Expression of a novel class of bacterial Ig-like proteins is required for IncHI plasmid conjugation.

Antimicrobial resistance (AMR) is currently one of the most important challenges to the treatment of bacterial infections. A critical issue to combat AMR is to restrict its spread. In several instances, bacterial plasmids are involved in the global spread of AMR. Plasmids belonging to the incompatibility group (Inc)HI are widespread in Enterobacteriaceae and most of them express multiple antibiotic resistance determinants. They play a relevant role in the recent spread of colistin resistance. We present in this report novel findings regarding IncHI plasmid conjugation. Conjugative transfer in liquid medium of an IncHI plasmid requires expression of a plasmid-encoded, large-molecular-mass protein that contains an Ig-like domain. The protein, termed RSP, is encoded by a gene (ORF R0009) that maps in the Tra2 region of the IncHI1 R27 plasmid. The RSP protein is exported outside the cell by using the plasmid-encoded type IV secretion system that is also used for its transmission to new cells. Expression of the protein reduces cell motility and enables plasmid conjugation. Flagella are one of the cellular targets of the RSP protein. The RSP protein is required for a high rate of plasmid transfer in both flagellated and nonflagellated Salmonella cells. This effect suggests that RSP interacts with other cellular structures as well as with flagella. These unidentified interactions must facilitate mating pair formation and, hence, facilitate IncHI plasmid conjugation. Due to its location on the outer surfaces of the bacterial cell, targeting the RSP protein could be a means of controlling IncHI plasmid conjugation in natural environments or of combatting infections caused by AMR enterobacteria that harbor IncHI plasmids.

Synthetic and Bio-Derived Surfactants Versus Microbial Biosurfactants in the Cosmetic Industry: An Overview.

This article includes an updated review of the classification, uses and side effects of surfactants for their application in the cosmetic, personal care and pharmaceutical industries. Based on their origin and composition, surfactants can be divided into three different categories: (i) synthetic surfactants; (ii) bio-based surfactants; and (iii) microbial biosurfactants. The first group is the most widespread and cost-effective. It is composed of surfactants, which are synthetically produced, using non-renewable sources, with a final structure that is different from the natural components of living cells. The second category comprises surfactants of intermediate biocompatibility, usually produced by chemical synthesis but integrating fats, sugars or amino acids obtained from renewable sources into their structure. Finally, the third group of surfactants, designated as microbial biosurfactants, are considered the most biocompatible and eco-friendly, as they are produced by living cells, mostly bacteria and yeasts, without the intermediation of organic synthesis. Based on the information included in this review it would be interesting for cosmetic, personal care and pharmaceutical industries to consider microbial biosurfactants as a group apart from surfactants, needing specific regulations, as they are less toxic and more biocompatible than chemical surfactants having formulations that are more biocompatible and greener.

Gene duplications in the E. coli genome: common themes among pathotypes.

BACKGROUND: Gene duplication underlies a significant proportion of gene functional diversity and genome complexity in both eukaryotes and prokaryotes. Although several reports in the literature described the duplication of specific genes in E. coli, a detailed analysis of the extent of gene duplications in this microorganism is needed. RESULTS: The genomes of the E. coli enteroaggregative strain 042 and other pathogenic strains contain duplications of the gene that codes for the global regulator Hha. To determine whether the presence of additional copies of the hha gene correlates with the presence of other genes, we performed a comparative genomic analysis between E. coli strains with and without hha duplications. The results showed that strains harboring additional copies of the hha gene also encode the yeeR irmA (aec69) gene cluster, which, in turn, is also duplicated in strain 042 and several other strains. The identification of these duplications prompted us to obtain a global map of gene duplications, first in strain 042 and later in other E. coli genomes. Duplications in the genomes of the enteroaggregative strain 042, the uropathogenic strain CFT073 and the enterohemorrhagic strain O145:H28 have been identified by a BLASTp protein similarity search. This algorithm was also used to evaluate the distribution of the identified duplicates among the genomes of a set of 28 representative E. coli strains. Despite the high genomic diversity of E. coli strains, we identified several duplicates in the genomes of almost all studied pathogenic strains. Most duplicated genes have no known function. Transcriptomic analysis also showed that most of these duplications are regulated by the H-NS/Hha proteins. CONCLUSIONS: Several duplicated genes are widely distributed among pathogenic E. coli strains. In addition, some duplicated genes are present only in specific pathotypes, and others are strain specific. This gene duplication analysis shows novel relationships between E. coli pathotypes and suggests that newly identified genes that are duplicated in a high percentage of pathogenic E. coli isolates may play a role in virulence. Our study also shows a relationship between the duplication of genes encoding regulators and genes encoding their targets.

