Exposure to sub-inhibitory concentrations of cefotaxime enhances the systemic colonization of Salmonella Typhimurium in BALB/c mice

It has been proposed that sub-inhibitory concentrations of antibiotics play a role in virulence modulation. In this study, we evaluated the ability of Salmonella enterica serovar Typhimurium (hereafter S. Typhimurium) to colonize systemically BALB/c mice after exposure to a sub-inhibitory concentration of cefotaxime (CTX). In vivo competition assays showed a fivefold increase in systemic colonization of CTX-exposed bacteria when compared to untreated bacteria. To identify the molecular mechanisms involved in this phenomenon, we carried out a high-throughput genetic screen. A transposon library of S. Typhimurium mutants was subjected to negative selection in the presence of a sub-inhibitory concentration of CTX and genes related to anaerobic metabolism, biosynthesis of purines, pyrimidines, amino acids and other metabolites were identified as needed to survive in this condition. In addition, an impaired ability for oxygen consumption was observed when bacteria were cultured in the presence of a sub-inhibitory concentration of CTX. Altogether, our data indicate that exposure to sub-lethal concentrations of CTX increases the systemic colonization of S. Typhimurium in BALB/c mice in part by the establishment of a fitness alteration conducive to anaerobic metabolism.     

Phenotypic Buffering in a Monogenean: Canalization and Developmental Stability in Shape and Size of the Haptoral Anchors of Ligophorus cephali (Monogenea: Dactylogyridae)

Phenotypic variation results from the balance between sources of variation and counteracting regulatory mechanisms. Canalization and developmental stability are two such mechanisms, acting at two different levels of regulation. The issue of whether or not they act concurrently as a common developmental buffering capacity has been subject to debate. We used geometric morphometrics to quantify the mechanisms that guarantee phenotypic constancy in the haptoral anchors of Ligophorus cephali. Canalization and developmental stability were appraised by estimating inter- and intra-individual variation, respectively, in size and shape of dorsal and ventral anchors. The latter variation was estimated as fluctuating asymmetry (FA) between anchor pairs. The general-buffering-capacity hypothesis was tested by two different methods based on correlations and Principal Components Analyses of the different components of size and shape variation. Evidence for FA in the dorsal and ventral anchors in both shape and size was found. Our analyses supported the hypothesis of a general developmental buffering capacity. The evidence was more compelling for shape than for size and, particularly, for the ventral anchors than for the dorsal ones. These results are in line with previous studies of dactylogyrids suggesting that ventral anchors secure a firmer, more permanent attachment, whereas dorsal anchors are more mobile. Because fixation to the host is crucial for survival in ectoparasites, we suggest that homeostatic development of the ventral anchors has been promoted to ensure the morphological constancy required for efficient attachment. Geometric morphometrics can be readily applied to other host-monogenean models, affording not only to disentangle the effects of canalization and developmental stability, as shown herein, but to further partition the environmental and genetic components of the former.     

Clade 8 and Clade 6 Strains of Escherichia coli O157:H7 from Cattle in Argentina have Hypervirulent-Like Phenotypes

The hemolytic uremic syndrome (HUS) whose main causative agent is enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a disease that mainly affects children under 5 years of age. Argentina is the country with the highest incidence of HUS in the world. Cattle are a major reservoir and source of infection with E. coli O157:H7. To date, the epidemiological factors that contribute to its prevalence are poorly understood. Single nucleotide polymorphism (SNP) typing has helped to define nine E. coli O157:H7 clades and the clade 8 strains were associated with most of the cases of severe disease. In this study, eight randomly selected isolates of EHEC O157:H7 from cattle in Argentina were studied as well as two human isolates. Four of them were classified as clade 8 through the screening for 23 SNPs; the two human isolates grouped in this clade as well, while two strains were closely related to strains representing clade 6. To assess the pathogenicity of these strains, we assayed correlates of virulence. Shiga toxin production was determined by an ELISA kit. Four strains were high producers and one of these strains that belonged to a novel genotype showed high verocytotoxic activity in cultured cells. Also, these clade 8 and 6 strains showed high RBC lysis and adherence to epithelial cells. One of the clade 6 strains showed stronger inhibition of normal water absorption than E. coli O157:H7 EDL933 in human colonic explants. In addition, two of the strains showing high levels of Stx2 production and RBC lysis activity were associated with lethality and uremia in a mouse model. Consequently, circulation of such strains in cattle may partially contribute to the high incidence of HUS in Argentina.     

