Biofilms, defined as aggregates of microorganisms embedded in a self-produced matrix of extracellular polymeric substances (EPS), are formed by most bacteria in both natural and pathogenic ecosystems. In aquaculture, biofilms pose a dual challenge: they confer recalcitrance to antimicrobials treatments and contribute to persistent infections by forming on facility surfaces such as tanks, nets, cages, and equipment. Tenacibaculum maritimum, the causative agent of tenacibaculosis, is responsible for significant economic losses in fish farming. Although the antibacterial activity of fish skin mucus against this pathogen has been evaluated in vitro, its effects on T. maritimum biofilms have not yet been determined. In this study, we provide a simple methodology for the in vitro formation and quantification of T. maritimum biofilms to monitor antibacterial properties of different compounds or substances, such as fish skin mucus. For this purpose, biofilm formation was assessed under varying culture volumes (200, 300, and 400 µL) and incubation times (24, 48, and 72 hours) in 48-well microplates. Then, the effects of gilthead seabream (Sparus aurata) skin mucus were evaluated on planktonic growth, biofilm formation, and biofilm dispersion, measuring both biomass and metabolic activity. Based on the tested volumes and incubation times, the optimal condition for biofilm formation was defined as 24 hours in MB at 25 ºC using 200 µL culture volume. These conditions supported the development of a biofilm (OD570>1.5 after crystal violet staining) while conserving time and mucus. Seabream mucus significantly impaired T. maritimum planktonic growth and biofilm formation in a concentration-dependent manner. Non-diluted mucus completely inhibited planktonic growth and biofilm metabolic activity, and reduced biofilm biomass by 81.16 ± 2.54%. In contrast, its effect on mature biofilms was limited, with reductions of approximately 50% in metabolic activity and 40% in biomass. This study provides a platform to assess how different fish culture conditions affect the host’s susceptibility to T. maritimum infections, which is crucial for preventing economic losses in fish farming. Additionally, it opens the door to studies analyzing the components of fish skin mucus responsible for its antibacterial activity, aiming to develop novel therapeutic compounds for targeting biofilms formed by this pathogen.

Genomic studies with Salmonella enterica serovar Typhimurium reveal a crucial role of horizontal gene transfer (HGT) in the acquisition of accessory cellular functions involved in host-interaction. Many virulence genes are located in genomic islands, plasmids and prophages. GreA and GreB proteins, Gre factors, interact transiently with the RNA polymerase alleviating backtracked complexes during transcription elongation. The overall effect of Gre factors depletion in Salmonella expression profile was studied. Both proteins are functionally redundant since only when both Gre factors were depleted a major effect in gene expression was detected. Remarkably, the accessory gene pool is particularly sensitive to the lack of Gre factors, with 18.6% of accessory genes stimulated by the Gre factors versus 4.4% of core genome genes. Gre factors involvement is particularly relevant for the expression of genes located in genomic islands. Our data reveal that Gre factors are required for the expression of accessory genes.

Cu addition to alloys for biomedical applications has been of great interest to reduce bacterial growth. In situ-alloyed Ti6Al4V(ELI)-3at.%Cu was successfully manufactured by laser powder bed fusion (L-PBF). Even so, post-heat treatments are required to avoid distortions and/or achieve required/desired mechanical and fatigue properties. The present study is focused on the investigation of microstructural changes in L-PBF Ti6Al4V(ELI)-3at.%Cu after stress relieving and annealing treatments, as well as their influence on osteoblast and bactericidal behavior. After the stress relieving treatment, a homogenously distributed β phase and CuTi2 intermetallic precipitates were observed over the αʹ matrix. The annealing treatment led to the increase in amount and size of both types of precipitates, but also to phase redistribution along α lamellas. Although microstructural changes were not statistically significant, such increase in β and CuTi2 content resulted in an increase in osteoblast proliferation after 14 days of cell culture. A significant bactericidal behavior of L-PBF Ti6Al4V(ELI)-3at.%Cu by means of ion release was found after the annealing treatment, provably due to the easier release of Cu ions from β phase. Biofilm formation was inhibited in all on Cu-alloyed specimens with stress relieving but also annealing treatment.