Effect of biosurfactant extract obtained from the corn-milling industry on probiotic bacteria in drinkable yogurt.

BACKGROUND: Recent studies have proven that biosurfactants (BS) obtained from controlled fermentation have shown surfactant and antimicrobial properties. In this work a biosurfactant extract obtained from a raw agroindustrial stream from the corn-milling industry was introduced into a drinkable probiotic yogurt containing Lactobacillus casei. RESULTS: The effect of the biosurfactant extract on the probiotic population was determined under different biosurfactant concentration, temperature, and time conditions. This extract was able to reduce the surface tension of water by 30 mN/m and it was observed that its addition to a drinkable probiotic yogurt did not negatively affect the biomass of L. casei during incubation. It also had a positive effect on the population of L. casei, increasing the growth of the probiotic bacterium in the yogurt under optimum temperature conditions for the growth of L. casei, in the range of 30-40 °C. Likewise, the biosurfactant extract did not modify the homofermentative pathway of L. casei; hence no acetic acid was detected in the presence of the biosurfactant extract in the drinkable yogurt. CONCLUSION: This is the first time that a biosurfactant extract, obtained from natural sources, has been introduced into a food product like a drinkable probiotic yogurt, producing a positive effect in the growth of probiotic bacterium. © 2018 Society of Chemical Industry.

Targeting plasmid-encoded proteins that contain immunoglobulin-like domains to combat antimicrobial resistance.

Antimicrobial resistance (AMR) poses a significant threat to human health. Although vaccines have been developed to combat AMR, it has proven challenging to associate specific vaccine antigens with AMR. Bacterial plasmids play a crucial role in the transmission of AMR. Our recent research has identified a group of bacterial plasmids (specifically, IncHI plasmids) that encode large molecular mass proteins containing bacterial immunoglobulin-like domains. These proteins are found on the external surface of the bacterial cells, such as in the flagella or conjugative pili. In this study, we show that these proteins are antigenic and can protect mice from infection caused by an AMR Salmonella strain harboring one of these plasmids. Furthermore, we successfully generated nanobodies targeting these proteins, that were shown to interfere with the conjugative transfer of IncHI plasmids. Considering that these proteins are also encoded in other groups of plasmids, such as IncA/C and IncP2, targeting them could be a valuable strategy in combating AMR infections caused by bacteria harboring different groups of AMR plasmids. Since the selected antigens are directly linked to AMR itself, the protective effect extends beyond specific microorganisms to include all those carrying the corresponding resistance plasmids.

Roles of Proteins Containing Immunoglobulin-Like Domains in the Conjugation of Bacterial Plasmids.

