Identification and functional characterization of fish IL-17 receptors suggest important roles in the response to nodavirus infection.

Th17 is a lymphocyte T helper (Th) subpopulation relevant in the control and regulation of the immune response characterized by the production of interleukin (IL)-17. This crucial cytokine family acts through their binding to the IL-17 receptors (IL-17R), having up to six members. Although the biology of fish Th17 is well-recognized, the molecular and functional characterization of IL-17 and IL-17R has been limited. Thus, our aim was to identify and characterize the IL-17R repertory and regulation in the two main Mediterranean cultured fish species, the gilthead seabream (Sparus aurata) and the European sea bass (Dicentrarchus labrax). Our in silico results showed the clear identification of six members in each fish species, from IL-17RA to IL-17RE-like, with well-conserved gene structure and protein domains with their human orthologues. All of them showed wide and constitutive transcription in naïve tissues but with highest levels in mucosal tissues, namely skin, gill or intestine. In leucocytes, T mitogens showed the strongest up-regulation in most of the il17 receptors though il17ra resulted in inhibition by most stimulants. Interestingly, in vivo nodavirus infection resulted in alterations on the transcription of il17 receptors. While nodavirus infection led to some increments in the il17ra, il17rb, il17rc and il17rd transcripts in the susceptible European sea bass, many down-regulations were observed in the resistant gilthead seabream. Our data identify the presence and conservation of six coding IL-17R in gilthead seabream and European sea bass as well as their differential regulation in vitro and upon nodavirus infection. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-024-00225-1.

Bacteriocins against biogenic amine-accumulating lactic acid bacteria in cheese: nisin A shows the broadest antimicrobial spectrum and prevents the formation of biofilms.

Cheese is a food in which toxic concentrations of biogenic amines (BA) may be reached, mainly as a consequence of the decarboxylation of determined amino acids by certain lactic acid bacteria (LAB). To maintain the food safety of cheese, environmentally friendly strategies are needed that specifically prevent the growth of BA-producing LAB and the accumulation of BA. The bacteriocins produced by LAB are natural compounds with great potential as food biopreservatives. This work examines the antimicrobial potential of 7 bacteriocin-containing, cell-free supernatants (CFS: coagulin A-CFS, enterocin A-CFS, enterocin P-CFS, lacticin 481-CFS, nisin A-CFS, nisin Z-CFS and plantaricin A-CFS) produced by LAB against 48 strains of the LAB species largely responsible for the accumulation of the most important BA in cheese, i.e., histamine, tyramine and putrescine. Susceptibility to the different CFS was strain-dependent. The histamine-producing species with the broadest sensitivity spectrum were Lentilactobacillus parabuchneri (the species mainly responsible for the accumulation of histamine in cheese) and Pediococcus parvulus. The tyramine-producing species with the broadest sensitivity spectrum was Enterococcus faecium, while Enterococcus faecalis and Enterococcus hirae were among the most sensitive putrescine producers. Nisin A-CFS was active against 31 of the 48 BA-producing strains (the broadest antimicrobial spectrum recorded). Moreover, commercial nisin A prevented biofilm formation by 67% of the BA-producing, biofilm-forming LAB strains. These findings underscore the potential of bacteriocins in the control of BA-producing LAB, and support the use of nisin A as a food grade biopreservative for keeping BA-producing LAB in check and reducing BA accumulation in cheese.

Regulation and distribution of European sea bass perforins point to their role in the adaptive cytotoxic response against NNV.

Cell-mediated cytotoxicity is a complex immune mechanism that involves the release of several killing molecules, being perforin (PRF) one of the most important effector players. Perforin is synthesized by T lymphocytes and natural killer cells in mammals and responsible for the formation of pores on the target cell membrane during the killing process. Although perforin has been extensively studied in higher vertebrates, this knowledge is very limited in fish. Therefore, in this study we have identified four prf genes in European sea bass (Dicentrarchus labrax) and evaluated their mRNA levels. All sea bass prf genes showed the typical and conserved domains of its human orthologue and were closely clustered by the phylogenetic analysis. In addition, all genes showed constitutive and ubiquitous tissular expression, being prf1.9 gene the most highly expressed in immune tissues. Subsequently, in vitro stimulation of head-kidney (HK) cells with phytohemagglutinin, a T-cell activator, showed an increase of all prf gene levels, except for prf1.3 gene. European sea bass HK cells increased the transcription of prf1.2 and prf1.9 during the innate cell-mediated cytotoxic activity against xenogeneic target cells. In addition, sea bass infected with nodavirus (NNV) showed a similar expression pattern of all prf in HK and brain at 15 days post-infection, except for prf1.3 gene and in the gonad. Finally, the use of a polyclonal antibody against PRF1.9 showed an increase of positive cells in HK, brain and gonad from NNV-infected fish. Taken together, the data seem to indicate that all prf genes, except prf1.3, appear to be involved in the European sea bass immunity, and probably in the cell-mediated cytotoxic response, with PRF1.9 playing the most important role against nodavirus. The involvement of the PRFs and the CMC activity in the vertical transmission success of the virus is also discussed.

