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1.
Nat Commun ; 12(1): 765, 2021 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-33536414

RESUMO

Chickens are the most common birds on Earth and colibacillosis is among the most common diseases affecting them. This major threat to animal welfare and safe sustainable food production is difficult to combat because the etiological agent, avian pathogenic Escherichia coli (APEC), emerges from ubiquitous commensal gut bacteria, with no single virulence gene present in all disease-causing isolates. Here, we address the underlying evolutionary mechanisms of extraintestinal spread and systemic infection in poultry. Combining population scale comparative genomics and pangenome-wide association studies, we compare E. coli from commensal carriage and systemic infections. We identify phylogroup-specific and species-wide genetic elements that are enriched in APEC, including pathogenicity-associated variation in 143 genes that have diverse functions, including genes involved in metabolism, lipopolysaccharide synthesis, heat shock response, antimicrobial resistance and toxicity. We find that horizontal gene transfer spreads pathogenicity elements, allowing divergent clones to cause infection. Finally, a Random Forest model prediction of disease status (carriage vs. disease) identifies pathogenic strains in the emergent ST-117 poultry-associated lineage with 73% accuracy, demonstrating the potential for early identification of emergent APEC in healthy flocks.


Assuntos
Infecções por Escherichia coli/prevenção & controle , Escherichia coli/genética , Evolução Molecular , Genoma Bacteriano/genética , Doenças das Aves Domésticas/prevenção & controle , Animais , Galinhas , Escherichia coli/classificação , Escherichia coli/patogenicidade , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/microbiologia , Genes Bacterianos , Variação Genética , Estudo de Associação Genômica Ampla/métodos , Genótipo , Humanos , Filogenia , Doenças das Aves Domésticas/diagnóstico , Doenças das Aves Domésticas/microbiologia , Virulência/genética
2.
PLoS One ; 15(10): e0239924, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33036018

RESUMO

The prevalence of carbapenem-resistant Enterobacterales (CRE) in the Arabian Peninsula is predicted to be high, as suggested from published case reports. Of particular concern, is carbapenem-resistant E. coli (CR-EC), due to the importance of this species as a community pathogen. Herein, we conducted a comprehensive molecular characterization of putative CR-EC strains from Oman. We aim to establish a baseline for future molecular monitoring. We performed whole-genome sequencing (WGS) for 35 putative CR-EC. Isolates were obtained from patients at multiple centers in 2015. Genetic relatedness was investigated using several typing approaches such as MLST, SNP calling, phylogroup and CRISPR typing. Maxiuium likelihood SNP-tree was performed by RAxML after variant calling and removal of recombination regions with Snippy and Gubbins, respectively. Resistance genes, plasmid replicon types, virulence genes, and prophage were also characterised. The online databases CGE, CRISPRcasFinder, Phaster and EnteroBase were used for the in silico analyses. Screening for mutations in genes regulating the expression of porins and efflux pump as well as mutations lead to fluoroquinolones resistance were performed with CLC Genomics Workbench. The genetic diversity suggests a polyclonal population structure with 21 sequence types (ST), of which ST38 being the most prevalent (11%). SNPs analysis revealed possible transmission episodes. Whereas, CRISPR typing helped to spot outlier strains belonged to phylogroups other than B2 which was CRISPR-free. The virulent phylogroups B2 and D were detected in 4 and 9 isolates, respectively. In some strains bacteriophages acted as vectors for virulence genes. Regarding resistance to ß-lactam, 22 were carbapenemase producers, 3 carbapenem non-susceptible but carbapenemase-negative, 9 resistant to expanded-spectrum cephalosporins, and one isolate with susceptibility to cephalosporins and carbapenems. Thirteen out of the 22 (59%) carbapenemase-producing isolates were NDM and 7 (23%) were OXA-48-like which mirrors the situation in Indian subcontinent. Two isolates co-produced NDM and OXA-48-like enzymes. In total, 80% (28/35) were CTX-M-15 producers and 23% (8/35) featured AmpC. The high-risk subclones ST131-H30Rx/C2, ST410-H24RxC and ST1193-H64RxC were detected, the latter associated with NDM. To our knowledge, this is the first report of ST1193-H64Rx subclone with NDM. In conclusion, strains showed polyclonal population structure with OXA-48 and NDM as the only carbapenemases in CR-EC from Oman. We detected the high-risk subclone ST131-H30Rx/C2, ST410-H24RxC and ST1193-H64RxC. The latter was reported with carbapenemase gene for the first time here.