Horizontal transfer of bacterial plasmids generates genetic variability and contributes to the dissemination of the genes that enable bacterial cells to develop antimicrobial resistance (AMR). Several aspects of the conjugative process have long been known, namely, those related to the proteins that participate in the establishment of cell-to-cell contact and to the enzymatic processes associated with the processing of plasmid DNA and its transfer to the recipient cell. In this work, we describe the roles of newly identified proteins that influence the conjugation of several plasmids. Genes encoding high-molecular-weight bacterial proteins that contain one or several immunoglobulin-like domains (Big) are located in the transfer regions of several plasmids that usually harbor AMR determinants. These Big proteins are exported to the external medium and target two extracellular organelles: the flagella and conjugative pili. The plasmid gene-encoded Big proteins facilitate conjugation by reducing cell motility and facilitating cell-to-cell contact by binding both to the flagella and to the conjugative pilus. They use the same export machinery as that used by the conjugative pilus components. In the examples characterized in this paper, these proteins influence conjugation at environmental temperatures (i.e., 25°C). This suggests that they may play relevant roles in the dissemination of plasmids in natural environments. Taking into account that they interact with outer surface organelles, they could be targeted to control the dissemination of different bacterial plasmids carrying AMR determinants. IMPORTANCE Transmission of a plasmid from one bacterial cell to another, in several instances, underlies the dissemination of antimicrobial resistance (AMR) genes. The process requires well-characterized enzymatic machinery that facilitates cell-to-cell contact and the transfer of the plasmid. Our paper identifies novel plasmid gene-encoded high-molecular-weight proteins that contain an immunoglobulin-like domain and are required for plasmid transmission. They are encoded by genes on different groups of plasmids. These proteins are exported outside the cell. They bind to extracellular cell appendages such as the flagella and conjugative pili. Expression of these proteins reduces cell motility and increases the ability of the bacterial cells to transfer the plasmid. These proteins could be targeted with specific antibodies to combat infections caused by AMR microorganisms that harbor these plasmids.

Changes in Blood Cell Deformability in Chorea-Acanthocytosis and Effects of Treatment With Dasatinib or Lithium.

Misshaped red blood cells (RBCs), characterized by thorn-like protrusions known as acanthocytes, are a key diagnostic feature in Chorea-Acanthocytosis (ChAc), a rare neurodegenerative disorder. The altered RBC morphology likely influences their biomechanical properties which are crucial for the cells to pass the microvasculature. Here, we investigated blood cell deformability of five ChAc patients compared to healthy controls during up to 1-year individual off-label treatment with the tyrosine kinase inhibitor dasatinib or several weeks with lithium. Measurements with two microfluidic techniques allowed us to assess RBC deformability under different shear stresses. Furthermore, we characterized leukocyte stiffness at high shear stresses. The results showed that blood cell deformability-including both RBCs and leukocytes - in general was altered in ChAc patients compared to healthy donors. Therefore, this study shows for the first time an impairment of leukocyte properties in ChAc. During treatment with dasatinib or lithium, we observed alterations in RBC deformability and a stiffness increase for leukocytes. The hematological phenotype of ChAc patients hinted at a reorganization of the cytoskeleton in blood cells which partly explains the altered mechanical properties observed here. These findings highlight the need for a systematic assessment of the contribution of impaired blood cell mechanics to the clinical manifestation of ChAc.

Comparative Analysis of the Thermal Conductivity of Handmade and Mechanical Bricks Used in the Cultural Heritage.

During interventions to improve the energy efficiency of cultural heritage, it is common to use methodologies that are used for current buildings with different thermal behaviour. For this reason, research has been carried out on the thermal behaviour of old brick walls by carrying out thermal flow tests in the laboratory on brickwork specimens, in order to compare the behaviour of handmade bricks and mechanical bricks from more than a century ago, and to analyse the relationship between the values of thermal conductivity, humidity, density and porosity, as well as to compare these results with those obtained by applying the procedure of the EN-1745 standard. It was concluded that bricks behave thermally differently, depending on the manufacturing process: handmade or mechanical, in both types of brick it was found that the higher the moisture content and density were, the higher the brick's thermal conductivity value. It has also been concluded that old bricks have thermal conductivity values different from those indicated in EN-1745 as a function of density, and that the ratio detected in these specimens in the dry state and in the wet state does not conform to the processes indicated in the standard. With regard to porosity, it is important to note that the greater the closed porosity, the lower the conductivity. It has been concluded that in order to intervene in cultural heritage buildings, it is necessary to carry out a specific study of the behaviour of the systems with which they were constructed.

Modulation of AggR levels reveals features of virulence regulation in enteroaggregative E. coli.