Campylobacter jejuni is a foodborne pathogen causing bacterial gastroenteritis, with the highest incidence reported in Europe. The prevalence of antibiotic resistance in C. jejuni, as well as in many other bacterial pathogens, has increased over the last few years. In this report, we describe the presence of a plasmid in a multi-drug-resistant C. jejuni strain isolated from a gastroenteritis patient. Mating experiments demonstrated the transference of this genetic element (pCjH01) among C. jejuni by plasmid conjugation. The pCjH01 plasmid was sequenced and assembled, revealing high similarity (97% identity) with pTet, a described tetracycline resistance encoding plasmid. pCjH01 (47.7 kb) is a mosaic plasmid composed of a pTet backbone that has acquired two discrete DNA regions. Remarkably, one of the acquired sequences carried an undescribed variant of the aadE-sat4-aphA-3 gene cluster, providing resistance to at least kanamycin and gentamycin. Aside from the antibiotic resistance genes, the cluster also carries genes coding for putative regulators, such as a sigma factor of the RNA polymerase and an antisigma factor. Homology searches suggest that Campylobacter exchanges genetic material with distant G-positive bacterial genera.

Bacterial nucleoid-associated proteins H-NS and Hha modulate gene expression in response to environmental factors. The N-terminal domain of H-NS is involved in homomeric and heteromeric protein–protein interactions. Homomeric interaction leads to the formation of dimers and higher oligomers. Heteromeric interactions with Hha-like proteins modify the modulatory properties of H-NS. In this study, we have used NMR and mutagenesis of the N-terminal domain of H-NS to identify the Hha-binding region around helices H1 and H2 of H-NS. Two conserved arginine residues, R12 and R15, located in the same side and in adjacent turns of helix H2 are shown to be involved in two different protein-protein interactions: R12 is essential for Hha binding and does not affect H-NS dimer formation, and R15 does not affect Hha binding but is essential for the proper folding of H-NS dimers. Our results demonstrate a close structural connection between Hha–H-NS interactions and H-NS dimerization that may be involved in a possible mechanism for the modulation of the H-NS regulatory activity by Hha.

Blood by-products are an untapped source of high-quality ingredients for aquafeeds, containing a broad variety of cytokines, hormones, growth factors, proteins, bioactive peptides, and amino acids. The effects of the spray-dried porcine plasma (SDPP), a type of processed animal protein on several immune parameters, were evaluated in sea bream using ex vivo and in vitro assays. In this study, fish were fed with two isoproteic, isolipidic, and isoenergetic diets: control diet (7% fish meal, FM) and SDPP diet (2% FM and 5% SDPP). At the end of the 92-days trial, those fed the SDPP diet were larger in body weight (p < 0.05) without differences in feed conversion ratio (p > 0.05). The ex vivo immune stimulation of splenocytes indicated that SDPP had a beneficial effect in promoting systemic immunity, since the surface cell marker (cd4), pro- (il-1β), and anti-inflammatory (tgf-β1) cytokines, and genes involved in humoral immunity (IgM) were up-regulated. The co-culture assays of skin mucus corroborated that SDPP enhanced the antibacterial capacity of mucus against V. anguillarum. In addition, main mucus biomarkers did not show significant differences, except for cortisol levels which were lower in the SDPP diet. The present study indicated that SDPP may be considered a functional ingredient in aquafeeds formulated with low FM levels.

Rdar biofilm formation of Salmonella typhimurium and Escherichia coli is a common ancient multicellular behavior relevant in cell–cell and inter-organism interactions equally, as in interaction with biotic and abiotic surfaces. With the expression of the characteristic extracellular matrix components amyloid curli fimbriae and the exopolysaccharide cellulose, the central hub for the delicate regulation of rdar morphotype expression is the orphan transcriptional regulator CsgD. Gre factors are ubiquitously interacting with RNA polymerase to selectively overcome transcriptional pausing. In this work, we found that GreA/GreB are required for expression of the csgD operon and consequently the rdar morphotype. The ability of the Gre factors to suppress transcriptional pausing and the 147 bp 5′-UTR of csgD are required for the stimulatory effect of the Gre factors on csgD expression. These novel mechanism(s) of regulation for the csgD operon might be relevant under specific stress conditions.