Assuntos
Carbapenêmicos/farmacologia , Infecções por Escherichia coli , Proteínas de Escherichia coli/genética , Escherichia coli , Resistência beta-Lactâmica/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Bases de Dados Genéticas , Escherichia coli/classificação , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Feminino , Genes Bacterianos , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Omã , Plasmídeos , Fatores de Virulência/genética , Adulto Jovem
3.
PLoS Negl Trop Dis ; 14(9): e0008613, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898134

RESUMO

Although enteroaggregative E. coli (EAEC) has been implicated as a common cause of diarrhea in multiple settings, neither its essential genomic nature nor its role as an enteric pathogen are fully understood. The current definition of this pathotype requires demonstration of cellular adherence; a working molecular definition encompasses E. coli which do not harbor the heat-stable or heat-labile toxins of enterotoxigenic E. coli (ETEC) and harbor the genes aaiC, aggR, and/or aatA. In an effort to improve the definition of this pathotype, we report the most definitive characterization of the pan-genome of EAEC to date, applying comparative genomics and functional characterization on a collection of 97 EAEC strains isolated in the course of a multicenter case-control diarrhea study (Global Enteric Multi-Center Study, GEMS). Genomic analysis revealed that the EAEC strains mapped to all phylogenomic groups of E. coli. Circa 70% of strains harbored one of the five described AAF variants; there were no additional AAF variants identified, and strains that lacked an identifiable AAF generally did not have an otherwise complete AggR regulon. An exception was strains that harbored an ETEC colonization factor (CF) CS22, like AAF a member of the chaperone-usher family of adhesins, but not phylogenetically related to the AAF family. Of all genes scored, sepA yielded the strongest association with diarrhea (P = 0.002) followed by the increased serum survival gene, iss (p = 0.026), and the outer membrane protease gene ompT (p = 0.046). Notably, the EAEC genomes harbored several genes characteristically associated with other E. coli pathotypes. Our data suggest that a molecular definition of EAEC could comprise E. coli strains harboring AggR and a complete AAF(I-V) or CS22 gene cluster. Further, it is possible that strains meeting this definition could be both enteric bacteria and urinary/systemic pathogens.


Assuntos
Aderência Bacteriana/genética , Infecções por Escherichia coli/epidemiologia , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/patogenicidade , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Adesinas Bacterianas/genética , Aderência Bacteriana/fisiologia , Estudos de Casos e Controles , Linhagem Celular , Pré-Escolar , Diarreia/microbiologia , Escherichia coli/classificação , Escherichia coli/isolamento & purificação , Genoma Bacteriano/genética , Genômica , Humanos , Lactente , Recém-Nascido , Transativadores/genética , Virulência/genética , Fatores de Virulência/genética , Sequenciamento Completo do Genoma
4.
Int J Food Microbiol ; 334: 108849, 2020 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-32906080

RESUMO

Frozen vegetables have previously been associated with outbreaks of listeriosis in both the USA and Europe. An outbreak of Listeria monocytogenes serogroup 4 caused 53 cases in five European countries between 2015 and 2018. Whole genome sequencing (WGS) indicated that frozen sweet corn from a producer in Hungary was the source of illness. However, limited data is available on the prevalence of Listeria in frozen produce. A study of frozen fruit and vegetables from catering and retail premises in England was therefore carried out to assess their microbiological quality with respect to Listeria and Escherichia coli. Between December 2018 and April 2019, 1050 frozen fruit and vegetable samples were collected. Of these, 99% were of a satisfactory or borderline microbiological quality. Eleven samples (1%) contained ≥100 cfu/g of Escherichia coli (considered unsatisfactory in products labelled as ready-to-eat). Listeria monocytogenes or other Listeria species were detected in six samples (2%) of fruit compared to 167 samples (24%) of vegetables and six samples (26%) of fruit and vegetable mixes, but none at a level of ≥100 cfu/g. Characterisation by WGS of 74 L. monocytogenes isolates identified ten genetic clusters indicating a common source. For 8 of the 10 clusters, the isolates came from homogenous food types: four were sweet corn, and there was one cluster each for beans, peas, peppers and broccoli. There were five genetic associations between isolates from frozen vegetables and from clinical cases of listeriosis, including two cultures from frozen beans that were indistinguishable from the 2015-2018 sweet corn outbreak strain. This study indicates that L. monocytogenes was present in 10% of frozen vegetables and even though products are generally not ready-to-eat and are intended to be cooked prior to consumption, these have the potential to cause illness. Clear cooking and handling instructions are therefore required on these products to ensure that the health of consumers is not put at risk, and appropriate Good Manufacturing Practice measures should be followed by all fruit and vegetable freezing plants in order to reduce contamination with Listeria during processing.


