Virulence traits and expression of bstA, fliC and sopE2 in Salmonella Dublin strains isolated from humans and animals in Brazil.

Salmonella Dublin is a strongly cattle-adapted serovar that has also been responsible for severe invasive infections in humans. Although invasive infections by non-typhoid Salmonella have increased in developed and in developing countries, in sub-Saharan Africa these infections have been frequently related to Salmonella Typhimurium strains from Sequence Type (ST) 313 that harbor a possible virulence marker, the bstA gene, broadly detected in S. Dublin strains. The aims of this study were to verify the frequency of bstA by PCR in 113 Salmonella Dublin strains isolated from humans (83) and animals (30) in Brazil and the expression by RT-PCR of bstA, sopE2 and fliC in six strains isolated from humans (4) and animals (2). Moreover, the invasion capacity in Caco-2 human epithelial cells and U937 human macrophages, plus in vivo virulence analysis in Galleria mellonella and the motility were verified for 20 S. Dublin strains isolated from humans (15) and animals (5). All studied strains presented the bstA gene. The relative expression rates ranged from 0.1 to 2.3 fold change for bstA and from no expression to 16.6 fold change for sopE2, while no expression was detected for fliC. The invasion in Caco-2 cells ranged from 54.0 to 88.9% and in U937 cells from 72.9 to 98.1% in the 20 strains studied. In addition, 17 strains presented a highly virulent profile in the G. mellonella model and 15 strains presented a non-motile profile. In conclusion, the presence and expression of bstA in the S. Dublin strains studied suggested that this gene may influence in the invasive characteristic of this serovar. The low expression of sopE2 in strains from human invasive cases suggested that its expression may not be a limiting factor to the invasion of S. Dublin strains. The absence of fliC expression and the low motility rates observed suggest that the flagella absence may favor the host immune system evasion by S. Dublin and the establishment of infection. Moreover, the high mortality rates observed in vivo in Galleria mellonella reinforce the pathogenic potential of S. Dublin strains.

Escherichia coli as a Multifaceted Pathogenic and Versatile Bacterium.

Genetic plasticity promotes evolution and a vast diversity in Escherichia coli varying from avirulent to highly pathogenic strains, including the emergence of virulent hybrid microorganism. This ability also contributes to the emergence of antimicrobial resistance. These hybrid pathogenic E. coli (HyPEC) are emergent threats, such as O104:H4 from the European outbreak in 2011, aggregative adherent bacteria with the potent Shiga-toxin. Here, we briefly revisited the details of these E. coli classic and hybrid pathogens, the increase in antimicrobial resistance in the context of a genetically empowered multifaceted and versatile bug and the growing need to advance alternative therapies to fight these infections.

Plasmids associated with heavy metal resistance and herbicide degradation potential in bacterial isolates obtained from two Brazilian regions.

The use of pesticides has been increasing due to the great agricultural production worldwide. The pesticides are used to eradicate pests and weeds; however, these compounds are classified as toxic to non-target organisms. Atrazine and diuron are herbicides widely used to control grassy and broadleaf weeds and weed control in agricultural crops and non-crop areas. Heavy metals are also important environmental contaminants that affect the ecological system. This study aimed to investigate the presence of herbicides-degrading genes and heavy metal resistance genes in bacterial isolates from two different soil samples from two Brazilian regions and to determine the genetic location of these genes. In this study, two isolates were obtained and identified as Escherichia fergusonii and Bacillus sp. Both isolates presented atzA, atzB, atzC, atzD, atzE, atzF, puhA, and copA genes and two plasmids each, being the major with ~ 60 Kb and a smaller with ~ 3.2 Kb. Both isolates presented the atzA-F genes inside the larger plasmid, while the puhA and copA genes were detected in the smaller plasmid. Digestion reactions were performed and showed that the ~ 60-Kb plasmid presented the same restriction profile using different restriction enzymes, suggesting that this plasmid harboring the complete degradation pathway to atrazine was found in both isolates. These results suggest the dispersion of these plasmids and the multi-herbicide degradation potential in both isolates to atrazine and diuron, which are widely used in different culture types worldwide.

Evaluation of different molecular and phenotypic methods for identification of environmental Burkholderia cepacia complex.