Enteroaggregative Escherichia coli (EAEC) strains are one of the diarrheagenic pathotypes. EAEC strains harbor a virulence plasmid (pAA2) that encodes, among other virulence determinants, the aggR gene. The expression of the AggR protein leads to the expression of several virulence determinants in both plasmids and chromosomes. In this work, we describe a novel mechanism that influences AggR expression. Because of the absence of a Rho-independent terminator in the 3'UTR, aggR transcripts extend far beyond the aggR ORF. These transcripts are prone to PNPase-mediated degradation. Structural alterations in the 3'UTR result in increased aggR transcript stability, leading to increased AggR levels. We therefore investigated the effect of increased AggR levels on EAEC virulence. Upon finding the previously described AggR-dependent virulence factors, we detected novel AggR-regulated genes that may play relevant roles in EAEC virulence. Mutants exhibiting high AggR levels because of structural alterations in the aggR 3'UTR show increased mobility and increased pAA2 conjugation frequency. Furthermore, among the genes exhibiting increased fold change values, we could identify those of metabolic pathways that promote increased degradation of arginine, fatty acids and gamma-aminobutyric acid (GABA), respectively. In this paper, we discuss how the AggR-dependent increase in specific metabolic pathways activity may contribute to EAEC virulence.

Characterization of extracellular and cell bound biosurfactants produced by Aneurinibacillus aneurinilyticus isolated from commercial corn steep liquor.

The presence of biosurfactants produced by a Bacillus strain in corn steep liquor (CSL), a wastewater stream of the corn milling process, has been recently discovered. However, the species responsible for their production has not been identified at the moment. Therefore, in this work, the Bacillus strain isolated from CSL, with capacity to produce biosurfactants, was subjected to amplification and sequence analysis of the 16S rRNA, being identified as Aneurinibacillus aneurinilyticus. This strain has been proved to be endospore forming and thermophile, what would explain its presence in the commercial CSL. It was observed that the strain under evaluation has the ability to produce both cell-bound and extracellular biosurfactant extracts, which were characterized in this work. The electrospray ionization mass spectrometry (ESI) analysis of the biosurfactant extracts revealed that the extracellular biosurfactant produced by Aneurinibacillus aneurinilyticus is composed by a mixture of lipopeptides, containing C16 and C18 fatty acids and amino acids, including valine, phenylalanine, proline, cysteine, histidine, aspartic acid/asparagine, alanine, glycine, leucine/isoleucine, with biomarkers between 1025-458 m/z. Conversely, the cell-bound biosurfactant extract produced by Aneurinibacillus aneurinilyticus was composed by the cyclic decapeptide gramicidin S, with a characteristic peak at 571 m/z, and lipopeptides with characteristic peaks between 1034-705 m/z, containing alanine, glycine, cysteine, serine, proline, aspartic acid/asparagine, similarly to the amino acid sequence of the extracellular biosurfactant extract.

Correction: López-Prieto et al. Fungistatic and Fungicidal Capacity of a Biosurfactant Extract Obtained from Corn Steep Water. Foods 2020, 9, 662.

The authors would like to make the following correction to the published paper [1]: a few words should be corrected in the abstract, pages 7 and 9 [...].

Water and temperature relations of Fusarium langsethiae strains and modelling of growth and T-2 and HT-2 mycotoxin production on oat-based matrices.

In the UK and Northern Europe, ripening oats can become contaminated with T-2 and HT-2 mycotoxins, produced mainly by Fusarium langsethiae. There are indicative levels related to the maximum limits for oat grain for these toxins. The objectives of this study were to examine the effect of interacting conditions of temperature (10-30 °C) and water activity (aw, 0.995-0.90) on (a) lag times prior to growth, (b) growth and (c) T-2 and HT-2 toxins by two strains of F. langsethiae isolated from oats in the UK and compare this with the type strain (Fl201059) which has been genomically sequenced, and (d) develop (and validated with published data) a probabilistic models for impacts of temperature × aw on growth and toxin production. All three strains had an optimum aw range and temperature of 0.995-0.98 and 25 °C for growth. For T-2 + HT-2 production these were 0.995 aw and 20 °C. Overall, the type strain produced higher amounts of T-2 + HT-2 with a HT-2/T-2 ratio of up to 76. Using this study data sets and those from the literature, probabilistic models were developed and validated for growth and T-2 + HT-2 toxin production in relation to temperature × aw conditions. These models, when applied in stored oats, will be beneficial in determining the conditions on the relative level of risk of contamination with these two toxins in the context of the EU indicative maximum levels.