Assuntos
Escherichia coli/isolamento & purificação , Alimentos em Conserva/microbiologia , Frutas/microbiologia , Listeria/isolamento & purificação , Verduras/microbiologia , Inglaterra , Escherichia coli/classificação , Escherichia coli/genética , Microbiologia de Alimentos , Doenças Transmitidas por Alimentos/microbiologia , Congelamento , Humanos , Listeria/classificação , Listeria/genética
5.
BMC Infect Dis ; 20(1): 544, 2020 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-32711470

RESUMO

BACKGROUND: This study aimed to identify ten different 16S rRNA methyltransferase genes (rmtA, rmtB, rmtC, rmtD, armA, rmtF, npmA, rmtH, rmtE and rmtG) and their coexisting ESBL and carbapenemase with the emergence of three E.coli clones within a single study centre. METHODS: A total of 329 non-duplicate E.coli isolates were studied to detect the presence of 16S rRNA methyltransferases along with ß-lactamases (TEM, SHV, OXA, VEB, GES, PER,CTX-M types, NDM, OXA-48,VIM, IMP and KPC) using PCR assay. Horizontal transferability were validated by transformation and conjugation analysis. Plasmid incompatibility typing and MLST analysis was also performed. RESULTS: A total of 117 isolates were found to be resistant to at least one of the aminoglycoside antibiotics. It was observed that 77 (65.8%) were positive for 16S rRNA methyltransferases. Among them thirty nine isolates were found to harbour only blaCTX-M-15, whereas combination of genes were observed in three isolates (blaVEB+ blaCTX-M-15 in 2 isolates and blaPER + blaCTX-M-15 in 1 isolate). blaNDM and blaOXA-48 like genes were found in 23 and 9 isolates, respectively. All the resistance genes were conjugatively transferable, and incompatibility typing showed multiple 16S rRNA methyltransferase genes were originated from a single Inc. I1 group. MLST analysis detected 3 clones of E.coliST4410, ST1341 and ST3906. CONCLUSION: The present study identified emergence of three clones of E.coli, resistant to aminoglycoside -cephalosporin- carbapenem. This warrants immediate measures to trace their transmission dynamics in order to slow down their spread in clinical setting.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Metiltransferases/genética , beta-Lactamases/genética , Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Carbapenêmicos/farmacologia , Cefalosporinas/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/classificação , Escherichia coli/efeitos dos fármacos , Genes Bacterianos/genética , Humanos , Índia , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus
6.
Sci Rep ; 10(1): 8094, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415168

RESUMO

Adherent-invasive Escherichia coli (AIEC) have been extensively implicated in Crohn's disease pathogenesis. Currently, AIEC is identified phenotypically, since no molecular marker specific for AIEC exists. An algorithm based on single nucleotide polymorphisms was previously presented as a potential molecular tool to classify AIEC/non-AIEC, with 84% accuracy on a collection of 50 strains isolated in Girona (Spain). Herein, our aim was to determine the accuracy of the tool using AIEC/non-AIEC isolates from different geographical origins and extraintestinal pathogenic E. coli (ExPEC) strains. The accuracy of the tool was significantly reduced (61%) when external AIEC/non-AIEC strains from France, Chile, Mallorca (Spain) and Australia (82 AIEC, 57 non-AIEC and 45 ExPEC strains in total) were included. However, the inclusion of only the ExPEC strains showed that the tool was fairly accurate at differentiating these two close pathotypes (84.6% sensitivity; 79% accuracy). Moreover, the accuracy was still high (81%) for those AIEC/non-AIEC strains isolated from Girona and Mallorca (N = 63); two collections obtained from independent studies but geographically close. Our findings indicate that the presented tool is not universal since it would be only applicable for strains from similar geographic origin and demonstrates the need to include strains from different origins to validate such tools.


Assuntos
Algoritmos , Aderência Bacteriana , Infecções por Escherichia coli/microbiologia , Escherichia coli/classificação , Escherichia coli/genética , Polimorfismo de Nucleotídeo Único , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/isolamento & purificação , Geografia , Humanos , Filogenia
7.
PLoS Negl Trop Dis ; 14(5): e0008274, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32357189

RESUMO

Multidrug-resistant Escherichia coli ST131 fimH30 responsible for extra-intestinal pathogenic (ExPEC) infections is globally distributed. However, the occurrence of a subclone fimH27 of ST131 harboring both ExPEC and enteroaggregative E. coli (EAEC) related genes and belonging to commonly reported O25:H4 and other serotypes causing bacteremia in African children remain unknown. We characterized 325 E. coli isolates causing bacteremia in Mozambican children between 2001 and 2014 by conventional multiplex polymerase chain reaction and whole genome sequencing. Incidence rate of EAEC bacteremia was calculated among cases from the demographic surveillance study area. Approximately 17.5% (57/325) of isolates were EAEC, yielding an incidence rate of 45.3 episodes/105 children-years-at-risk among infants; and 44 of isolates were sequenced. 72.7% (32/44) of sequenced strains contained simultaneously genes associated with ExPEC (iutA, fyuA and traT); 88.6% (39/44) harbored the aggregative adherence fimbriae type V variant (AAF/V). Sequence type ST-131 accounted for 84.1% (37/44), predominantly belonging to serotype O25:H4 (59% of the 37); 95.6% (35/44) harbored fimH27. Approximately 15% (6/41) of the children died, and five of the six yielded ST131 strains (83.3%) mostly (60%; 3/5) due to serotypes other than O25:H4. We report the emergence of a new subclone of ST-131 E. coli strains belonging to O25:H4 and other serotypes harboring both ExPEC and EAEC virulence genes, including agg5A, associated with poor outcome in bacteremic Mozambican children, suggesting the need for prompt recognition for appropriate management.