The gene cluster associated with strong biofilm-formation capacity by histamine-producing Lentilactobacillus parabuchneri encodes a sortase-mediated pilus and is located on a plasmid.

Histamine is a biogenic amine synthesized through the enzymatic decarboxylation of the amino acid histidine. It can accumulate at high concentrations in foods through the metabolism of certain bacteria, sometimes leading to adverse reactions in consumers. In cheese, histamine can accumulate at toxic levels; Lentilactobacillus parabuchneri has been identified the major cause of this problem. Previous studies have shown some L. parabuchneri strains to form biofilms on different surfaces, posing a contamination risk during cheese production, particularly for cheeses that are processed post-ripening (e.g., grating or slicing). The food contamination they cause can result in economic losses and even foodborne illness if histamine accumulates in the final product. The aim of the present work was to identify the genes of L. parabuchneri involved in biofilm formation, and to determine their function. The genomes of six strains with different biofilm-production capacities (strong, moderate and weak) were sequenced and analysed. A cluster of four genes, similar to those involved in sortase-mediated pilus formation, was identified in the strong biofilm-producers, suggesting it to have a role in surface adhesion. Cloning and heterologous expression in Lactococcus cremoris NZ9000 confirmed its functionality and involvement in adhesion and, therefore, in biofilm formation. PacBio sequencing showed this cluster to be located on a 33.4 kb plasmid, which might increase its chances of horizontal transmission. These findings provide insight into the genetic factors associated with biofilm formation in histamine-producing L. parabuchneri, and into the risks associated with this bacterium in cheese production.

Cytotoxic activity and gene expression during in vitro adaptive cell-mediated cytotoxicity of head-kidney cells from betanodavirus-infected European sea bass.

Cell-mediated cytotoxicity (CMC) is essential in eradicating virus-infected cells, involving CD8+ T lymphocytes (CTLs) and natural killer (NK) cells, through the activation of different pathways. This immune response is well-studied in mammals but scarcely in teleost fish. Our aim was to investigate the adaptive CMC using head-kidney (HK) cells from European sea bass infected at different times with nodavirus (NNV), as effector cells, and the European sea bass brain cell line (DLB-1) infected with different NNV genotypes, as target cells. Results showed low and unaltered innate cytotoxic activity through the infection time. However, adaptive CMC against RGNNV and SJNNV/RGNNV-infected target cells increased from 7 to 30 days post-infection, peaking at 15 days, demonstrating the specificity of the cytotoxic activity and suggesting the involvement of CTLs. At transcriptomic level, we observed up-regulation of genes related to T cell activation, perforin/granzyme and Fas/FasL effector pathways as well as apoptotic cell death. Further studies are necessary to understand the adaptive role of European sea bass CTLs in the elimination of NNV-infected cells.

Characterization of the Biofilms Formed by Histamine-Producing Lentilactobacillus parabuchneri Strains in the Dairy Environment.

Lentilactobacillus parabuchneri, a lactic acid bacterium, is largely responsible for the production and accumulation of histamine, a toxic biogenic amine, in cheese. L. parabuchneri strains can form biofilms on the surface of industry equipment. Since they are resistant to cleaning and disinfection, they may act as reservoirs of histamine-producing contaminants in cheese. The aim of this study was to investigate the biofilm-producing capacity of L. parabuchneri strains. Using the crystal violet technique, the strains were first categorized as weak, moderate or strong biofilm producers. Analysis of their biofilm matrices revealed them to be mainly composed of proteins. Two strains of each category were then selected to analyze the influence on the biofilm-forming capacity of temperature, pH, carbon source, NaCl concentration and surface material (i.e., focusing on those used in the dairy industry). In general, low temperature (8 °C), high NaCl concentrations (2-3% w/v) and neutral pH (pH 6) prevented biofilm formation. All strains were found to adhere easily to beech wood. These findings increase knowledge of the biofilm-forming capacity of histamine-producing L. parabuchneri strains and how their formation may be prevented for improving food safety.

