RESUMO
BACKGROUND: The primary aim of our study was to investigate the association between intubation timing and hospital mortality in critically ill patients with coronavirus disease 2019 (COVID-19)-associated respiratory failure. We also analysed both the impact of such timing throughout the first four pandemic waves and the influence of prior noninvasive respiratory support on outcomes. METHODS: This is a secondary analysis of a multicentre, observational and prospective cohort study that included all consecutive patients undergoing invasive mechanical ventilation due to COVID-19 from across 58 Spanish intensive care units (ICUs) participating in the CIBERESUCICOVID project. The study period was between 29 February 2020 and 31 August 2021. Early intubation was defined as that occurring within the first 24â h of ICU admission. Propensity score matching was used to achieve a balance across baseline variables between the early intubation cohort and those patients who were intubated after the first 24â h of ICU admission. Differences in outcomes between early and delayed intubation were also assessed. We performed sensitivity analyses to consider a different time-point (48â h from ICU admission) for early and delayed intubation. RESULTS: Of the 2725 patients who received invasive mechanical ventilation, a total of 614 matched patients were included in the analysis (307 for each group). In the unmatched population, there were no differences in mortality between the early and delayed groups. After propensity score matching, patients with delayed intubation presented higher hospital mortality (27.3% versus 37.1%; p=0.01), ICU mortality (25.7% versus 36.1%; p=0.007) and 90-day mortality (30.9% versus 40.2%; p=0.02) compared with the early intubation group. Very similar findings were observed when we used a 48-h time-point for early or delayed intubation. The use of early intubation decreased after the first wave of the pandemic (72%, 49%, 46% and 45% in the first, second, third and fourth waves, respectively; first versus second, third and fourth waves p<0.001). In both the main and sensitivity analyses, hospital mortality was lower in patients receiving high-flow nasal cannula (HFNC) (n=294) who were intubated earlier. The subgroup of patients undergoing noninvasive ventilation (n=214) before intubation showed higher mortality when delayed intubation was set as that occurring after 48â h from ICU admission, but not when after 24â h. CONCLUSIONS: In patients with COVID-19 requiring invasive mechanical ventilation, delayed intubation was associated with a higher risk of hospital mortality. The use of early intubation significantly decreased throughout the course of the pandemic. Benefits of such an approach occurred more notably in patients who had received HFNC.
Assuntos
COVID-19 , Ventilação não Invasiva , Insuficiência Respiratória , Humanos , Estudos Prospectivos , Pandemias , Intubação Intratraqueal/efeitos adversos , Respiração Artificial/efeitos adversos , Insuficiência Respiratória/terapia , Insuficiência Respiratória/etiologia , Unidades de Terapia IntensivaRESUMO
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.
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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).
Assuntos
Queijo/microbiologia , Citosol/química , Histamina/biossíntese , Lactobacillus/fisiologia , Animais , Queijo/análise , Regulação Bacteriana da Expressão Gênica , Histidina/análise , Histidina/metabolismo , Histidina Descarboxilase/genética , Concentração de Íons de Hidrogênio , Lactobacillus/genética , Lactobacillus/isolamento & purificação , Lactobacillus/metabolismo , Viabilidade MicrobianaRESUMO
Biogenic amines (BA) - nitrogenous compounds of low molecular weight - are the result of metabolism of certain amino acids. They are biologically present in all living organisms and play essential physiological roles. However, their accumulation in foodstuffs due to the metabolic activity of certain microorganisms represents a toxicological risk. Containing such microorganisms, and with an abundance of precursor substrate amino acids, fermented foods in general, and cheeses in particular, provide an ideal matrix for the accumulation of these toxic compounds. Unfortunately, the main microorganisms responsible for BA accumulation are members of the lactic acid bacteria (LAB) group, which are also essential for the development of the organoleptic characteristics of the final product. The methods used to reduce the BA content of cheese, such as milk pasteurization, commonly fail to do so, and affect desirable non-BA-producing LAB as well. Bacteriophages have been proposed as biotechnological tools for diminishing the presence of undesirable microorganisms in dairy products. Given their specificity, they could be used to target the population of BA-producing bacteria. In this work, we aimed to explore the use of Enterococcus faecalis infecting phages as a tool to reduce the content of BA in dairy products. For this, we proceeded to the isolation and characterization of E. faecalis bacteriophage 156, a member of the family Myoviridae. Its genome was sequenced and compared with that of E. faecalis family Myoviridae phages available in public databases. Its capacity to decrease the accumulation of the BA tyramine and putrescine in an experimental laboratory-scale cheese model was proven.