ppGpp is an intracellular sensor that, in response to different types of stress, coordinates the rearrangement of the gene expression pattern of bacteria to promote adaptation and survival to new environmental conditions. First described to modulate metabolic adaptive responses, ppGpp modulates the expression of genes belonging to very diverse functional categories. In Escherichia coli, ppGpp regulates the expression of cellular factors that are important during urinary tract infections. Here, we characterize the role of this alarmone in the regulation of the hlyCABDII operon of the UPEC isolate J96, encoding the toxin α-hemolysin that induces cytotoxicity during infection of bladder epithelial cells. ppGpp is required for the expression of the α-hemolysin encoded in hlyCABDII by stimulating its transcriptional expression. Prototrophy suppressor mutations in a ppGpp-deficient strain restore the α-hemolysin expression from this operon to wild-type levels, confirming the requirement of ppGpp for its expression. ppGpp stimulates hlyCABDII expression independently of RpoS, RfaH, Zur, and H-NS. The expression of hlyCABDII is promoted at 37 °C and at low osmolarity. ppGpp is required for the thermoregulation but not for the osmoregulation of the hlyCABDII operon. Studies in both commensal and UPEC isolates demonstrate that no UPEC specific factor is strictly required for the ppGpp-mediated regulation described. Our data further support the role of ppGpp participating in the coordinated regulation of the expression of bacterial factors required during infection.

Horizontal gene transfer (HGT) by plasmid conjugation is a major driving force in the spread of antibiotic resistance among Enterobacteriaceae. Most of the conjugation studies are based on calculation of conjugation ratios (number of transconjugants/number of donors) after viable counting of transconjugant and donor cells. The development of robust, fast and reliable techniques for in situ monitoring and quantification of conjugation ratios might accelerate progress in understanding the impact of this cellular process in the HGT. The IncHI1 plasmids, involved in multiresistance phenotypes of relevant pathogens such as Salmonella and E. coli, are distinguished by the thermosensitivity of their conjugative transfer. Conjugation mediated by IncHI1 plasmids is more efficient at temperatures lower than 30 °C, suggesting that the transfer process takes place during the environmental transit of the bacteria. In this report, we described a methodology to monitor in situ the conjugation process during agar surface matings of the IncHI1 plasmid R27 and its derepressed derivative drR27 at different temperatures. A three-color-labeling strategy was used to visualize the spatial distribution of transconjugants within the heterogeneous environment by epifluorescence and confocal microscopy. Moreover, the fluorescent labelling was also used to quantify conjugation frequencies in liquid media by flow cytometry.

Campylobacter, a major cause of food-borne gastroenteritis worldwide, colonize the gastrointestinal tract of a wide range of animals, being birds the main reservoir. The mechanisms involved in the interaction of Campylobacter with the different hosts are poorly understood. The cytolethal distending toxin, encoded in the cdtABC operon, is considered a pivotal virulence factor during human infection. Differences in the prevalence of cdtABC genes in Campylobacter isolates from three distinct origins (wild birds, broiler chickens and humans) prompted us to further characterize their allelic variability. The sequence of cdtABC is highly conserved among broiler and human isolates. A high diversity of cdtABC alleles was found among wild bird isolates, including several alleles that do not produce any functional CDT. These results suggest that specific variants of the cdtABC operon might define the host range of specific Campylobacter jejuni isolates. Moreover, our data indicate that PCR methodology is inaccurate to characterize the prevalence of the cdt genes, since negative PCR detection can be the result of divergences in the sequence used for primer design rather than indicating the absence of a specific gene.