Enterococcus faecium Bacteriophage vB_EfaH_163, a New Member of the Herelleviridae Family, Reduces the Mortality Associated with an E. faecium vanR Clinical Isolate in a Galleria mellonella Animal Model.

The rise of antimicrobial resistant (AMR) bacteria is a major health concern, especially with regard to members of the ESKAPE group, to which vancomycin-resistant (VRE) Enterococcus faecium belongs. Phage therapy has emerged as a novel alternative for the treatment of AMR infections. This, however, relies on the isolation and characterisation of a large collection of phages. This work describes the exploration of human faeces as a source of new E. faecium-infecting phages. Phage vB_EfaH_163 was isolated and characterised at the microbiological, genomic, and functional levels. vB_EfaH_163 phage, a new member of Herelleviridae, subfamily Brockvirinae, has a dsDNA genome of 150,836 bp that does not harbour any virulence factors or antibiotic resistance genes. It infects a wide range of E. faecium strains of different origins, including VRE strains. Interestingly, it can also infect Enterococcus faecalis strains, even some that are linezolid-resistant. Its capacity to control the growth of a clinical VRE isolate was shown in broth culture and in a Galleria mellonella animal model. The discovery and characterisation of vB_EfaH_163 increases the number of phages that might be used therapeutically against AMR bacteria.

Investigating the biotechnological potential of lactic acid bacteria strains isolated from different Algerian dairy and farm sources.

Currently, consumption of spontaneously fermented milks is common in Algeria, making it a feasible source of diverse lactic acid bacteria (LAB) with the potential to be used as adjunct cultures to improve quality and safety of fermented dairy products. In this context, to select eligible indigenous strains which could be applied as bioprotective and/or starter cultures, the present study aimed to characterize the genomic variability, biotechnological potential, and safety of thirty-eight LAB isolated from Algerian dairy and farm sources of western Algeria. The isolates were unequivocally identified by 16S rRNA gene and fingerprint-based methods. The following species were identified: Enterococcus faecium (n = 15), Enterococcus durans (n = 2), Enterococcus hirae (n = 2), Enterococcus lactis (n = 1), Lactiplantibacillus plantarum (n = 6), Lactococcus lactis (n = 4), Levilactobacillus brevis (n = 3), Lacticaseibacillus paracasei (n = 3), Lacticaseibacillus rhamnosus (n = 1), and Pediococcus acidilactici (n = 1). Among the strains, three of them, L. lactis LGMY8, Lb. plantarum LGMY30 and Lb. paracasei LGMY31 were safe and showed some valuable biotechnological properties, such as high acidification, proteolytic activity, EPS production, and inhibition of undesirable bacteria that made them powerful candidates to be used as starter.

Effect of operational parameters on photocatalytic degradation of ethylparaben using rGO/TiO2 composite under UV radiation.

The objective of this study was to analyze the influence of different operational variables (catalyst loading, initial EtP concentration, medium pH, the presence of anions and radical scavengers) on the performance of ethylparaben (EtP) photodegradation catalyzed with an rGO/TiO2 composite. EtP was selected for study after analyzing the effect of paraben chain length on its catalytic photodegradation, finding that the photodegradation rate constant values of methyl-, ethyl-, and butyl-paraben are 0.050, 0.096, and 0.136 min-1, respectively. This indicates that the photodegradation rate constant of parabens is higher with longer alkyl chain, which augments its oxidation capacity. The percentage removal of EtP at 40 min increases from 66.3 to 98.6 % when the composite dose rises from 100 to 700 mg/L; however, an additional increase in the composite dose to 1000 mg/L does not substantively improve the photodegradation rate or percentage EtP removal (98.9 %). A rise in the initial EtP concentration from 15 to 100 mg/L reduces the percentage of degradation from 100 to 76.4 %. The percentage EtP degradation is lower with higher solution pH. The presence of HCO3- or Cl- anions in the medium reduces the degradation performance. Results obtained using positive hole and hydroxyl radical scavengers demonstrate that positive holes play an important role in EtP degradation. No degradation product evidences toxicity against the cultured human embryonic kidney cell line HEK-293.

