptible, 94 resistant (non-ESBL) and 98 Extended Spectrum Beta-lactamases- (ESBL)-producing E. coli isolates. RESULTS: The ESBL population was dominated vastly by ST131 (51 %), ST38 (9 %) and ST69 (6 %). In the resistant group ST69 (18 %), ST73 (11 %) and ST131 (15 %) were the largest clusters. In the susceptible population more STs and a-MLVA codes were identified compared to the other groups and ST73 and ST95 were found as the only clusters with 16 % and 6 %, respectively. Ninety-eight per cent of the ESBL-producing E. coli isolates were CTX-M-producers. CONCLUSION: ST131 dominated the population of community-associated uropathogenic ESBL-producing E. coli, but was less frequent among non-ESBL-producing E. coli. The fully susceptible E. coli population was a much more diverse group than the resistant and ESBL-producing E. coli populations. Overall, these findings suggest that dominant ESBL-producing lineages are derived from UPEC lineages already established in the general UPEC population.


Assuntos
Infecções por Escherichia coli/microbiologia , Testes de Sensibilidade Microbiana/métodos , Tipagem de Sequências Multilocus/métodos , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/classificação , Escherichia coli Uropatogênica/efeitos dos fármacos , Infecções Comunitárias Adquiridas/microbiologia , DNA Bacteriano/genética , Humanos , Repetições Minissatélites , Filogenia , Urina/microbiologia , Escherichia coli Uropatogênica/genética , Resistência beta-Lactâmica
6.
Antimicrob Agents Chemother ; 59(6): 3578-81, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25779586

RESUMO

Genome alignment of a macrolide, lincosamide, and streptogramin B (MLSB)-resistant Staphylococcus fleurettii strain with an MLSB-susceptible S. fleurettii strain revealed a novel 11,513-bp genomic island carrying the new erythromycin resistance methylase gene erm(45). This gene was shown to confer inducible MLSB resistance when cloned into Staphylococcus aureus. The erm(45)-containing island was integrated into the housekeeping gene guaA in S. fleurettii and was able to form a circular intermediate but was not transmissible to S. aureus.


Assuntos
Antibacterianos/farmacologia , Ilhas Genômicas/genética , Lincosamidas/farmacologia , Macrolídeos/farmacologia , Staphylococcus/genética , Estreptogramina B/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Staphylococcus/efeitos dos fármacos
7.
J Clin Microbiol ; 52(12): 4305-8, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25297335

RESUMO

spa typing of methicillin-resistant Staphylococcus aureus (MRSA) has traditionally been done by PCR amplification and Sanger sequencing of the spa repeat region. At Hvidovre Hospital, Denmark, whole-genome sequencing (WGS) of all MRSA isolates has been performed routinely since January 2013, and an in-house analysis pipeline determines the spa types. Due to national surveillance, all MRSA isolates are sent to Statens Serum Institut, where the spa type is determined by PCR and Sanger sequencing. The purpose of this study was to evaluate the reliability of the spa types obtained by 150-bp paired-end Illumina WGS. MRSA isolates from new MRSA patients in 2013 (n = 699) in the capital region of Denmark were included. We found a 97% agreement between spa types obtained by the two methods. All isolates achieved a spa type by both methods. Nineteen isolates differed in spa types by the two methods, in most cases due to the lack of 24-bp repeats in the whole-genome-sequenced isolates. These related but incorrect spa types should have no consequence in outbreak investigations, since all epidemiologically linked isolates, regardless of spa type, will be included in the single nucleotide polymorphism (SNP) analysis. This will reveal the close relatedness of the spa types. In conclusion, our data show that WGS is a reliable method to determine the spa type of MRSA.


Assuntos
Staphylococcus aureus Resistente à Meticilina/classificação , Staphylococcus aureus Resistente à Meticilina/genética , Tipagem Molecular/métodos , Análise de Sequência de DNA/métodos , Proteína Estafilocócica A/genética , Dinamarca/epidemiologia , Humanos , Epidemiologia Molecular/métodos , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia
11.
J Antimicrob Chemother ; 65(3): 460-4, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20097787