Tools are required for quick and easy preliminary evaluation of functional feeds efficiency on fisheries. The analysis of skin mucus biomarkers is a recent alternative approach providing a faster feed-back from the laboratory which is characterized by being less invasive, more rapid and with reduced costs. The effect of replacing fishmeal and fish protein hydrolysates by means of two porcine by-products, the porcine spray-dried plasma (SDPP) and pig protein hydrolysate (PPH), in compound diets (50.4% crude protein, 16.2% crude protein, 22.1 MJ/kg feed) was evaluated in juvenile meagre (Argyrosomus regius) during a two-months period. To determine the impact of these dietary replacements, growth and food performance were measured together with digestive enzymes activities and filet proximal composition. Additionally, skin mucus was collected and characterized by determining main mucus biomarkers (protein, glucose, lactate, cortisol, and antioxidant capacity) and its antibacterial properties, measured by the quick in vitro co-culture challenges. In comparison to the control group, the inclusion of PPH and SDPP, in meagre diets reduced growth (7.4-8.8% in body weight), increased feed conversion ratios (9.0-10.0%), results that were attributed to a reduction in feed intake values (24.2-33.0%) (P < 0.05). Porcine blood by-products did not modify the activity of gastric and pancreatic digestive enzymes as well as those involved in nutrient absorption (alkaline phosphatase) nor liver oxidative stress condition (P > 0.05). In contrast, a reduction in fillet lipid content associated to an increase in fillet protein levels were found in fish fed SDPP and PPH diets (P < 0.05). As compared to the control diet, the dietary replacement did not alter the levels of the skin mucus biomarkers related to stress (cortisol and antioxidant capacity) or nutritional status (soluble protein, glucose and lactate) (P > 0.05). Interestingly, regardless of the worst performance in somatic growth, meagre fed diets containing both tested porcine by-products showed a significantly improved antibacterial capacity of their skin mucus. This enhancement was more prominent for fish fed with the PPH diet, which may be attributed to a higher content of immunomodulatory bioactive compounds in PPH. Further research will be necessary to provide insights on how the inclusion of SDPP and PPH, at the expense of dietary fishmeal and fish protein hydrolysates, affects feed intake and growth performance in meagre. However, the use of skin mucus biomarkers has been demonstrated to be an excellent methodology for a preliminary characterization of the functional feeds, in particular for their prophylactic properties by the study of mucus antibacterial activity.

Campylobacter jejuni causes campylobacteriosis, a bacterial gastroenteritis with high incidence worldwide. Moreover, C. jejuni infection can trigger the polyneuropathic disorder denominated Guillain-Barré syndrome (GBS). The C. jejuni strains that can elicit GBS carry either wlaN or cgtB, coding both genes for a β-1,3-galactosyltransferase enzyme that is required for the production of sialylated lipooligosaccharide (LOSSIAL). We described a differential prevalence of the genes wlaN and cgtB in C. jejuni isolates from three different ecological niches: humans, broiler chickens, and wild birds. The distribution of both genes, which is similar between broiler chicken and human isolates and distinct when compared to the wild bird isolates, suggests a host-dependent distribution. Moreover, the prevalence of the wlaN and cgtB genes seems to be restricted to some clonal complexes. Gene sequencing identified the presence of new variants of the G- homopolymeric tract within the wlaN gene. Furthermore, we detected two variants of a G rich region within the cgtB gene, suggesting that, similarly to wlaN, the G-tract in the cgtB gene mediates the phase variation control of cgtB expression. Caco-2 cell invasion assays indicate that there is no evident correlation between the production of LOSSIAL and the ability to invade eukaryotic cells.

Conjugation of R27 plasmid is thermoregulated, being promoted at 25°C and repressed at 37°C. Previous studies identified plasmid-encoded regulators, HtdA, TrhR and TrhY, that control expression of conjugation-related genes (tra). Moreover, the nucleoid-associated protein H-NS represses conjugation at non-permissive temperature. A transcriptomic approach has been used to characterize the effect of temperature on the expression of the 205 R27 genes. Many of the 35 tra genes, directly involved in plasmid-conjugation, were upregulated at 25°C. However, the majority of the non-tra R27 genes—many of them with unknown function—were more actively expressed at 37°C. The role of HtdA, a regulator that causes repression of the R27 conjugation by counteracting TrhR/TrhY mediated activation of tra genes, has been investigated. Most of the R27 genes are severely derepressed at 25°C in an htdA mutant, suggesting that HtdA is involved also in the repression of R27 genes other than the tra genes. Interestingly, the effect of htdA mutation was abolished at non-permissive temperature, indicating that the HtdA-TrhR/TrhY regulatory circuit mediates the environmental regulation of R27 gene expression. The role of H-NS in the proposed model is discussed.