GABA-Producing Lactococcus lactis Strains Isolated from Camel's Milk as Starters for the Production of GABA-Enriched Cheese.

The multiple health benefits attributed to the bioactive compound γ-aminobutyric acid (GABA) have prompted the food industry to investigate the development of functional GABA-rich foods via the use of GABA-producing microorganisms. This study reports the isolation of six GABA-producing Lactococcus lactis strains from camel's milk; this is the first time that such microorganisms have been isolated from milk. The sequencing and in silico analysis of their genomes, and the characterisation of their technological and safety properties, confirmed their potential as starters. Experimental cheeses made with all six strains (individually) accumulated GABA at concentrations of up to 457 mg/kg. These GABA-producing L. lactis strains could be used as starter cultures for the manufacture of functional GABA-enriched cheeses that provide health benefits to consumers.

Conjugative DNA Transfer From E. coli to Transformation-Resistant Lactobacilli.

Lactic acid bacteria (LAB) belonging to the genus classically known as Lactobacillus, recently split into 25 different genera, include many relevant species for the food industry. The well-known properties of lactobacilli as probiotics make them an attractive model also for vaccines and therapeutic proteins delivery in humans. However, scarce tools are available to accomplish genetic modification of these organisms, and most are only suitable for laboratory strains. Here, we test bacterial conjugation as a new tool to introduce genetic modifications into many biotechnologically relevant laboratory and wild type lactobacilli. Using mobilizable shuttle plasmids from a donor Escherichia coli carrying either RP4 or R388 conjugative systems, we were able to get transconjugants to all tested Lactocaseibacillus casei strains, including many natural isolates, and to several other genera, including Lentilactobacillus parabuchneri, for which no transformation protocol has been reported. Transconjugants were confirmed by the presence of the oriT and 16S rRNA gene sequencing. Serendipitously, we also found transconjugants into researcher-contaminant Staphylococcus epidermidis. Conjugative DNA transfer from E. coli to S. aureus was previously described, but at very low frequencies. We have purified this recipient strain and used it in standard conjugation assays, confirming that both R388 and RP4 conjugative systems mediate mobilization of plasmids into S. epidermidis. This protocol could be assayed to introduce DNA into other Gram-positive microorganisms which are resistant to transformation.

Physicochemical characteristics of calcined MnFe2O4 solid nanospheres and their catalytic activity to oxidize para-nitrophenol with peroxymonosulfate and n-C7 asphaltenes with air.

Manganese ferrite solid nanospheres (MSNs) were prepared by a solvothermal method and calcined at various temperatures up to 500 °C. Their surface area, morphology, particle size, weight change during calcination, surface coordination number of metal ions, oxidation state, crystal structure, crystallite size, and magnetic properties were studied. The MSNs were used as catalysts to activate potassium peroxymonosulfate (PMS) for the oxidative degradation of para-nitrophenol (PNP) from water and for the oxidation of n-C7 asphaltenes in flowing air at atmospheric (0.084 MPa) and high pressure (6 MPa). Mn was in oxidation states (II) and (III) at calcination temperature of 200 °C, and the crystalline structure corresponded to jacobsite. Mn was in oxidation states (III) and (IV) at 350 °C and in oxidation states (II), (III), and (IV) at 500 °C, and the crystalline structure was maghemite at both temperatures. MSN catalysts generated hydroxyl (HO·) and sulfate (SO4·-) radicals in the PMS activation and generated HO· radicals in the n-C7 asphaltene oxidation. In both reactions, the best catalyst was MSN calcined at 350 °C (MSN350), because it has the highest concentration of Mn(III) in octahedral B sites, which are directly exposed to the catalyst surface, and the largest total and lattice oxygen contents, favoring oxygen mobility for Mn redox cycles. The MSN350 sample reduces the decomposition temperature of n-C7 asphaltenes from 430 to 210 °C at 0.084 MPa and from 370 to 200 °C at 6.0 MPa. In addition, it reduces the effective activation energy by approximately 77.6% in the second combustion (SC) region, where high-temperature oxidation reactions take place.

