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% foram classificados como E. coli enterotoxigênica (ETEC), 2,5% como E. coli produtora de shiga toxina (STEC) e 43,8%, 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% foram obtidos de amostras clínicas, 8,57% de leitões sadios e 12,8% de amostras ambientais.

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% were classified as enterotoxigenic E. coli (ETEC), 2.5% were shiga toxin-producing E. coli (STEC), and 43.8% 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% of clinical isolates, 8.57% of non-diarrheic feces, and 12.8% of environment.

RESUMO A suinocultura moderna tem propiciado a obtenção de índices produtivos positivos, entretanto tem predisposto os suínos a um grande número de doenças. A ocorrência dessas enfermidades estimulou o uso indiscriminado das drogas antimicrobianas na prevenção de infecções.Escherichia coli é um dos principais patógenos da suinocultura e se caracteriza pela alta resistência aos agentes antimicrobianos. A habilidade deste patógeno na transmissão horizontal da resistência aos antimicrobianos decorre de vários mecanismos genéticos e possui sérias implicações à saúde pública. Dentre os problemas associados à disseminação da resistência múltipla aos antimicrobianos, podemos citar a contaminação do homem e dos animais por bactérias patogênicas de difícil controle terapêutico, principalmente por meio dos alimentos e de ambiente contaminados. Esta revisão tem como objetivo abordar aspectos relevantes de E. coli relativos ao seu potencial patogênico em suínos e à sua resistência às drogas antimicrobianas. Além disso, também apresenta algumas das alternativas aos usos desses fármacos na suinocultura.

ABSTRACT Modern porcine-raising practice has provided positive results in pork production, however this practice predisposes the animals to a large number of diseases. The occurrence of infectious diseases has stimulated pork producers to engage in the indiscriminate use of antimicrobial drugs. Escherichia coli is one of the most important pathogens in swine production systems and has been characterized by multi-resistance to antimicrobial drugs. The ability of this bacteria in the horizontal spread of antibiotic resistance is associated to several genetic mechanisms and has many public health implications. Among the problems associated to the spread of multi-resistance are the contamination of humans and animals by pathogenic bacteria of difficult therapeutic control, mainly by contamination of their food and environment. This review aims to discuss important aspects related to the pathogenic potential of E. coli in swine and its resistance to antimicrobial drugs. In addition, it presents some alternatives to the use of antimicrobial drugs in swine production.

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.

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.

Com o objetivo de melhorar os sistemas de transferência gênica e mutagênese para Azospirillum brasilense, a técnica de mutagênese através do uso de um gene marcador ("gene-cartridge mutagenesis") foi utilizada para substituir a região genômica de A. brasilense correspondente ao gene nifD por um segmento de DNA do transposon Tn5 contendo o gene que confere resistência ao antibiótico canamicina. A construção foi transferida para a linhagem de A. brasilense por eletrotransformação. Doze colônias transformantes foram isoladas com o plasmídeo suicida pSUP202 servindo como vetor. Dessas, somente quatro não possuíam o vetor integrado no cromossomo da bactéria. Independentemente da integração ou não do vetor, as 12 colônias foram deficientes na redução do gás acetileno, evidenciando o fenótipo Nif -. Quatro mutantes Nif - foram analisados através da técnica de Southern blot, utilizando-se seis diferentes fragmentos contendo genes nif, de resistência à canamicina e do vetor como sondas. Os resultados sugerem a ocorrência de eventos recombinacionais variados no genoma dos mutantes. A combinação entre a disrupção gênica através da técnica de mutagênese utilizada e eletrotransformação foi, provavelmente, a causa principal do rearranjo genômico ocorrido nessas bactérias.

In order to improve the gene transfer/mutagenesis system for Azospirillum brasilense, gene-cartridge mutagenesis was used to replace the nifD gene with the Tn5 kanamycin resistance gene. The construct was transferred to A. brasilense by electrotransformation. Of the 12 colonies isolated using the suicide plasmid pSUP202 as vector, only four did not show vector integration into the chromosome. Nevertheless, all 12 colonies were deficient in acetylene reduction, indicating an Nif- phenotype. Four Nif- mutants were analyzed by Southern blot, using six different probes spanning the nif and Km r genes and the plasmid vector. Apparently, several recombination events occurred in the mutant genomes, probably caused mainly by gene disruption owing to the mutagenesis technique used: resistance gene-cartridge mutagenesis combined with electrotransformation.

NifA protein activates transcription of nitrogen fixation operons by the alternative sigma54 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.