RESUMO
PURPOSE: To assess incidence, related factors and characteristics of safety incidents associated with the whole process of airway management and mechanical ventilation (MV) in Spanish ICUs. MATERIALS AND METHODS: Observational, prospective, 7â¯days cross-sectional multicenter study. Airway and MV related incidents were reported using structured questionnaire. Type, characteristics, severity, avoidability and contributing factors of the incidents were assessed. RESULTS: Participant ICUs: 104. Inclusion of 1267 patients; 745 (59%) suffered one or more incidents. Incidents reported: 2492 (59% non-harm-events, 41% adverse events). Individual risk of suffering at least one incident: 66.6%. Incidence ratio (median) of incidents: 2 per 100 patient-hours. 73.7% of incidents were related to MV process, 9.5% to tracheostomy, 6.2% to non-invasive MV, 5.4% to weaning/extubation, 4.4% to intubation and 0.8% to prone position. Temporary damage was produced in 12% incidents, while 0.8% was related to permanent injuries, risk to the patient's life or contributed to death. Incidents were considered avoidable in 73.5% of cases. 98% of all incidents had 1 or more contributing factors. CONCLUSIONS: MV is a risk process in critical patients. Although most incidents did not harm patients, some caused damage and a few were related to the patient's death or permanent damage. Preventability is high.
Assuntos
Doença Iatrogênica/epidemiologia , Unidades de Terapia Intensiva/estatística & dados numéricos , Respiração Artificial/efeitos adversos , Traqueostomia/efeitos adversos , Adulto , Idoso , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Espanha/epidemiologiaRESUMO
This work reports a Lactobacillus rossiae strain (L. rossiae D87) isolated from sourdough that synthesizes putrescine - a biogenic amine that raises food safety and spoilage concerns - from arginine via the ornithine decarboxylase (ODC) pathway. The odc and potE genes were identified and sequenced. These genes respectively encode ornithine decarboxylase (Odc), which participates in the decarboxylation of ornithine to putrescine, and the ornithine/putrescine exchanger (PotE), which exchanges ornithine for putrescine. Transcriptional analysis showed that odc and potE form an operon that is regulated transcriptionally by ornithine in a dose-dependent manner. To explore the possible role of the ODC pathway as an acid stress resistance mechanism for this bacterium, the effect of acidic pHs on its transcriptional regulation and on putrescine biosynthesis was analysed. Acidic pHs induced the transcription of the odc-potE genes and the production of putrescine over that seen at neutral pH. Further, putrescine production via the ODC system improved the survival of L. rossiae D87 by counteracting the acidification of the cytoplasm when the cells were subjected to acidic conditions. These results suggest the ODC pathway of L. rossiae D87 provides a biochemical defence mechanism against acidic environments.
Assuntos
Arginina/metabolismo , Lactobacillus/metabolismo , Putrescina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Pão/microbiologia , Regulação Bacteriana da Expressão Gênica , Concentração de Íons de Hidrogênio , Lactobacillus/genética , Lactobacillus/isolamento & purificação , Família Multigênica , Óperon , Ornitina/metabolismo , Ornitina Descarboxilase/genética , Ornitina Descarboxilase/metabolismo , FilogeniaRESUMO
BACKGROUND: To assess the impact of a real-time random safety tool on structure, process and outcome indicators. METHODS: Prospective study conducted over a period of 12 months in two adult patient intensive care units. Safety rounds were conducted three days a week ascertaining 37 safety measures (grouped into 10 blocks). In each round, 50% of the patients and 50% of the measures were randomized. The impact of this safety tool was analysed on indicators of structure (safety culture, healthcare protocols), process (improvement proportion related to tool application, IPR) and outcome (mortality, average stay, rate of catheter-related bacteraemias and rate of ventilator-associated pneumonia, VAP). RESULTS: A total of 1214 patient-days were analysed. Structure indicators: the use of the safety tool was associated with an increase in the safety climate and the creation/modification of healthcare protocols (sedation/analgesia and weaning). Process indicators: Twelve of the 37 measures had an IPR > 10%; six showed a progressive decrease in the IPR over the study period. Nursing workloads and patient severity on the day of analysis were independently associated with a higher IPR in half of the blocks of variables. Outcome indicators: A significant decrease in the rate of VAP was observed. CONCLUSIONS: The real-time random safety tool improved the care process and adherence to clinical practice guidelines and was associated with an improvement in structure, process and outcome indicators.