Methotrexate Gold Nanocarriers: Loading and Release Study: Its Activity in Colon and Lung Cancer Cells.

In the present study, the synthesis of gold nanoparticles (AuNPs) loaded with methotrexate (MTX) has been carried out in order to obtain controlled size and monodispersed nanocarriers of around 20 nm. The characterization study shows metallic AuNPs with MTX polydispersed on the surface. MTX is linked by the replacement of citrate by the MTX carboxyl group. The drug release profiles show faster MTX release when it is conjugated, which leads to the best control of plasma concentration. Moreover, the enhanced release observed at pH 5 could take advantage of the pH gradients that exist in tumor microenvironments to achieve high local drug concentrations. AuNP-MTX conjugates were tested by flow cytometry against lung (A-549) and colon (HTC-116) cancer cell lines. Results for A-549 showed a weaker dose-response effect than for colon cancer ones. This could be related to the presence of folate receptors in line HTC-116 in comparison to line A-549, supporting the specific uptake of folate-conjugated AuNP-MTX by folate receptor positive tumor cells. Conjugates exhibited considerably higher cytotoxic effects compared with the effects of equal doses of free MTX. Annexin V-PI tests sustained the cell death mechanism of apoptosis, which is normally disabled in cancer cells.

Polyphasic Characterisation of Non-Starter Lactic Acid Bacteria from Algerian Raw Camel's Milk and Their Technological Aptitudes.

RESEARCH BACKGROUND: Consumption of spontaneously fermented camel´s milk is common in Algeria, making it a feasible source of diverse lactic acid bacteria (LAB) with the potential to be used as adjunct cultures to improve quality and safety of fermented dairy products. EXPERIMENTAL APPROACH: Twelve raw camel´s milk samples were used as a source of indigenous LAB, which were further characterised by examining39 phenotypic traits with technological relevance. RESULTS AND CONCLUSIONS: Thirty-five non-starter LAB (NSLAB) were isolated from 12 Algerian raw camel's milk samples and they were microbiologically, biochemically and genetically characterised. Some isolates showed proteolytic activity, acidifying capacity, the ability to use citrate, and to produce dextran and acetoin. Ethanol, acetaldehyde, methyl acetate, acetoin and acetic acid were the major volatile compounds detected. Cluster analysis performed using the unweighted group with arithmetic average (UPGMA) method, and based on the thirty-nine phenotypic characteristics investigated, reflected the microbial diversity that can be found in raw camel´s milk. NOVELTY AND SCIENTIFIC CONTRIBUTION: The isolated strains, from a non-typical source, showed interesting technological traits to be considered as potential adjunct cultures. Cluster analysis based on the examined phenotypic characteristics proved to be a useful tool for the typification of isolates when no genetic information is available. These findings may be of use towards an industrialised production of camel's milk dairy products.

The biogenic amine tryptamine, unlike ß-phenylethylamine, shows in vitro cytotoxicity at concentrations that have been found in foods.

ß-phenylethylamine and tryptamine are biogenic amines (BA) often found in foods. In general, BA are assumed to be toxic and their accumulation in food is not recommended. However, present knowledge regarding the toxicity of ß-phenylethylamine and tryptamine is limited; more information is needed if qualitative and quantitative risk assessments of foods are to be successfully conducted. This study describes a real-time analysis of ß-phenylethylamine and tryptamine toxicity on a human intestinal epithelial cell line. Both BA caused cell necrosis and apoptosis, although the former was the main mode of action of ß-phenylethylamine, and the latter the main mode of action of tryptamine. Only tryptamine was cytotoxic at concentrations found in BA-rich foods. The results presented in this work may contribute to establish legal limits for ß-phenylethylamine and tryptamine in food.

Hemodiafiltration with ultrafiltrate regeneration reduces free light chains without albumin loss in multiple myeloma patients.