Evaluation of the catalytic specificity,biochemical properties,and milk clotting abilities of an aspartic peptidase from <Emphasis Type="Italic">Rhizomucor miehei</Emphasis>

In this study, we detail the specificity of an aspartic peptidase from Rhizomucor miehei and evaluate the effects of this peptidase on clotting milk using the peptide sequence of k-casein (Abz-LSFMAIQ-EDDnp) and milk powder. Molecular mass of the peptidase was estimated at 37 kDa, and optimum activity was achieved at pH 5.5 and 55 °C. The peptidase was stable at pH values ranging from 3 to 5 and temperatures of up 45 °C for 60 min. Dramatic reductions in proteolytic activity were observed with exposure to sodium dodecyl sulfate, and aluminum and copper (II) chloride. Peptidase was inhibited by pepstatin A, and mass spectrometry analysis identified four peptide fragments (TWSISYGDGSSASGILAK, ASNGGGGEYIFGGYDSTK, GSLTTVPIDNSR, and GWWGITVDRA), similar to rhizopuspepsin. The analysis of catalytic specificity showed that the coagulant activity of the peptidase was higher than the proteolytic activity and that there was a preference for aromatic, basic, and nonpolar amino acids, particularly methionine, with specific cleavage of the peptide bond between phenylalanine and methionine. Thus, this peptidase may function as an important alternative enzyme in milk clotting during the preparation of cheese.     

HmuS and HmuQ of <Emphasis Type="Italic">Ensifer</Emphasis>/<Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> degrade heme in vitro and participate in heme metabolism in vivo

Ensifer meliloti is a nitrogen-fixing symbiont of the alfalfa legume able to use heme as an iron source. The transport mechanism involved in heme acquisition in E. meliloti has been identified and characterized, but the fate of heme once inside the cell is not known. In silico analysis of E. meliloti 1021 genome revealed no canonical heme oxygenases although two genes encoding putative heme degrading enzymes, smc01518 and hmuS, were identified. SMc01518 is similar to HmuQ of Bradyrhizobium japonicum, which is weakly homologous to the Staphylococcus aureus IsdG heme-degrading monooxygenase, whereas HmuS is homolog to Pseudomonas aeruginosa PhuS, a protein reported as a heme chaperone and as a heme degrading enzyme. Recombinant HmuQ and HmuS were able to bind hemin with a 1:1 stoichiometry and displayed a K_d value of 5 and 4 µM, respectively. HmuS degrades heme in vitro to the biliverdin isomers IX-β and IX-δ in an equimolar ratio. The HmuQ recombinant protein degrades heme to biliverdin IX-δ only. Additionally, in this work we demonstrate that humS and hmuQ gene expression is regulated by iron and heme in a RirA dependent manner and that both proteins are involved in heme metabolism in E. meliloti in vivo.     

A Heterologous Multiepitope DNA Prime/Recombinant Protein Boost Immunisation Strategy for the Development of an Antiserum against Micrurus corallinus (Coral Snake) Venom

BackgroundEnvenoming by coral snakes (Elapidae: Micrurus), although not abundant, represent a serious health threat in the Americas, especially because antivenoms are scarce. The development of adequate amounts of antielapidic serum for the treatment of accidents caused by snakes like Micrurus corallinus is a challenging task due to characteristics such as low venom yield, fossorial habit, relatively small sizes and ophiophagous diet. These features make it difficult to capture and keep these snakes in captivity for venom collection. Furthermore, there are reports of antivenom scarcity in USA, leading to an increase in morbidity and mortality, with patients needing to be intubated and ventilated while the toxin wears off. The development of an alternative method for the production of an antielapidic serum, with no need for snake collection and maintenance in captivity, would be a plausible solution for the antielapidic serum shortage.ConclusionHere we describe that the genetic immunisation with a synthetic multiepitope gene followed by booster doses with recombinant protein is a promising approach to develop an alternative antielapidic serum against M. corallinus venom without the need of collection and the very challenging maintenance of these snakes in captivity.     

Eggshell Bacterial Load Is Related to Antimicrobial Properties of Feathers Lining Barn Swallow Nests

The use of feathers to line bird’s nests has traditionally been interpreted as having a thermoregulatory function. Feather-degrading bacteria growing on feathers lining nests may have antimicrobial properties, which may provide an additional benefit to lining nests with feathers. We test the hypothesis that the production of antimicrobial substances by feather bacteria affects the microbiological environment of the nest, and therefore the bacterial density on eggshells and, indirectly, hatching success. These effects would be expected to differ between nests lined with pigmented and white feathers, because bacteria grow differently on feathers of different colors. We experimentally manipulated the composition of pigmented and unpigmented feathers in nests of the barn swallow (Hirundo rustica) and studied the antimicrobial properties against the keratin-degrading bacterium Bacillus licheniformis of bacteria isolated from feathers of each color. Analyzed feathers were collected at the end of the incubation period, and antimicrobial activity was defined as the proportion of bacteria from the feathers that produce antibacterial substances effective against B. licheniformis. Our experimental manipulation affected antimicrobial activity, which was higher in nests with only white feathers at the beginning of incubation. Moreover, white feathers showed higher antimicrobial activity than black ones. Interestingly, antimicrobial activity in feathers of one of the colors correlated negatively with bacterial density on feather of the opposite color. Finally, antimicrobial activity of white feathers was negatively related to eggshell bacterial load. These results suggest that antimicrobial properties of feathers in general and of white feathers in particular affect the bacterial environment in nests. This environment in turn affects the bacterial load on eggshells, which may affect hatching success.