Campylobacter is the causal agent of campylobacteriosis in humans, a self-limiting gastroenteritis. Campylobacteriosis is a zoonosis, commonly transmitted from contaminated chicken meat by either direct consumption or cross contamination during food manipulation. Presence of plasmids encoding for resistance to antibiotics such as tetracycline is common among Campylobacter isolates. In this report, we studied the effect of the temperature in the conjugation frequency of several tet(O) carrying plasmids, providing tetracycline resistance to the recipient cells. The conjugation frequency from donor cells carrying three previously characterized plasmids (pCjA13, pCjA9 and pTet) and from two clinical isolates was determined. Two temperatures, 37 and 42 °C, mimicking the conditions encountered by C. jejuni in the human and broiler chicken gastrointestinal tracts, respectively, were assessed. Our results clearly indicate that the conjugation process is promoted at high temperature. Accordingly, the transcriptional expression of some putative conjugative apparatus genes is thermoregulated, being induced at 42 °C. The two plasmids present in the clinical isolates were sequenced and assembled. Both plasmids are highly related among them and to the pTet plasmid. The high identity of the genes putatively involved in the conjugation process among the plasmids is in agreement with the similar behavior regarding the temperature dependency of the conjugative process. This report suggest that conjugation of plasmids carrying antibiotic resistance genes occurs preferentially at temperatures that resemble the gastrointestinal tract of birds, the main reservoir of C. jejuni.

SummaryPlasmid conjugation is a major horizontal gene transfer mechanism. The acquisition of a plasmid may cause a perturbation of the cell functions in addition to provide advantageous properties for the recipient cell, such as the gaining of antibiotic resistances. The interplay between plasmid and chromosomal functions has been studied using the IncHI1 plasmid R27. Plasmids of the incompatibility group HI1, isolated from several Gram‐negative pathogens, are associated with the spread of multidrug resistance. Their conjugation is tightly regulated by temperature, being repressed at temperatures within the host (37°C). In this report, we described that at permissive temperature, when conjugation of plasmid R27 is prompted, a reduction in the motility of the cells is observed. This reduction is mediated by the plasmid‐encoded regulators TrhR/TrhY, which together with HtdA form a plasmid‐borne regulatory circuit controlling R27 conjugation. TrhR/TrhY, required to induce R27 conjugation, is responsible for the downregulation of the flagella synthesis and the consequent decrease in motility. TrhR/TrhY repress, direct or indirectly, the expression of the specific flagellar sigma subunit FliA and, consequently, the expression of all genes located bellow in the flagellar expression cascade.

Campylobacter jejuni is the causal agent of the food-borne infection with the highest incidence in Europe. Both poultry and wild birds are a major reservoir. To gain insight into the population structure, virulence potential, and antimicrobial resistance (AMR), a collection of 150 isolates from three different ecological niches (broilers, wild birds, and human patients) was studied. Despite the high genetic diversity found, the population structure defined two distinct clusters, one formed mostly by broiler and human isolates and another one by most wild bird isolates. The ST-21 complex exhibits highest prevalence (in humans and broilers), followed by ST-1275 complex (only in wild birds). The ST-48, -45, and -354 complexes were found in all three niches, but represent only 22 out of 150 studied strains. A higher occurrence of AMR and multidrug resistance was detected among broiler and human isolates. Moreover, significant differences were found in the distribution of certain putative virulence genes. Remarkably, many wild bird strains were negative for either cdtA, cdtB, or cdtC from the canonical strain 81-176, whereas all broiler and human strains were positive. These data suggest that the different variants of the cdt genes might be relevant for the efficient colonization of certain hosts by C. jejuni. Our study contributes to the understanding of the role of the diverse Campylobacter reservoirs in the transmission of campylobacteriosis to humans.