Assuntos
Adesinas de Escherichia coli/genética , Bacteriemia/microbiologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Escherichia coli/classificação , Fímbrias Bacterianas/genética , Genótipo , Transativadores/genética , Adolescente , Bacteriemia/epidemiologia , Criança , Pré-Escolar , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/epidemiologia , Feminino , Humanos , Incidência , Lactente , Recém-Nascido , Masculino , Moçambique/epidemiologia , Reação em Cadeia da Polimerase , Sorogrupo , Sequenciamento Completo do Genoma
8.
Appl Environ Microbiol ; 86(13)2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32358002

RESUMO

Food safety is a new area for novel applications of metagenomics analysis, which not only can detect and subtype foodborne pathogens in a single workflow but may also produce additional information with in-depth analysis capabilities. In this study, we applied a quasimetagenomic approach by combining short-term enrichment, immunomagnetic separation (IMS), multiple-displacement amplification (MDA), and nanopore sequencing real-time analysis for simultaneous detection of Salmonella and Escherichia coli in wheat flour. Tryptic soy broth was selected for the 12-h enrichment of samples at 42°C. Enrichments were subjected to IMS using beads capable of capturing both Salmonella and E. coli MDA was performed on harvested beads, and amplified DNA fragments were subjected to DNA library preparation for sequencing. Sequencing was performed on a portable device with real-time basecalling adaptability, and resulting sequences were subjected to two parallel pipelines for further analysis. After 1 h of sequencing, the quasimetagenomic approach could detect all targets inoculated at approximately 1 CFU/g flour to the species level. Discriminatory power was determined by simultaneous detection of dual inoculums of Salmonella and E. coli, absence of detection in control samples, and consistency in microbial flora composition of the same flour samples over several rounds of experiments. The total turnaround time for detection was approximately 20 h. Longer sequencing for up to 15 h enabled serotyping for many of the samples with more than 99% genome coverage, which could be subjected to other appropriate genetic analysis pipelines in less than a total of 36 h.IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) and Salmonella are of serious concern in low-moisture foods, including wheat flour and its related products, causing illnesses, outbreaks, and recalls. The development of advanced detection methods based on molecular principles of analysis is essential to incorporate into interventions intended to reduce the risk from these pathogens. In this work, a quasimetagenomic method based on real-time sequencing analysis and assisted by magnetic capture and DNA amplification was developed. This protocol is capable of detecting multiple Salmonella and/or E. coli organisms in the sample within less than a day, and it can also generate sufficient whole-genome sequences of the target organisms suitable for subsequent bioinformatics analysis. Multiplex detection and identification were accomplished in less than 20 h and additional whole-genome analyses of different nature were attained within 36 h, in contrast to the several days required in previous sequencing pipelines.


Assuntos
Escherichia coli/isolamento & purificação , Farinha/microbiologia , Microbiologia de Alimentos/métodos , Salmonella enterica/isolamento & purificação , Sorotipagem/métodos , Escherichia coli/classificação , Separação Imunomagnética/métodos , Fenômenos Magnéticos , Metagenômica/métodos , Sequenciamento por Nanoporos/métodos , Salmonella enterica/classificação , Triticum
9.
Klin Lab Diagn ; 65(6): 368-374, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32459896

RESUMO

Were studied the genes encoding the virulence factors of 221 strains: E. coli O6:H1 (194) and E. coli O25:H4 (27), isolated in 2014-2018 from stool samples of children and adults examined according to epidemic indications. Molecular methods included PCR with hybridization-fluorescence and electrophoresis detection of amplified products. The strains did not have virulence genes for diarrheagenic E. coli (DEC) pathogroups EPEC, ETEC, EIEC, EHEC, EAggEC, and belonged to the phylogenetic group B2. They contained from four to eight genes encoding virulence factors of ExPEC: E. coli O6:H1 - pap (68,6%), sfa (87,6%), fimH (96,4%), hly (62,4%), cnf (74,7%), iutA (97,9%), fyuA (95,9%), chu (100%); E. coli O25:H4 - pap (66,7%), afa (22,2%), fimH (100%), hly (44,4%), cnf (44,4%), iutA (100%) , fyuA (100%), chu (100%). The antimicrobial susceptibility testing to 6 classes of antimicrobials (beta-lactams, fluoroquinolones, aminoglycosides, nitrofurantoin, sulfanilamide, trimethoprim / sulfamethoxazole) according the EUCAST. 60,3% of E. coli O6:H1 were sensitive to antibiotics, E. coli O25:H4 remained sensitive to carbapenems and nitrofurans. Extended-spectrum cephalosporins resistance was due to the production ESBL (CTX-M). The 57,1% resistant strains of E. coli O6:H1 and 100% of E. coli O25:H4 strains belonged to the MDR phenotype. The XDR phenotype had one in five MDR strains of E. coli O6:H1 and E. coli O25:H4. All strains of E. coli O25:H4 belonged to ST131. Given the important role of E. coli in human pathology, detection of virulence genes should be performed to confirm the etiological significance of the isolated strain.


