Virulence factors, antimicrobial resistance, and plasmid content of Escherichia coli isolated in swine commercial farms / Fatores de virulência, resistência aos antimicrobianos, presença de plasmídeos em Escherichia coli isoladas de amostras clínicas e ambientais de suínos

Virulence factors and antimicrobial resistance patterns of Escherichia coli isolates were evaluated. A total of 80 E. coli isolates were evaluated, being 64 from clinical samples (intestinal content and fragments of organs from diarrheic piglets), seven from feces of clinically healthy piglets and sows, and nine environmental samples (five from facilities, two from feed, one from insect, and one from waste). Molecular characterization was performed by PCR detection of fimbriae and toxin genes and plasmid content determination. The isolates were also characterized according to their resistance or sensitivity to the following drugs: ampicillin, trimethoprim:sulfamethoxazole, tetracycline, amikacine, colistin, norfloxacin, florfenicol, enrofloxacin, cefalexin, trimethoprim, neomycin, chloramphenicol, and gentamicin. From 80 E. coli isolates, 53.8 percent were classified as enterotoxigenic E. coli (ETEC), 2.5 percent were shiga toxin-producing E. coli (STEC), and 43.8 percent showed a non specific pattern and were unclassified. One fecal isolate from non-diarrheic piglet was classified as ETEC by PCR. Clinical isolates showed resistance mainly for tetracycline and trimethoprim:sulfamethoxazole. Plasmidial DNA was observed in 70 isolates, being 78.5 percent of clinical isolates, 8.57 percent of non-diarrheic feces, and 12.8 percent of environment.

Os fatores de virulência e a resistência aos antimicrobianos foram avaliados em Escherichia coli. Um total de 80 isolados de E. coli, sendo 64 de amostras clínicas (conteúdo intestinal e fragmentos de órgãos de leitões diarreicos), sete das fezes de porcas e leitões saudáveis e nove de amostras ambientais (cinco de instalações, dois de alimentos, um de inseto e um de esterqueira). A caracterização molecular feita pela PCR objetivou detectar fimbrias e toxinas, bem como a determinação do conteúdo de plasmídeos. Os isolados foram caracterizados quanto à resistência ou sensibilidade às seguintes drogas: ampicilina, sulfazotrim, tetraciclina, amikacina, colistina, norfloxacina, florfenicol, enrofloxacina, cefalexina, trimetoprim, neomicina, cloranfenicol e gentamicina. Dos 80 isolados, 53,8 por cento foram classificados como E. coli enterotoxigênica (ETEC), 2,5 por cento como E. coli produtora de shiga toxina (STEC) e 43,8 por cento, por não apresentarem padrão específico, não foram classificadas. Pela PCR, um isolado de fezes de suíno sem diarreia foi classificado como ETEC. Os isolados das amostras clínicas foram principalmente resistentes à tetraciclina e à sulfazotrim. Em 70 isolados, observaram-se DNA plasmidial, destes 78,5 por cento foram obtidos de amostras clínicas, 8,57 por cento de leitões sadios e 12,8 por cento de amostras ambientais.

Sequencing and promoter analysis of the nifENXorf3orf5fdxAnifQ operon from Azospirillum brasilense Sp7

A 40-kb DNA region containing the major cluster of nif genes has been isolated from the Azospirillum brasilense Sp7 genome. In this region three nif operons have been identified: nifHDKorf1Y, nifENXorf3orf5fdxAnifQ and orf2nifUSVorf4. The operons containing nifENX and nifUSV genes are separated from the structural nifHDKorf1Y operon by about 5 kb and 10 kb, respectively. The present study shows the sequence analysis of the 6045-bp DNA region containing the nifENX genes. The deduced amino acid sequences from the open reading frames were compared to the nif gene products of other diazotrophic bacteria and indicate the presence of seven ORFs, all reading in the same direction as that of the nifHDKorf1Y operon. Consensus sigma54 and NifA-binding sites are present only in the promoter region upstream of the nifE gene. This promoter is activated by NifA protein and is approximately two-times less active than the nifH promoter, as indicated by the ß-galactosidase assays. This result suggests the differential expression of the nif genes and their respective products in Azospirillum

Partial characterization of nif genes from the bacterium Azospirillum amazonense

Azospirillum amazonense revealed genomic organization patterns of the nitrogen fixation genes similar to those of the distantly related species A. brasilense. Our work suggests that A. brasilense nifHDK, nifENX, fixABC operons and nifA and glnB genes may be structurally homologous to the counterpart genes of A. amazonense. This is the first analysis revealing homology between A. brasilense nif genes and the A. amazonense genome. Sequence analysis of PCR amplification products revealed similarities between the amino acid sequences of the highly conserved nifD and glnB genes of A. amazonense and related genes of A. brasilense and other bacteria. However, the A. amazonense non-coding regions (the upstream activator sequence region and the region between the nifH and nifD genes) differed from related regions of A. brasilense even in nitrogenase structural genes which are highly conserved among diazotrophic bacteria. The feasibility of the 16S ribosomal RNA gene-based PCR system for specific detection of A. amazonense was shown. Our results indicate that the PCR primers for 16S rDNA defined in this article are highly specific to A. amazonense and can distinguish this species from A. brasilense

Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense

NifA protein activates transcription of nitrogen fixation operons by the alternative s54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS) located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST)-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.

The nifHDK operon in the free-living nitrogen-fixin bacteria azospirillum brasiliense sequentially comprises genes H, D, K, an 353 BP orf and gene Y

The complete nucleotide sequence of the nitrogenase structural genes form Azospirillum brasilense was determined. Two additional open reading frames of 353 and 683 base pairs were detected downstream of the nifK gene. Structures resembling the consensus nif promoter and NifA-binding motif were found only upstream from the nifH region and an inverted repeat structure locate downstream of the nifY gene may be a potential stem-and-loop transcriptional terminator. The nif structural genes of Azospirillum brasiliense are transcribed as a transcription unit and organized as nifHDK orf1 Y, NifH, NifD and NifK polypeptides share significant sequence identies when compared to nif structural gene products from other organisms. The three polypeptides are characterized by the presence of highly conserved cysteine residues which may play a role in binding the iron-sulfur cluster

Construction of a gene library from Azospirillum brasilense and characterization of a recombinant containing the nif structural genes

1.We have constructed a gene library, from Azospirillum brasilense using the vector EMBL4. 2. A recombinant containing the nif structural genes from A. brasilense was isolated and characterized. This recombinant contains a DNA insert of about 15 kilobases (KB) which gives rise to five fragments after cleavage with EcoRI. Only one of the DNA fragments (6.5 Kb) hybridized to the nifHDK genes of Klebsiella pneumoniae. 3 The organization of the nif genes in this DNA fragment was determined using different DNA segments containing the nifH, nifK or nifD genes of K. pneumoniae as probes