Salmonella is one of the most frequently reported causes of foodborne illness worldwide. Non-typhoidal serovars cause gastroenteritis in humans. Salmonella can grow on surfaces forming biofilms, contributing to its persistence since biofilms are difficult to eradicate due to the high resistance to antimicrobials and disinfectants. It has been described that there are two crucial biofilm promoting factors in Salmonella: curli and cellulose. The expression of both factors is coordinately regulated by the transcriptional regulator CsgD. Most biofilm studies of Salmonella have been performed by growing bacteria in low osmolarity rich medium and low temperature (25°C). In such conditions, the biofilm is formed at the air-liquid interface (pellicle biofilm). Remarkably, when Salmonella grow in minimal medium, biofilm formation switches from the air-liquid interface to the solid-liquid interface (bottom biofilm). In this report, the switching between pellicle and bottom biofilm has been characterized. Our data indicate that curli, but not cellulose, is crucial for the formation of both kinds of biofilms. In minimal medium, conditions promoting formation of bottom biofilm, a high transcriptional expression of csgD and consequently of the genes involved in the synthesis of curli and cellulose was detected. The nutritional status of the cells seems to be pivotal for the spatial distribution of the biofilms formed. When bacteria is growing in minimal medium the addition of amino acids downregulates the expression of csgB and causes the switch between bottom and pellicle biofilm. The crosstalk between general metabolism and biofilm formation is also highlighted by the fact that the metabolic sensor cAMP modulates the type of biofilm generated by Salmonella. Moreover, cAMP regulates transcriptional expression of csgD and stimulates pellicle biofilm formation, suggesting that the physiological conditions define the type of biofilm formed by Salmonella. The consequences of the switching between pellicle and bottom biofilm during either infection or survival in natural environments remain undercover.

In this report, we describe a High-Throughput Screening (HTS) to identify compounds that inhibit biofilm formation or cause the disintegration of an already formed biofilm using the Salmonella Enteritidis 3934 strain. Initially, we developed a new methodology for growing Salmonella biofilms suitable for HTS platforms. The biomass associated with biofilm at the solid-liquid interface was quantified by staining both with resazurin and crystal violet, to detect living cells and total biofilm mass, respectively. For a pilot project, a subset of 1120 extracts from the Fundación MEDINA's collection was examined to identify molecules with antibiofilm activity. This is the first validated HTS assay of microbial natural product extracts which allows for the detection of four types of activities which are not mutually exclusive: inhibition of biofilm formation, detachment of the preformed biofilm and antimicrobial activity against planktonic cells or biofilm embedded cells. Currently, several extracts have been selected for further fractionation and purification of the active compounds. In one of the natural extracts patulin has been identified as a potent molecule with antimicrobial activity against both, planktonic cells and cells within the biofilm. These findings provide a proof of concept that the developed HTS can lead to the discovery of new natural compounds with antibiofilm activity against Salmonella and its possible use as an alternative to antimicrobial therapies and traditional disinfectants.

Plasmids of the incompatibility group HI1 (IncHI1) have been isolated from several Gram-negative pathogens and are associated with the spread of multidrug resistance. Their conjugation is tightly regulated and it is inhibited at temperatures higher than 30°C, indicating that conjugation occurs outside warm-blooded hosts. Using R27, the prototype of IncHI1 plasmids, we report that plasmid transfer efficiency in E. coli strongly depends on the physiological state of the donor cells. Conjugation frequency is high when cells are actively growing, dropping sharply when cells enter the stationary phase of growth. Accordingly, our transcriptomic assays show significant downregulation of numerous R27 genes during the stationary phase, including several tra (transfer) genes. Growth phase-dependent regulation of tra genes transcription is independent of H-NS, a silencer of horizontal gene transfer, and ppGpp and RpoS, regulators of the stationary phase, but highly dependent on the plasmid-encoded regulatory circuit TrhR/TrhY-HtdA. The metabolic sensor cAMP, whose synthesis is chromosomally encoded, is also involved in the growth phase regulation of R27 conjugation by modulating htdA expression. Our data suggest that the involvement of regulators encoded by both chromosome and plasmid are required for efficient physiological control of IncHI1 plasmid conjugation.