The correct identification of different genera and bacterial species is essential, especially when these bacteria cause infections and appropriate therapies need to be chosen. Bacteria belonging to the Burkholderia cepacia complex are considered important opportunistic pathogens, causing different types of infections in immunocompromised, principally in patients with cystic fibrosis. Twenty-one isolates were obtained from different soil samples and identified by sequencing of 16S rRNA, 23S rRNA, recA gene, MLST and by VITEK 2 and MALDI-TOF MS systems. Then, statistical analyses were performed. VITEK 2 and MALDI-TOF MS systems showed different bacterial genera. Sequencing of the 16S rRNA, 23S rRNA gene and amplification of recA gene showed that all the isolates belong to the B. cepacia complex. Sequencing of the recA gene showed a predominance of B. cenocepacia. The PCR of the recA gene showed a high specificity when it is necessary to identify the bacteria belonging to the B. cepacia complex in comparison with 16S and 23S rRNA genes sequencing. MLST analyzes showed a diversity of STs, which have not yet been correlated to the species. Phenotypic identification was not suitable for the identification of these pathogens since in many cases different genera have been reported, including identification by using MALDI-TOF MS.

Change in the antimicrobial resistance profile of Pseudomonas aeruginosa from soil after exposure to herbicides.

The extensive use of pesticides represents a risk to human health and to the environment. This study aimed to investigate if the exposure to atrazine and diuron, two herbicides widely used in Brazil, could induce changes in the susceptibility profile to aztreonam, colistin and polymyxin B antimicrobials in isolates of P. aeruginosa obtained from soil samples by using the determination of minimum inhibitory concentration (MIC) test. Three isolates had an increase of MIC to aztreonam after exposure to both herbicides and one isolate did not show any MIC change. The MexAB-OprM efflux pump has already been upregulated in these isolates and the herbicides atrazine and diuron did not increase MexAB-OprM overexpression. Therefore, the decrease in aztreonam susceptibility was not directly related to this pump, suggesting that probably other mechanisms should be involved.

Buttiauxella chrysanthemi sp. nov., isolated from a chrysanthemum plantation in Brazil.

A novel Gram-negative, rod-shaped, non-motile bacterium, designated C1BT was isolated from a soil sample of a chrysanthemum plantation in Campinas, Brazil. Strain C1BT formed white colonies on BHI medium, it produces acid from D-lactose, D-mannose, D-arabinose, but does not produce from D-adonitol, m-inositol, D-melibiose, D-raffinose and D-sorbitol and it is negative for lysine and ornithine decarboxylase, phenylalanine deaminase, and citrate. Phylogenetic analysis based on 16S rRNA and rpoB genes sequences showed that strain C1BT has a similarity of 98.2 and 96.8% with different species of Buttiauxella genus. Major fatty acids were C16:0, summed features 4 (C16:1 ω7c and iso-C15:0 2OH), summed features 7 (C18:1 ω7c, C18:1 ω9t, and/or C18:1 ω12t), C17:0 cyclo, summed features 3 (iso-C16:1 I and C14:0 3OH) and C14:0. The mole percent of G+C was 49.6 mol%. Based on these results, a new species Buttiauxella chrysanthemi is proposed. The type strain is C1BT (= CPQBA 1120/15T = CMRVSP5791T).

Analysis of the Caulobacter crescentus Zur regulon reveals novel insights in zinc acquisition by TonB-dependent outer membrane proteins.

BACKGROUND: Intracellular zinc concentration needs to be maintained within strict limits due to its toxicity at high levels, and this is achieved by a finely regulated balance between uptake and efflux. Many bacteria use the Zinc Uptake Regulator Zur to orchestrate zinc homeostasis, but little is known regarding the transport of this metal across the bacterial outer membrane. RESULTS: In this work we determined the Caulobacter crescentus Zur regulon by global transcriptional and in silico analyses. Among the genes directly repressed by Zur in response to zinc availability are those encoding a putative high affinity ABC uptake system (znuGHI), three TonB-dependent receptors (znuK, znuL and znuM) and one new putative transporter of a family not yet characterized (zrpW). Zur is also directly involved in the activation of a RND and a P-type ATPase efflux systems, as revealed by ß-galactosidase and site-directed mutagenesis assays. Several genes belonging to the Fur regulon were also downregulated in the zur mutant, suggesting a putative cross-talk between Zur and Fur regulatory networks. Interestingly, a phenotypic analysis of the znuK and znuL mutants has shown that these genes are essential for growth under zinc starvation, suggesting that C. crescentus uses these TonB-dependent outer membrane transporters as key zinc scavenging systems. CONCLUSIONS: The characterization of the C. crescentus Zur regulon showed that this regulator coordinates not only uptake, but also the extrusion of zinc. The uptake of zinc by C. crescentus in conditions of scarcity of this metal is highly dependent on TonB-dependent receptors, and the extrusion is mediated by an RND and P-type ATPase transport systems. The absence of Zur causes a disturbance in the dynamic equilibrium of zinc intracellular concentration, which in turn can interfere with other regulatory networks as seen for the Fur regulon.