Assuntos
Infecções por Escherichia coli/diagnóstico , Escherichia coli/classificação , Sorotipagem , Adulto , Criança , Escherichia coli/efeitos dos fármacos , Genes Bacterianos , Humanos , Testes de Sensibilidade Microbiana , Filogenia , Fatores de Virulência/genética , beta-Lactamases
10.
J Med Microbiol ; 69(5): 685-688, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32375948

RESUMO

Carbapenem resistance in Enterobacteriaceae has become an increasingly worrying threat. So far, no epidemiological data regarding NDM-producing enterobacterial isolates has been available on these strains in West Africa. The aim of this study was to seek for carbapenemase-producing Enterobacteriaceae clinical strains isolated in Bamako Teaching Hospital in Mali. Of 50 strains isolated between May 2016 and September 2016, we found a ST448 E. coli harbouring an IncX3 plasmid with bla NDM-5 embedded in the ΔISAba125-ble MBL structure. This study reports the first description of NDM-5 in Mali isolated in an undescribed ST E. coli in West Africa.


Assuntos
Infecções por Escherichia coli/microbiologia , Escherichia coli/genética , beta-Lactamases/genética , Escherichia coli/classificação , Escherichia coli/isolamento & purificação , Humanos , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , beta-Lactamases/biossíntese
11.
Poult Sci ; 99(4): 2125-2135, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32241498

RESUMO

Applying broiler litter containing extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (E. coli) to arable land poses a potential risk for humans to get colonized by contact with contaminated soil or vegetables. Therefore, an inactivation of these bacteria before land application of litter is crucial. We performed 2 short-term litter storage trials (one in summer and winter, respectively), each covering a time span of 5 D to investigate the effectiveness of this method for inactivation of ESBL-producing E. coli in chicken litter. Surface and deep litter samples were taken from a stacked, ESBL-positive chicken litter heap in triplicates in close sampling intervals at the beginning and daily for the last 3 D of the experiments. Samples were analyzed quantitatively and qualitatively for ESBL-producing E. coli, total E. coli, and enterococci. Selected isolates were further characterized by whole-genome sequencing (WGS). In the depth of the heap ESBL-producing E. coli were detected quantitatively until 72 h and qualitatively until the end of the trial in winter. In summer detection was possible quantitatively up to 36 h and qualitatively until 72 h. For surface litter samples a qualitative detection of ESBL-producing E. coli was possible in all samples taken in both trials. In the deep samples a significant decrease in the bacterial counts of over 2 Log10 was observed for total E. coli in the winter and for total E. coli and enterococci in the summer. Genetic differences of the isolates analyzed by WGS did not correlate with survival advantage. In conclusion, short-term storage of chicken litter stacked in heaps is a useful tool for the reduction of bacterial counts including ESBL-producing E. coli. However, incomplete inactivation was observed at the surface of the heap and at low ambient temperatures. Therefore, an extension of the storage period in winter as well as turning of the heap to provide aerobic composting conditions should be considered if working and storage capacities are available on the farms.


Assuntos
Clima , Escherichia coli/fisiologia , Esterco/microbiologia , Viabilidade Microbiana , Tempo (Meteorologia) , Proteínas de Bactérias/metabolismo , Escherichia coli/classificação , Fazendas , Alemanha , Estações do Ano , beta-Lactamases/metabolismo
12.
Klin Lab Diagn ; 65(4): 251-257, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32227732

RESUMO

Escherichia coli is characterized by a wide intraspecific diversity. The species includes both commensals and pathogens that cause diarrhea and extra-intestinal diseases. Pathogenic strains differ from non-pathogenic ones by the presence of virulence factors and their genes. The phylogenetic structure of the species is represented by four main groups (A, B1, B2, D), which differ in their prevalence among residents of different geographical regions. Pathogenic members of the species have been studied in detail, while non-pathogenic strains have not received such attention. This report presents the results of a study of 511 E. coli strains isolated from the gut microbiota of children without diarrhea and urinary tract infections, aged from 1 month to 17 years, living in St. Petersburg. The main phylogenetic groups were determined by PCR, and E. coli virulence genes associated with diarrhea and extra-intestinal diseases were identified. Results: population structure of E. coli is represented by the following groups: A-33.3%, B1-6.7%, B2-34.0%, D-26%. In the studied population 2.5% of strains belonded to EPEC and 4.5% to EAggEC. EPEC virulence genes were more often detected in strains of phylogroup B1, and EAggEC virulence genes in isolates of phylogroup D. The prevalence of extra - intestinal virulence genes was as follows: pap - 29.5%; sfa - 19.8%; afa - 3.3%; hly - 20.9%; cnf - 17.4%; aer-20.0%. The pap, sfa, hly, and cnf genes were detected mostly in the B2 phylogenetic group. Obtained data shows the similarity of E. coli phylogenetic groups structure in St. Petersburg with E. coli populations isolated from residents of Paris and Sydney. Analysis of the virulence genes prevalence showed the dependence of their presence on the genetic background bacteria.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Microbioma Gastrointestinal , Filogenia , Fatores de Virulência/genética , Adolescente , Criança , Pré-Escolar , Escherichia coli/classificação , Infecções por Escherichia coli/microbiologia , Humanos , Lactente , Virulência
13.
Vet Microbiol ; 243: 108619, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32273005