RESUMO

OBJECTIVES: To establish the prevalence of the AmpC beta-lactamase phenotype in clinical isolates of Escherichia coli and characterize the genetic resistance mechanisms causing the observed phenotype. METHODS: Clinical E. coli (n = 74) with reduced susceptibility to third-generation cephalosporins and resistance to cefoxitin were collected from the Department of Clinical Microbiology at Hvidovre Hospital, Denmark, in 2006. The AmpC disc test was used to confirm expression of AmpC, and test-positive strains were selected for further antimicrobial susceptibility testing and molecular characterization. Hyperproduction of AmpC beta-lactamase was confirmed by isoelectric focusing (IEF). The presence of a plasmid-mediated ampC gene (pAmpC) was detected by multiplex PCR. The promoter and the entire reading frame of the chromosomal ampC gene were sequenced to identify promoter mutations associated with hyperproduction and gene mutations associated with extended-spectrum AmpC (ESAC) beta-lactamase activity. RESULTS: Twenty-four isolates exhibited a positive AmpC disc test. IEF confirmed AmpC expression in all isolates except one. Four isolates contained a bla(CMY-2) gene. These were not clonally related by multilocus sequence typing (MLST). The remaining isolates all had mutations or insertions in the promoter region, which could explain increased expression of the chromosomal AmpC enzyme. Mutations in the ampC gene associated with extended activity were rare and did not cause resistance to cefepime. Sequencing of ampC showed that most isolates were not clonally related. CONCLUSIONS: E. coli expressing an AmpC phenotype occur sporadically and cause significant resistance to cephalosporins. The majority of these are hyperproducing chromosomal ampC although some isolates have acquired pAmpC.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Infecções por Escherichia coli/epidemiologia , Proteínas de Escherichia coli/biossíntese , Escherichia coli/enzimologia , Resistência beta-Lactâmica , beta-Lactamases/biossíntese , beta-Lactamas/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Dinamarca/epidemiologia , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Humanos , Ponto Isoelétrico , Fenótipo , Plasmídeos , Reação em Cadeia da Polimerase , Prevalência , Análise de Sequência de DNA , beta-Lactamases/química , beta-Lactamases/genética
12.
Microbiologyopen ; 8(12): e941, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31573735

RESUMO

Pivmecillinam (amdinocillin pivoxil) is the recommended first-choice antibiotic used to treat urinary tract infections (UTIs) in Denmark. The frequency of mutation to mecillinam (MEC) resistance is described as high in vitro; however, treatment of UTI has a good clinical response and prevalence of mecillinam resistance in Escherichia coli remains low despite many years of use. We describe occurrence of in vivo mecillinam resistance in a clinical isolate of ESBL-producing E. coli following pivmecillinam treatment. The identified phenotypic differences in the mecillinam resistant isolate compared with the original mecillinam susceptible isolate were a full-length LPS with O-antigen (O25), mecillinam resistance and a lower MIC for ceftazidime. Regarding genotype, the resistant isolate differed with a mutation in blaCTX-M-15 to blaCTX-M-127 , loss of a part of a plasmid and a genomic island, respectively, and insertion of a transposase in wbbL, causing the rough phenotype. The observed mecillinam resistance is expected to be caused by the mutation in blaCTX-M-15 with additional contribute from the serotype shift. We continue to recommend the use of pivmecillinam as first-line treatment for UTI.


Assuntos
Andinocilina Pivoxil/uso terapêutico , Andinocilina/farmacologia , Farmacorresistência Bacteriana , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , beta-Lactamases/genética , Andinocilina Pivoxil/farmacologia , Escherichia coli/classificação , Infecções por Escherichia coli/diagnóstico , Genoma Bacteriano , Genômica/métodos , Humanos , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Mutação , Filogenia , Sequenciamento Completo do Genoma
14.
Environ Microbiol Rep ; 9(4): 428-440, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28574206

RESUMO

Whole genome sequencing is increasing used in epidemiology, e.g. for tracing outbreaks of food-borne diseases. This requires in-depth understanding of pathogen emergence, persistence and genomic diversity along the food production chain including in food processing plants. We sequenced the genomes of 80 isolates of Listeria monocytogenes sampled from Danish food processing plants over a time-period of 20 years, and analysed the sequences together with 10 public available reference genomes to advance our understanding of interplant and intraplant genomic diversity of L. monocytogenes. Except for three persisting sequence types (ST) based on Multi Locus Sequence Typing being ST7, ST8 and ST121, long-term persistence of clonal groups was limited, and new clones were introduced continuously, potentially from raw materials. No particular gene could be linked to the persistence phenotype. Using time-based phylogenetic analyses of the persistent STs, we estimate the L. monocytogenes evolutionary rate to be 0.18-0.35 single nucleotide polymorphisms/year, suggesting that the persistent STs emerged approximately 100 years ago, which correlates with the onset of industrialization and globalization of the food market.


Assuntos
Contaminação de Alimentos/análise , Listeria monocytogenes/isolamento & purificação , Biodiversidade , Manipulação de Alimentos/instrumentação , Genoma Bacteriano , Listeria monocytogenes/classificação , Listeria monocytogenes/genética , Tipagem de Sequências Multilocus , Filogenia
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