RESUMO
The genus Arcobacter includes species considered emerging food and waterborne pathogens. Despite Arcobacter has been linked to the presence of faecal pollution, few studies have investigated its prevalence in wastewater, and the only isolated species were Arcobacter butzleri and Arcobacter cryaerophilus. This study aimed to establish the prevalence of Arcobacter spp. at a WWTP using in parallel two culturing methods (direct plating and culturing after enrichment) and a direct detection by m-PCR. In addition, the genetic diversity of the isolates was established using the ERIC-PCR genotyping method. Most of the wastewater samples (96.7%) were positive for Arcobacter and a high genetic diversity was observed among the 651 investigated isolates that belonged to 424 different ERIC genotypes. However, only few strains persisted at different dates or sampling points. The use of direct plating in parallel with culturing after enrichment allowed recovering the species A. butzleri, A. cryaerophilus, Arcobacter thereius, Arcobacter defluvii, Arcobacter skirrowii, Arcobacter ellisii, Arcobacter cloacae, and Arcobacter nitrofigilis, most of them isolated for the first time from wastewater. The predominant species was A. butzleri, however, by direct plating predominated A. cryaerophilus. Therefore, the overall predominance of A. butzleri was a bias associated with the use of enrichment.
Assuntos
Arcobacter/isolamento & purificação , Técnicas Bacteriológicas/métodos , Biodiversidade , Águas Residuárias/microbiologia , Purificação da Água/métodos , Aerobiose , Reação em Cadeia da Polimerase , Especificidade da EspécieRESUMO
Forty-eight Aeromonas isolates from rainbow trout previously identified by the 16S rDNA-RFLP technique were re-identified using 2 housekeeping genes (gyrB and rpoD). After sequencing the prevalences of the species were A. veronii (29.2%), A. bestiarum (20.8%), A. hydrophila (16.7%), A. sobria (10.4%), A. media (8.3%), A. popoffii (6.2%), A. allosaccharophila (2.1%), A. caviae (2.1%), A. salmonicida (2.1%) and one isolate (2.1%) belongs to a candidate new species "Aeromonas lusitana". Coincident identification results to the 16S rDNA-RFLP technique were only obtained for 68.8% of the isolates. PCR amplification of the enterobacterial repetitive intergenic consensus (ERIC-PCR) indicated that the 48 isolates belonged to 33 different ERIC genotypes. Several genotypes were isolated from different farms and organs in the same fish, indicating a systemic dissemination of the bacteria. The presence of genes (blaIMP, blaCphA/IMIS, blaTEM, blaSHV and intI1) that encode extended-spectrum beta-lactamases (ESBLs), metallo-beta-lactamases (MBLs) and class 1 integrons were studied by PCR. Only 39.6% (19/48) of the strains showed the presence of one or more resistance genes. The gene blaCphA/IMIS was detected in 29.2% of the isolates, followed by the intI1 (6.2%) and blaSHV (4.2%) genes. The variable region of class 1 integrons of the 3 positive isolates was sequenced revealing the presence of the gene cassette aadA1 (aminoglycoside transferase) that plays a role in streptomycin/spectinomycin resistance.
Assuntos
Aeromonas/isolamento & purificação , Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Integrons/genética , Oncorhynchus mykiss , beta-Lactamases/metabolismo , Aeromonas/genética , Animais , Antibacterianos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Incidência , beta-Lactamases/genéticaRESUMO
A study employing a polyphasic taxonomic approach was undertaken to clarify the position of 12 isolates recovered from sewage samples. These isolates were recognized as a potential novel species because a new and specific pattern was produced with the 16S rRNA-RFLP Arcobacter identification method. The sequences of the 16S rRNA gene not only supported the classification of these novel strains as members of the genus Arcobacter, but also showed that they formed a separate phylogenetic line. Strain SW28-11(T), chosen as the representative of these strains, showed 16S rRNA gene sequence similarity of 95.6â% with the closest related species Arcobacter nitrofigilis. The phylogenetic position of the novel strains was further confirmed by analysis of the housekeeping genes hsp60, rpoB and, for the first time, gyrB. The latter proved to be an excellent additional gene for establishing the phylogeny of this genus. These data, together with phenotypic characterization, revealed that this group of isolates represent a novel species of the genus Arcobacter. The name Arcobacter defluvii sp. nov., is proposed, with the type strain SW28-11(T) (â=âCECT 7697(T)â=âLMG 25694(T)).
Assuntos
Arcobacter/classificação , Arcobacter/isolamento & purificação , Esgotos/microbiologia , Arcobacter/genética , Arcobacter/metabolismo , Técnicas de Tipagem Bacteriana , Chaperonina 60/genética , Análise por Conglomerados , DNA Girase/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , RNA Polimerases Dirigidas por DNA/genética , Dados de Sequência Molecular , Tipagem Molecular , Filogenia , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
A comparative study of 109 Aeromonas clinical isolates belonging to the 3 species most frequently isolated from patients with diarrhea in Mexico and Spain was performed to investigate the distribution of 3 prominent toxin genes and the gene encoding flagellin of lateral flagella; 4 well-established virulence factors in the genus Aeromonas. The aerolysin-hemolysin toxin genes were the most prevalent, being present in 89% of the total isolates. The ast toxin gene was conspicuously absent from the Aeromonas caviae and Aeromonas veronii groups but was present in 91% of the Aeromonas hydrophila isolates. Both the alt toxin gene and the lafA flagellin gene also had a low incidence in A. caviae and A. veronii. Differences in the prevalence of alt and lafA were observed between isolates from Mexico and Spain, confirming genus heterogeneity according to geographic location. Carriage of multiple toxin genes was primarily restricted to A. hydrophila isolates, suggesting that A. caviae and A. veronii isolates circulating in Mexico and Spain possess a limited array of virulence genes. Enterobacterial repetitive intergenetic consensus - polymerase chain reaction showed that the Aeromonas populations sampled lack dominant clones and were genetically heterogeneous, with A. caviae being the most diverse species. Further surveys of virulence determinants in genetically heterogeneous populations of Aeromonas isolates circulating worldwide are required to enhance the understanding of their capacity to cause disease.
Assuntos
Aeromonas/genética , Aeromonas/patogenicidade , Infecções por Bactérias Gram-Negativas/microbiologia , Aeromonas/isolamento & purificação , Southern Blotting/métodos , Análise por Conglomerados , Diarreia/microbiologia , Variação Genética , Humanos , México , Reação em Cadeia da Polimerase/métodos , Espanha , Virulência/genéticaRESUMO
Aeromonas hydrophila strains recovered from clinical samples and ambient sources were phenotypically and genetically identified. In addition, the distribution of putative virulence factors was assayed. To determine the genetic diversity of these strains, random amplification of polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus (ERIC)-PCR markers were used. The discriminatory ability of the techniques, using Simpson's index, was 0.96 for both methods. The most consistent dendrogram was obtained when RAPD and ERIC data were combined. The genetic diversity revealed a high intra-specific genetic diversity (h=0.364+/-0.024 and I=0.538+/-0.030). The strains showed a tendency to cluster according to their origin of isolation (best-cut test 0.80 and bootstrap values >50%). The present study demonstrates and quantifies the high intra-specific diversity within this species and reveals a clear differentiation of strains according to their ecological origin. The distribution of virulence-related genes confirm that A. hydrophila is a genetically heterogeneous species that harbour ecotypes which have different pathogenic potential to human and other animals.