BACKGROUND: Acute kidney injury (AKI) occurs in 12-20% of multiple myeloma (MM) patients. Several studies have shown a reduction of free light chains (FLC) using hemodialysis with High-Cut-Off membranes. However, this technique entails albumin loss. Hemodiafiltration with ultrafiltrate regeneration is a technique that includes a process of adsorption. The aim of this study was to evaluate the effectiveness of hemodiafiltration with ultrafiltrate regeneration in reducing FLC levels without causing albumin loss. METHODS: This is an observational study (2012 to 2018) including nine patients with MM (5 kappa, 4 lambda) and AKI. All patients were treated with chemotherapy and hemodiafiltration with ultrafiltrate regeneration. Blood Samples (pre and post-dialysis) and ultrafiltrate were collected pre and post-resin at 5 min after initiation of the session and 5 min before the end of the procedure. RESULTS: The serum levels of kappa and lambda were reduced by a 57.6 ± 10% and 33.5 ± 25% respectively. Serum albumin concentration remained unchanged after the procedure. In the ultrafiltrate, the mean FLC reduction ratio shortly after initiation of the dialysis procedure was: 99.2 and 97.06% for kappa and lambda respectively, and only 0.7% for albumin; and at the end of the session the percent reduction was: 63.7 and 33.62% for kappa and lambda respectively, and 0.015% for albumin. Patients clinical outcome was: 33.3% recovered renal function, 22.2% died during the first year and 44.4% required maintenance dialysis. CONCLUSIONS: Hemodiafiltration with ultrafiltrate regeneration reduces FLC levels without producing a significant loss of albumin; and, FLC removal is maintained throughout the session. Therefore, hemodiafiltration with ultrafiltrate regeneration may be considered an effective adjunctive therapy in patients with MM.

Histamine production in Lactobacillus vaginalis improves cell survival at low pH by counteracting the acidification of the cytosol.

Histamine, one of the most toxic and commonly encountered biogenic amines (BA) in food, is produced by the microbial decarboxylation of histidine. It may accumulate at high concentrations in fish and fermented food. Cheese has some of the highest histamine concentrations, the result of the histidine-decarboxylase activity of certain lactic acid bacteria (LAB). The present work describes the nucleotide sequence and transcriptional organization of the gene cluster responsible for histamine biosynthesis (the HDC cluster) in Lactobacillus vaginalis IPLA 11064 isolated from cheese. The influence of histidine availability and pH on histamine production and the expression of the HDC cluster genes is also examined. As expected, the results suggest that the production of histamine under acidic conditions improves cell survival by maintaining the cytosol at an appropriate pH. However, the transcriptional regulation of the HDC cluster is quite different from that described in other dairy histamine-producing LAB, probably due to the lack of a termination-antitermination system in the histidyl-tRNA synthetase gene (hisS).

Isolation and Characterization of Enterococcus faecalis-Infecting Bacteriophages From Different Cheese Types.

Enterococci are a diverse group of Gram-positive, lactic acid bacteria (LAB). They are found in many environments, including fermented foods, in which they could constitute a health threat since they produce biogenic amines, which consumption can lead to intoxication. Moreover, enterococci has also emerged as an important hospital-acquired pathogens via its acquisition of antimicrobial resistance. Bacteriophages possess features that make them optimal biotechnological weapons for controlling bacterial growth in disease and food spoilage contexts. However, no silver bullet bacteriophage exists that can eliminate all the undesirable bacteria in a complex environment. Rather, a combination of phages with different host ranges would be required which implies the need for large collections of diverse phages. This work reports the isolation of several Enterococcus faecalis-infecting bacteriophages from different types of cheese, along with the range of E. faecalis strains of diverse origin (from dairy to clinical environments) they are able to infect. The isolated phages showed a large diversity regarding the number and origin of strains they infect. Some of these phages were subjected to morphological and genomic characterization which confirmed their diversity and showed they belong to different families and genera. The present findings increase the potential arsenal for the bacteriophage-based biocontrol of harmful E. faecalis populations.

Construction and characterization of a double mutant of Enterococcus faecalis that does not produce biogenic amines.

Enterococcus faecalis is a lactic acid bacterium characterized by its tolerance of very diverse environmental conditions, a property that allows it to colonize many different habitats. This species can be found in food products, especially in fermented foods where it plays an important role as a biopreservative and influences the development of organoleptic characteristics. However, E. faecalis also produces the biogenic amines tyramine and putrescine. The consumption of food with high concentrations of these compounds can cause health problems. The present work reports the construction, via homologous recombination, of a double mutant of E. faecalis in which the clusters involved in tyramine and putrescine synthesis (which are located in different regions of the chromosome) are no longer present. Analyses showed the double mutant to grow and adhere to intestinal cells normally, and that the elimination of genes involved in the production of tyramine and putrescine has no effect on the expression of other genes.