Sequelae following infantile haemangiomas treated with propranolol.

BACKGROUND: Oral propranolol accelerates the involution of infantile haemangiomas (IHs). However, it is not clear whether IHs treated with oral propranolol are associated with fewer sequelae than when left untreated. OBJECTIVES: To quantify and describe sequelae associated with IHs treated with oral propranolol, and to explore whether treated IHs are associated with fewer sequelae than untreated IHs. MATERIALS & METHODS: This multicentre, retrospective, cohort study included patients with IH treated with oral propranolol ≥2 mg/kg for at least six months, with photographic images available at baseline and at age 4-5 years. A historical comparison cohort comprised 185 patients with untreated IHs. Main outcomes/measures were: IH features, treatment characteristics and type/degree of sequelae. RESULTS: Oral propranolol, most commonly at 2 mg/kg/day (mean duration: nine months), was initiated in 171 patients (mean age: 6.02 months). After treatment, 125 of 171 (73.1%) IHs were associated with no/minimal sequelae. The most common sequelae were telangiectasia (78%), fibrofatty tissue (37%) and anetodermic skin (28%). Deep IHs were associated with significantly fewer sequelae than other subtypes. Ulceration appeared to increase the likelihood of severe sequelae. IHs with a stepped border was associated with more severe sequelae than those with a progressive border (44% versus 27%, p < 0.05). Treated IHs resolved without sequelae or were associated with a sequela that did not need correction in 27.7% more cases than untreated IHs (RR: 1.61; p < 0.001). CONCLUSION: Among IHs treated with oral propranolol, 73% resolved without, or were associated with minimal sequelae. Deep IHs were associated fewer sequelae than other subtypes. Oral propranolol decreased the likelihood of IH sequelae requiring correction.

Gene Duplications in the Genomes of Staphylococci and Enterococci.

Gene duplications are a feature of bacterial genomes. In the present work we analyze the extent of gene duplications in the genomes of three microorganisms that belong to the Firmicutes phylum and that are etiologic agents of several nosocomial infections: Staphylococcus aureus, Enterococcus faecium, and Enterococcus faecalis. In all three groups, there is an irregular distribution of duplications in the genomes of the strains analyzed. Whereas in some of the strains duplications are scarce, hundreds of duplications are present in others. In all three species, mobile DNA accounts for a large percentage of the duplicated genes: phage DNA in S. aureus, and plasmid DNA in the enterococci. Duplicates also include core genes. In all three species, a reduced group of genes is duplicated in all strains analyzed. Duplication of the deoC and rpmG genes is a hallmark of S. aureus genomes. Duplication of the gene encoding the PTS IIB subunit is detected in all enterococci genomes. In E. faecalis it is remarkable that the genomes of some strains encode duplicates of the prgB and prgU genes. They belong to the prgABCU cluster, which responds to the presence of the peptide pheromone cCF10 by expressing the surface adhesins PrgA, PrgB, and PrgC.

Fungistatic and Fungicidal Capacity of a Biosurfactant Extract Obtained from Corn Steep Water.

Biosurfactants are surface-active compounds that are produced by microorganisms, which in addition to their surfactant capacity, can possess interesting antimicrobial activities that are used in their incorporation into the agrifood industry. In this work, the preservative capacity of a novel biosurfactant extract obtained from a residual stream of the corn-milling industry was evaluated against two different fungi (Aspergillus brasiliensis and Candida albicans) under different biosurfactant concentrations (0.33-0.99 mg/mL), temperatures (4-40 °C), and incubation times (5-11 days). All the assays started with the same concentration of fungi (2 × 106 CFU/mL). The results showed that temperature played an important role in the bactericidal and fungistatic effects of this biosurfactant extract. It was observed that at a low biosurfactant concentration (0.33 mg/mL) and low or high temperatures in the range tested, this biosurfactant extract possessed an important fungicidal effect (complete inhibition) on A. brasiliensis, while at intermediate temperatures, it achieved a fungistatic effect (50% of inhibition). Regarding C. albicans, it was observed that this strain was more resistant than A. brasiliens, although it was possible to achieve growth inhibitions of 76.3% at temperatures of 40 °C after 8 days of incubation with a biosurfactant concentration of 0.99 mg/mL. This work supports the possible application of biosurfactants extracted from corn steep water as preservatives and antimicrobial agents against fungal contaminations on agrifood products.