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Tyramine and histamine are the biogenic amines (BA) most commonly found at high concentrations in food; they may even appear together at toxic concentrations. The present work examines, via real-time cell analysis, whether histamine and tyramine show synergistic toxicity towards intestinal cell cultures. Employing a constant equipotency ratio, their interaction was examined via the combination index (CI) method of Chou & Talalay. Co-treatment with tyramine and histamine was associated with a stronger cytotoxic effect than was treatment with either BA or on its own. Indeed, a synergistic interaction (CI<1) was observed in the range of concentrations found in foods. The results also show that histamine, at concentrations below the legal limit, increases the cytotoxicity of tyramine at concentrations frequently reached in some foods. The synergistic cytotoxicity of tyramine and histamine should be taken into account when establishing legal limits designed to ensure consumer safety.
Assuntos
Aminas Biogênicas/toxicidade , Células Epiteliais/efeitos dos fármacos , Histamina/toxicidade , Intestinos/citologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Análise de Alimentos , Células HT29/efeitos dos fármacos , Humanos , Intestinos/efeitos dos fármacos , Tiramina/toxicidadeRESUMO
Lactococcus lactis subsp. cremoris CECT 8666 is a lactic acid bacterium that synthesizes the biogenic amine putrescine from agmatine via the agmatine deiminase (AGDI) pathway. The AGDI genes cluster includes aguR. This encodes a transmembrane protein that functions as a one-component signal transduction system, the job of which is to sense the agmatine concentration of the medium and accordingly regulate the transcription of the catabolic operon aguBDAC. The latter encodes the proteins necessary for agmatine uptake and its conversion into putrescine. This work reports the effect of extracellular pH on putrescine biosynthesis and on the genetic regulation of the AGDI pathway. Increased putrescine biosynthesis was detected at acidic pH (pH5) compared to neutral pH. Acidic pH induced the transcription of the catabolic operon via the activation of the aguBDAC promoter PaguB. However, the external pH had no significant effect on the activity of the aguR promoter PaguR, or on the transcription of the aguR gene. The transcriptional activation of the AGDI pathway was also found to require a lower agmatine concentration at pH5 than at neutral pH. Finally, the following of the AGDI pathway counteracted the acidification of the cytoplasm under acidic external conditions, suggesting it to provide protection against acid stress.
Assuntos
Proteínas de Bactérias/genética , Citosol/metabolismo , Lactococcus lactis/metabolismo , Putrescina/biossíntese , Ácidos/metabolismo , Agmatina/metabolismo , Proteínas de Bactérias/metabolismo , Citosol/química , Regulação Bacteriana da Expressão Gênica , Homeostase , Concentração de Íons de Hidrogênio , Hidrolases/genética , Hidrolases/metabolismo , Lactococcus lactis/química , Lactococcus lactis/genética , Óperon , Transcrição GênicaRESUMO
The reduction of NaCl in food is a public health priority; high NaCl intakes have been associated with serious health problems. However, it is reported that reducing the NaCl content of cheeses may lead to an increase in the content of biogenic amines (BAs). The present work examines the effect of NaCl on the accumulation of putrescine (one of the BAs often detected at high concentration in cheese) in experimental Cabrales-like cheeses containing Lactococcus lactis subsp. cremoris CECT 8666, a dairy strain that catabolises agmatine to putrescine via the agmatine deiminase (AGDI) pathway. The genes responsible for this pathway are grouped in the AGDI cluster. This comprises a regulatory gene (aguR) (transcribed independently), followed by the catabolic genes that together form an operon (aguBDAC). Reducing the NaCl concentration of the cheese led to increased putrescine accumulation. In contrast, increasing the NaCl concentration of both pH-uncontrolled and pH-controlled (pH 6) cultures of L. lactis subsp. cremoris CECT 8666 significantly inhibited its growth and the production of putrescine. Such production appeared to be inhibited via a reduction in the transcription of the aguBDAC operon; no effect on the transcription of aguR was recorded. The present results suggest that low-sodium cheeses are at risk of accumulating higher concentrations of putrescine.