RESUMO

Antimicrobial resistance is a "One Health" issue that requires improved knowledge of the presence and abundance of resistant bacteria in the environment. Extended-spectrum cephalosporins (ESCs) are critically important antibiotics (CIAs), and resistance to these CIAs is often encoded by beta-lactamase genes borne on conjugative plasmids. We thus decided to characterise 21 plasmids of ESC-resistant Escherichia coli randomly selected from isolates previously obtained from river water collected in a rural area in western France. The plasmids encoding ESC resistance were sequenced to investigate the diversity of the genes encoding ESC resistance and their genetic context. Sequences revealed that eleven IncI1 pMLST3 plasmids carried the blaCTX-M-1 and sul2 genes, and some of them also had the tet(A), aadA5 or dfrA17 genes. The blaCTX-M-1 gene was also detected on an IncN plasmid. Five plasmids obtained from four rivers contained blaCTX-M-14, either on IncI1 or on IncFII plasmids. Two strains from two rivers contained blaCTX-M-15 on IncN pMLST7 plasmids, with qnrS1 and dfrA14 genes. One plasmid contained the blaCTX-M-55, a blaTEM-1B-like, and fosA genes. One plasmid contained the blaCMY-2 gene. The diversity of the genes and plasmids of the resistant bacteria isolated from French rivers is probably related to the various animal and human origins of the isolated bacteria.


Assuntos
Antibacterianos/farmacologia , Resistência às Cefalosporinas/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Plasmídeos/genética , Rios/microbiologia , Escherichia coli/classificação , França , Transferência Genética Horizontal , Variação Genética , Tipagem de Sequências Multilocus , beta-Lactamases/genética
14.
Vet Microbiol ; 243: 108629, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32273008

RESUMO

INTRODUCTION: The prevalence of Extended-Spectrum Beta-Lactamase (ESBL)-producing Enterobacteriaceae is increasing worldwide and the Agri-Food sector acts as a reservoir of clinically relevant ESBL genes. Our study aimed at detecting and characterizing ESBL-producing Enterobacteriaceae responsible for intestinal colonization of Brazilian bovines. MATERIAL AND METHODS: ESBL-producing E. coli isolates were recovered from fecal samples of healthy cattle in Northwest Brazil. Antimicrobial susceptibility was determined by disk diffusion. Resistance and virulence genes were identified by PCR and amplicons were sequenced, clonality was assessed by PFGE and MLST, and plasmids were characterized by replicon typing, S1-PFGE and Southern blot hybridizations. Transferability of ESBL genes was assessed by conjugation assay. RESULTS: A total of 40 ESBL-producing E. coli were characterized, which originated from 34/191 animals (17.8 %) and 15/22 farms (68.2 %). The blaCTX-M-8 gene was the most frequent ESBL gene (62.5 %), followed by blaSHV-2a (20.0 %), blaCTX-M-2 (10.0 %), and blaCTX-M-15 (7.5 %). The blaCTX-M-8 gene was localized on the IncI1/pST113 plasmid in multiple E. coli sequence types across unrelated animals and farms. DISCUSSION: We report the first characterization and a high prevalence of ESBL-producing E. coli in the beef cattle sector in Brazil, which is mainly supported by the spread of an epidemic IncI1/pST113/blaCTX-M-8 plasmid. Since Brazil is one of the biggest beef meat exporters worldwide, the spread of this ESBL plasmid across other sectors, countries and continents should be considered with attention.