Can a Corn-Derived Biosurfactant Improve Colour Traits of Wine? First Insight on Its Application during Winegrape Skin Maceration versus Oenological Tannins.

In winemaking, oenological tannins are used to preserve wine colour by enhancing the antioxidant activity, taking part in copigmentation, and forming polymeric pigments with anthocyanins. As a novel processing aid, in this study, a biosurfactant extract was evaluated as a solubilizing and stabilizing agent of anthocyanins in red wine. The biosurfactant extract under evaluation was obtained from a fermented residual stream of the corn milling industry named corn steep liquor (CSL). Two red winegrape varieties (Vitis vinifera L. cv. Aglianico and Cabernet sauvignon) were studied for anthocyanin content and profile, and colour traits, during simulated skin maceration for 7 days at 25 °C, as well as polymerization and copigmentation at the end of maceration. A model wine solution was used as a control, which was added either with the CSL biosurfactant or with four different oenological tannins (from grape skin, grape seed, quebracho, and acacia). The results showed that CSL biosurfactant addition improved the colour properties of skin extracts by the formation of more stable compounds mainly through copigmentation interactions. These preliminary results highlighted that the effectiveness of CSL biosurfactant is variety-dependent; however, there is no significant protection of individual anthocyanin compounds as observed for delphinidin and petunidin forms using quebracho tannin.

Characterization and Cytotoxic Effect of Biosurfactants Obtained from Different Sources.

In this work, five biosurfactant extracts, obtained from different sources, all of them with demonstrated antimicrobial properties, were characterized and subjected to a cytotoxic study using mouse fibroblast cells (NCTC clone 929). Biosurfactant extracts obtained directly from corn steep water (CSW) showed similar surfactant characteristics to those of the extracellular biosurfactant extract produced by Bacillus isolated from CSW and grown in tryptic soy broth, observing that they are amphoteric, consisting of viscous and yellowish liquid with no foaming capacity. Contrarily, cell-bound biosurfactant extracts produced from Lactobacillus pentosus or produced by Bacillus sp isolated from CSW are nonionic, consisting of a white powder with foaming capacity. All the biosurfactants possess a similar fatty acid composition. The cytotoxic test revealed that the extracts under evaluation, at a concentration of 1 g/L, were not cytotoxic for fibroblasts (fibroblast growth > 90%). The biosurfactant extract obtained from CSW with ethyl acetate, at 1 g/L, showed the highest cytotoxic effect but above the cytotoxicity limit established by the UNE-EN-ISO10993-5. It is remarkable that the cell-bound biosurfactant produced by L. pentosus, at a concentration of 1 g/L, promoted the growth of the fibroblast up to 113%.

A Multifunctional Biosurfactant Extract Obtained from Corn Steep Water as Bactericide for Agrifood Industry.

The increase of crop production along with stricter requirements on food security have augmented the demand of new and eco-friendly bactericides. Most of the bactericides used at the moment consist of persistent organic substances, representing a risk for environmental and human health. For instance, agriculture bactericides used for crop protection includes copper-based, dithiocarbamate and amide bactericides, which are not biodegradable, resulting in the necessity of further research about the production of new active principles that attack microorganisms without producing any harmful effect on human health or environment. The biosurfactant extract evaluated in this work as a bactericide, is obtained from corn steep water, a residual stream of corn wet milling industry, which is fermented spontaneously by probiotic lactic acid bacteria that possess the capacity to produce biosurfactants. In previous works, it has been demonstrated that this biosurfactant extract is able to promote the growth of Lactobacillus casei in drinkable yogurts, though its antimicrobial activity against pathogenic strains has not been evaluated at the moment. The results obtained in this work have proved that this biosurfactant extract is effective as bactericide against Pseudomonas aeruginosa and Escherichia coli, at concentrations of 1 mg/mL, opening the door to its use in agrifood formulations for reducing the use of chemical pesticides and preservatives.