Assuntos
Queijo/microbiologia , Lactococcus lactis/crescimento & desenvolvimento , Lactococcus lactis/metabolismo , Putrescina/biossíntese , Cloreto de Sódio/farmacologia , Agmatina/metabolismo , Hidrolases/genética , Hidrolases/metabolismo , Lactococcus lactis/genética , Cloreto de Sódio/efeitos adversos , Cloreto de Sódio/análiseRESUMO
The decarboxylation of histidine -carried out mainly by some gram-positive bacteria- yields the toxic dietary biogenic amine histamine (Ladero et al. 2010 ã10.2174/157340110791233256ã [1], Linares et al. 2016 ãhttp://dx.doi.org/10.1016/j.foodchem.2015.11.013ãã [2]). The reaction is catalyzed by a pyruvoyl-dependent histidine decarboxylase (Linares et al. 2011 ã10.1080/10408398.2011.582813ã [3]), which is encoded by the gene hdcA. In order to locate conserved regions in the hdcA gene of Gram-positive bacteria, this article provides a nucleotide sequence alignment of all the hdcA sequences from Gram-positive bacteria present in databases. For further utility and discussion, see ãhttp://dx.doi.org/ 10.1016/j.foodcont.2015.11.035ãã [4].
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A selective culture medium containing acid-hydrolyzed gliadins as the sole nitrogen source was used in the search for sourdough-indigenous lactic acid bacteria (LAB) with gliadin-metabolizing activity. Twenty gliadin-degrading LAB strains were isolated from 10 sourdoughs made in different ways and from different geographical regions. Fifteen of the 20 isolated strains were identified as Lactobacillus casei, a species usually reported as subdominant in sourdough populations. The other 5 gliadin-degrading strains belonged to the more commonly encountered sourdough species Leuconostoc mesenteroides and Lactobacillus plantarum. All these strains were shown to be safe in terms of their resistance to antimicrobial agents. When individually incubated with the α2-gliadin-derived immunotoxic 33-mer peptide (97.5 ppm), half of the L. casei strains metabolized at least 50% of it within 24 h. One strain metabolized 82% of the 33-mer peptide within 8 h and made it fully disappear within 12 h. These results reveal for the first time the presence in sourdough of proteolytic L. casei strains with the capacity to individually metabolize the coeliac-disease-related 33-mer peptide.
Assuntos
Pão/microbiologia , Gliadina/metabolismo , Lacticaseibacillus casei/metabolismo , Fragmentos de Peptídeos/metabolismo , Fermentação , Hidrólise , Lacticaseibacillus casei/isolamento & purificação , Lactobacillus plantarum/metabolismo , Peptídeos/metabolismoRESUMO
Lactococcus lactis subsp. cremoris CECT 8666 (formerly GE2-14) is a dairy strain that catabolizes agmatine (a decarboxylated derivative of arginine) into the biogenic amine putrescine by the agmatine deiminase (AGDI) pathway [1]. The AGDI cluster of L. lactis is composed by five genes aguR, aguB, aguD, aguA and aguC. The last four genes are responsible for the deamination of agmatine to putrescine and are co-transcribed as a single policistronic mRNA forming the catabolic operon aguBDAC[1]. aguR encodes a transmembrane protein that functions as a one-component signal transduction system that senses the agmatine concentration of the medium and accordingly regulates the transcription of aguBDAC[2], which is also transcriptionally regulated by carbon catabolic repression (CCR) via glucose, but not by other sugars such as lactose and galactose [1], [3]. Here we report the transcriptional profiling of the aguR gene deletion mutant (L. lactis subsp. cremoris CECT 8666 ∆aguR) [2] compared to the wild type strain, both grown in M17 medium with galactose as carbon source and supplemented with agmatine. The transcriptional profiling data of AguR-regulated genes were deposited in the Gene Expression Omnibus (GEO) database under accession no. GSE59514.