Assuntos
Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Plasmídeos/genética , beta-Lactamases/genética , Animais , Antibacterianos/farmacologia , Brasil/epidemiologia , Bovinos/microbiologia , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/classificação , Fazendas/estatística & dados numéricos , Fezes/microbiologia , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Virulência/genética
15.
Int J Mol Sci ; 21(3)2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-32023871

RESUMO

The need for a comparative analysis of natural metagenomes stimulated the development of new methods for their taxonomic profiling. Alignment-free approaches based on the search for marker k-mers turned out to be capable of identifying not only species, but also strains of microorganisms with known genomes. Here, we evaluated the ability of genus-specific k-mers to distinguish eight phylogroups of Escherichia coli (A, B1, C, E, D, F, G, B2) and assessed the presence of their unique 22-mers in clinical samples from microbiomes of four healthy people and four patients with Crohn's disease. We found that a phylogenetic tree inferred from the pairwise distance matrix for unique 18-mers and 22-mers of 124 genomes was fully consistent with the topology of the tree, obtained with concatenated aligned sequences of orthologous genes. Therefore, we propose strain-specific "barcodes" for rapid phylotyping. Using unique 22-mers for taxonomic analysis, we detected microbes of all groups in human microbiomes; however, their presence in the five samples was significantly different. Pointing to the intraspecies heterogeneity of E. coli in the natural microflora, this also indicates the feasibility of further studies of the role of this heterogeneity in maintaining population homeostasis.


Assuntos
Doença de Crohn/genética , Código de Barras de DNA Taxonômico/métodos , Infecções por Escherichia coli/genética , Escherichia coli/genética , Genes Bacterianos , Genoma Bacteriano , Microbiota , Algoritmos , Estudos de Casos e Controles , Biologia Computacional , Doença de Crohn/microbiologia , Escherichia coli/classificação , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Humanos , Metagenoma
16.
BMC Genomics ; 21(1): 138, 2020 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-32041522

RESUMO

BACKGROUND: We investigated the association of symptoms and disease severity of shigellosis patients with genetic determinants of infecting Shigella and entero-invasive Escherichia coli (EIEC), because determinants that predict disease outcome per individual patient could be used to prioritize control measures. For this purpose, genome wide association studies (GWAS) were performed using presence or absence of single genes, combinations of genes, and k-mers. All genetic variants were derived from draft genome sequences of isolates from a multicenter cross-sectional study conducted in the Netherlands during 2016 and 2017. Clinical data of patients consisting of binary/dichotomous representation of symptoms and their calculated severity scores were also available from this study. To verify the suitability of the methods used, the genetic differences between the genera Shigella and Escherichia were used as control. RESULTS: The isolates obtained were representative of the population structure encountered in other Western European countries. No association was found between single genes or combinations of genes and separate symptoms or disease severity scores. Our benchmark characteristic, genus, resulted in eight associated genes and > 3,000,000 k-mers, indicating adequate performance of the algorithms used. CONCLUSIONS: To conclude, using several microbial GWAS methods, genetic variants in Shigella spp. and EIEC that can predict specific symptoms or a more severe course of disease were not identified, suggesting that disease severity of shigellosis is dependent on other factors than the genetic variation of the infecting bacteria. Specific genes or gene fragments of isolates from patients are unsuitable to predict outcomes and cannot be used for development, prioritization and optimization of guidelines for control measures of shigellosis or infections with EIEC.


Assuntos
Disenteria Bacilar/diagnóstico , Disenteria Bacilar/microbiologia , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/microbiologia , Escherichia coli/genética , Shigella/genética , Estudos Transversais , Escherichia coli/classificação , Escherichia coli/isolamento & purificação , Marcadores Genéticos , Estudo de Associação Genômica Ampla , Humanos , Filogenia , Shigella/classificação , Shigella/isolamento & purificação
17.
Arch Microbiol ; 202(5): 1173-1179, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32076735

RESUMO

Escherichia coli strains are part of the normal biota of humans and animals; however, several clinical reports have implicated E. coli as the etiological agent of diarrhea in humans and companion animals. Thus, the aim of the present study was to know if companion dogs in the city of San Luis Potosi are colonized with virulent potentially harmful E. coli strains. Rectal swabs from 30 dogs, 13 with and 17 without diarrhea were analyzed. Phylogenetic and virulence genes analysis was performed to the E. coli isolates. Additionally, the Kirby-Bauer test was used to analyze the sensitivity to 32 different antimicrobials from 14 families. Eighty-five isolates were identified as E. coli and detected in 97% of healthy and diarrheic dog samples. E. coli isolates from healthy dogs carried several virulence genes, in contrast with those from diarrheic animals that presented only eaeA. In healthy dogs, phylogenetic analysis showed that 57% and 43% of E. coli isolates belonged to commensal (A and B1) and virulent (B2 and D) groups respectively. Meanwhile, diarrheic dogs showed that 69% of the isolates were identified as virulent B2 and D phylogroups. Moreover, E. coli resistant to ß-lactams, aminoglycosides, tetracycline, quinolones, and folate inhibitors were detected in both groups of dogs. The presence of E. coli with eaeA virulence gene in diarrheic dogs, suggest that these strains are associated with the animal´s condition. Finally, major attention must be drawn to the careful handling of dogs because of their capability to harbor and disseminate virulent E. coli strains.