RESUMO
In food, the biogenic amine (BA) histamine is mainly produced by histidine decarboxylation catalysed by microbial histidine decarboxylase. The consumption of foods containing high concentrations of histamine can trigger adverse neurological, gastrointestinal and respiratory reactions. Indeed, histamine is one of the most toxic of all BAs, and is often detected in high concentration in cheese. However, little is known about the microorganisms responsible for its accumulation in this food. In the present work, 25 histamine-producing Lactobacillus vaginalis strains were isolated from a blue-veined cheese (the first time that histamine-producing strains of this species have been isolated from any food). The restriction profiles of their genomes were analysed by PFGE, and seven lineages identified. The presence of the histidine decarboxylase gene (hdcA) was confirmed by PCR. The nucleotide sequence and genetic organisation of the histamine biosynthesis gene cluster (HDC) and its flanking regions are described for a representative strain (L. vaginalis IPLA11050).
Assuntos
Técnicas de Tipagem Bacteriana , Queijo/microbiologia , Histamina/biossíntese , Histidina Descarboxilase/genética , Lactobacillus/classificação , Família Multigênica , Sequência de Bases , Queijo/análise , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Microbiologia de Alimentos , Lactobacillus/genética , Lactobacillus/isolamento & purificação , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Enterococcus faecalis is one of the most controversial species of lactic acid bacteria. Some strains are used as probiotics, while others are associated with severe and life-threatening nosocomial infections. Their pathogenicity depends on the acquisition of multidrug resistance and virulence factors. Gelatinase, which is required in the first steps of biofilm formation, is an important virulence determinant involved in E. faecalis pathogenesis, including endocarditis and peritonitis. The gene that codes for gelatinase (gelE) is controlled by the Fsr quorum-sensing system, whose encoding genes (fsrA, fsrB, fsrC, and fsrD) are located immediately upstream of gelE. The integration of a DNA fragment into the fsr locus of a derived mutant of E. faecalis V583 suppressed the gelatinase activity and prevented biofilm formation. Sequence analysis indicated the presence of IS256 integrated into the fsrC gene at nucleotide position 321. Interestingly, IS256 is also associated with biofilm formation in Staphylococcus epidermidis and Staphylococcus aureus. This is the first description of an insertion sequence that prevents biofilm formation in E. faecalis.
Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes , Infecção Hospitalar/microbiologia , Elementos de DNA Transponíveis , Enterococcus faecalis/enzimologia , Enterococcus faecalis/fisiologia , Gelatinases/metabolismo , Infecções por Bactérias Gram-Positivas/microbiologia , Proteínas de Bactérias/genética , Enterococcus faecalis/genética , Gelatinases/genética , Humanos , Mutagênese Insercional , Percepção de QuorumRESUMO
Lactococcus lactis is the lactic acid bacterial (LAB) species most widely used as a primary starter in the dairy industry. However, several strains of L. lactis produce the biogenic amine putrescine via the agmatine deiminase (AGDI) pathway. We previously reported the putrescine biosynthesis pathway in L. lactis subsp. cremoris GE2-14 to be regulated by carbon catabolic repression (CCR) via glucose but not lactose (Linares et al., 2013). The present study shows that both these sugars repress putrescine biosynthesis in L. lactis subsp. lactis T3/33, a strain isolated from a Spanish artisanal cheese. Furthermore, we demonstrated that both glucose and lactose repressed the transcriptional activity of the aguBDAC catabolic genes of the AGDI route. Finally, a screening performed in putrescine-producing dairy L. lactis strains determined that putrescine biosynthesis was repressed by lactose in all the L. lactis subsp. lactis strains tested, but in only one L. lactis subsp. cremoris strain. Given the obvious importance of the lactose-repression in cheese putrescine accumulation, it is advisable to consider the diversity of L. lactis in this sense and characterize consequently the starter cultures to select the safest strains.
Assuntos
Repressão Catabólica , Queijo/microbiologia , Lactococcus lactis/metabolismo , Lactose/metabolismo , Putrescina/biossíntese , Animais , Bovinos , Glucose/metabolismo , Leite/microbiologiaRESUMO
Enterococcus faecalis is a commensal bacterium of the human gut that requires the ability to pass through the stomach and therefore cope with low pH. E. faecalis has also been identified as one of the major tyramine producers in fermented food products, where they also encounter acidic environments. In the present work, we have constructed a non-tyramine-producing mutant to study the role of the tyramine biosynthetic pathway, which converts tyrosine to tyramine via amino acid decarboxylation. Wild-type strain showed higher survival in a system that mimics gastrointestinal stress, indicating that the tyramine biosynthetic pathway has a role in acid resistance. Transcriptional analyses of the E. faecalis V583 tyrosine decarboxylase cluster showed that an acidic pH, together with substrate availability, induces its expression and therefore the production of tyramine. The protective role of the tyramine pathway under acidic conditions appears to be exerted through the maintenance of the cytosolic pH. Tyramine production should be considered important in the adaptability of E. faecalis to acidic environments, such as fermented dairy foods, and to survive passage through the human gastrointestinal tract.
Assuntos
Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Tiramina/biossíntese , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Concentração de Íons de Hidrogênio , Viabilidade Microbiana/efeitos dos fármacos , Família Multigênica , Tirosina Descarboxilase/biossíntese , Tirosina Descarboxilase/genéticaRESUMO
Lactococcus lactis is the most important starter culture organism used in the dairy industry. Although L. lactis species have been awarded Qualified Presumption of Safety status by the European Food Safety Authority, and Generally Regarded as Safe status by the US Food and Drug Administration, some strains can produce the biogenic amine putrescine. One such strain is L. lactis subsp. cremoris CECT 8666 (formerly L. lactis subsp. cremoris GE2-14), which was isolated from Genestoso cheese. This strain catabolizes agmatine to putrescine via the agmatine deiminase (AGDI) pathway, which involves the production of ATP and two ammonium ions. The present work shows that the availability of agmatine and its metabolization to putrescine allows for greater bacterial growth (in a biphasic pattern) and causes the alkalinization of the culture medium in a dose-dependent manner. The construction of a mutant lacking the AGDI cluster (L. lactis CECT 8666 Δagdi) confirmed the latter's direct role in putrescine production, growth, and medium alkalinization. Alkalinization did not affect the putrescine production pattern and was not essential for increased bacterial growth.
Assuntos
Proteínas de Bactérias/metabolismo , Meios de Cultura/química , Hidrolases/metabolismo , Lactococcus lactis/crescimento & desenvolvimento , Putrescina/biossíntese , Compostos de Amônio/metabolismo , Proteínas de Bactérias/genética , Queijo/análise , DNA Bacteriano/genética , Fermentação , Inocuidade dos Alimentos , Concentração de Íons de Hidrogênio , Hidrolases/genética , Lactococcus lactis/genética , Família Multigênica , MutaçãoRESUMO
Prolyl endopeptidases (PEP) (EC 3.4.21.26), a family of serine proteases with the ability to hydrolyze the peptide bond on the carboxyl side of an internal proline residue, are able to degrade immunotoxic peptides responsible for celiac disease (CD), such as a 33-residue gluten peptide (33-mer). Oral administration of PEP has been suggested as a potential therapeutic approach for CD, although delivery of the enzyme to the small intestine requires intrinsic gastric stability or advanced formulation technologies. We have engineered two food-grade Lactobacillus casei strains to deliver PEP in an in vitro model of small intestine environment. One strain secretes PEP into the extracellular medium, whereas the other retains PEP in the intracellular environment. The strain that secretes PEP into the extracellular medium is the most effective to degrade the 33-mer and is resistant to simulated gastrointestinal stress. Our results suggest that in the future, after more studies and clinical trials, an engineered food-grade Lactobacillus strain may be useful as a vector for in situ production of PEP in the upper small intestine of CD patients.