Assuntos
Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/transmissão , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Animais de Estimação/microbiologia , Animais , Antibacterianos/farmacologia , Diarreia/microbiologia , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Cães , Escherichia coli/classificação , Humanos , Filogenia , Virulência/genética , Fatores de Virulência/genética
18.
Sci Rep ; 10(1): 3495, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-32103089

RESUMO

Oral lichen planus (OLP) is a chronic T cell-mediated inflammatory disease of unknown etiology. We previously proposed that the intracellular bacteria detected in OLP lesions are important triggering factors for T cell infiltration. This study aimed to identify OLP-associated bacterial species through the characterization of intratissue bacterial communities of OLP lesions. Seven pairs of bacterial communities collected from the mucosal surface and biopsied tissues of OLP lesions were analyzed by high-throughput sequencing of the 16S rRNA gene. The intratissue bacterial communities were characterized by decreased alpha diversity but increased beta diversity compared with those on the mucosal surface. While the relative abundance of most taxa was decreased within the tissues, that of Escherichia coli was significantly increased. Four E. coli strains were isolated from additional OLP biopsies and verified as K12 strains by whole-genome sequencing. The distribution of E. coli in sections of control (n = 12) and OLP (n = 22) tissues was examined by in situ hybridization. E. coli was detected in most OLP tissues, suggesting its potential role in the pathogenesis of OLP. The oral E. coli strains isolated from OLP tissues will be useful to investigate their role as triggering factors for T cell infiltration.


Assuntos
Escherichia coli/isolamento & purificação , Líquen Plano Bucal/patologia , Microbiota , Idoso , Apoptose , Linhagem Celular , Escherichia coli/classificação , Escherichia coli/genética , Escherichia coli/fisiologia , Feminino , Humanos , Líquen Plano Bucal/microbiologia , Masculino , Pessoa de Meia-Idade , Membrana Mucosa/microbiologia , Filogenia , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Sequenciamento Completo do Genoma
19.
Nat Commun ; 11(1): 378, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31953385

RESUMO

Bacteriophages constitute an important part of the human gut microbiota, but their impact on this community is largely unknown. Here, we cultivate temperate phages produced by 900 E. coli strains isolated from 648 fecal samples from 1-year-old children and obtain coliphages directly from the viral fraction of the same fecal samples. We find that 63% of strains hosted phages, while 24% of the viromes contain phages targeting E. coli. 150 of these phages, half recovered from strain supernatants, half from virome (73% temperate and 27% virulent) were tested for their host range on 75 E. coli strains isolated from the same cohort. Temperate phages barely infected the gut strains, whereas virulent phages killed up to 68% of them. We conclude that in fecal samples from children, temperate coliphages dominate, while virulent ones have greater infectivity and broader host range, likely playing a role in gut microbiota dynamics.


Assuntos
Colífagos/fisiologia , Escherichia coli/virologia , Fezes/virologia , Proteínas de Transporte , Colífagos/classificação , Colífagos/genética , Colífagos/isolamento & purificação , Escherichia coli/classificação , Escherichia coli/isolamento & purificação , Fezes/microbiologia , Microbioma Gastrointestinal , Genoma Viral , Especificidade de Hospedeiro , Humanos , Lactente , Lisogenia , Especificidade da Espécie
20.
Sci Rep ; 10(1): 36, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31913346

RESUMO

Extraintestinal multidrug resistant Escherichia coli sequence type (ST) 131 is a worldwide pandemic pathogen and a major cause of urinary tract and bloodstream infections. The role of this pandemic lineage in multidrug resistance plasmid dissemination is still scarce. We herein performed a meta-analysis on E. coli ST131 whole-genome sequence (WGS) databases to unravel ST131 plasmidome and specifically to decipher CTX-M encoding plasmids-clade associations. We mined 880 ST131 WGS data and proved that CTX-M-27-encoding IncF[F1:A2:B20] (Group1) plasmids are strictly found in clade C1, whereas CTX-M-15-encoding IncF[F2:A1:B-] (Group2) plasmids exist only in clade C2 suggesting strong plasmid-clade adaptations. Specific Col-like replicons (Col156, Col(MG828), and Col8282) were also found to be clade C1-associated. BLAST-based search revealed that Group1 and Group2 plasmids are narrow-host-range and restricted to E.coli. Among a collection of 20 newly sequenced Israeli ST131 CTX-M-encoding plasmids (2003-2016), Group1 and Group2 plasmids were dominant and associated with the expected clades. We found, for the first time in ST131, a CTX-M-15-encoding phage-like plasmid group (Group3) and followed its spread in the WGS data. This study offers a comprehensive way to decipher plasmid-bacterium associations and demonstrates that the CTX-M-encoding ST131 Group1 and Group2 plasmids are clade-restricted and presumably less transmissible, potentially contributing to ST131 clonal superiority.


Assuntos
Infecções por Escherichia coli/microbiologia , Escherichia coli/genética , Genoma Bacteriano , Plasmídeos/genética , Escherichia coli/classificação , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/genética , Humanos , Pandemias , Filogenia , Replicon , Sequenciamento Completo do Genoma
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