Reversing the biofilm-inducing effect of two xanthones from Garcinia mangostana by 3-methyl-2(5H)-furanone and N-butyryl-D-L homoserine lactone.

BACKGROUND: According to the WHO, 12 bacteria cause numerous human infections, including Enterobacteriaceae Klebsiella pneumoniae, and thus represent a public health problem. Microbial resistance is associated with biofilm formation; therefore, it is critical to know the biofilm-inducing potential of various compounds of everyday life. Likewise, the reversibility of biofilms and the modulation of persister cells are important for controlling microbial pathogens. In this work, we investigated the biofilm-inducing effects of xanthones from Garcinia mangostana on Klebsiella pneumoniae. Furthermore, we investigated the reversal effect of 3-methyl-2(5H)-furanone and the formation of persister cells induced by xanthones and their role in modulating the biofilm to the antibiotic gentamicin. METHODS: To analyze the biofilm-inducing role of xanthones from Garcinia mangostana, cultures of K. pneumoniae containing duodenal probe pieces were treated with 0.1-0.001 µM α- and γ-mangostin, and the biofilm levels were measured using spectrophotometry. To determine biofilm reversion, cultures treated with xanthones, or gentamicin were mixed with 3-methyl-2(5H)-furanone or N-butyryl-DL-homoserine lactone. The presence of K. pneumoniae persister cells was determined by applying the compounds to the mature biofilm, and the number of colony-forming units was counted. RESULTS: The xanthones α- and γ-mangostin increased K. pneumoniae biofilm production by 40% with duodenal probes. However, 3-methyl-2(5H)-furanone at 0.001 µΜ reversed biofilm formation by up to 60%. Moreover, adding the same to a culture treated with gentamicin reduced the biofilm by 80.5%. This effect was highlighted when 3-methyl-2(5H)-furanone was administered 6 h later than xanthones. At high concentrations of α-mangostin, persister K. pneumoniae cells in the biofilm were about 5 - 10 times more abundant than cells, whereas, with γ-mangostin, they were about 100 times more. CONCLUSION: Two xanthones, α- and γ-mangostin from G. mangostana, induced biofilm formation in K. pneumoniae and promoted persister cells. However, the biofilm formation was reversed by adding 3-methyl-2(5H)-furanone, and even this effect was achieved with gentamicin. In addition, this compound controlled the persister K. pneumoniae cells promoted by α-mangostin. Thus, synthetic, and natural biofilm-inducing compounds could harm human health. Therefore, avoiding these substances and looking for biofilm inhibitors would be a strategy to overcome microbial resistance and recover antibiotics that are no longer used.

Biodégradation of cyanide via recombinant cyanide dehydratase from Bacillus pumilus expressed heterologously in Escherichia coli / Biodegradación de cianuro por la enzima cianuro dihidratasa de Bacillus pumilus expresada heterologamente en Escherichia coli

ABSTRACT Despite its high toxicity, cyanide is used in several industrial processes, and as a result, large volumes of cyanide wastewater need to be treated prior to discharge. Enzymatic degradation of industrial cyanide wastewater by cyanide dihydratase, which is capable of converting cyanide to ammonia and formate, is an attractive alternative to conventional chemical methods of cyanide decontamination. However, the main impediment to the use of this enzyme for the biodegradation of cyanide is its intolerance to the alkaline pH at which cyanide waste is kept and its low thermoresistance. In the present study, the catalytic properties of whole Escherichia coli cells overexpressing a cyanide dihydratase gene from Bacillus pumilus were compared to those of the purified enzyme under conditions similar to those found in industrial cyanide wastewater. In addition, the capacity of whole cells to degrade free cyanide in contaminated wastewater resulting from the gold mining process was also determined. The characteristics of intracellular enzyme, relative to purified enzyme, included increased thermostability (>60% activity at 50°C), as well as greater tolerance to heavy metals, and to a lesser extent pH (20% activity remaining at pH 9.0) On the other hand, enzymatic degradation of 70% of free cyanide (initial concentration 528 mM) in the industrial sample was achieved only after dilution. Nevertheless, the increased thermostability observed for intracellular CynD suggest that whole cells of E. coli overexpressing CynD are suited for process that operate at elevated temperatures (50°C), a limitation observed for the purified enzyme.

RESUMEN A pesar de su alta toxicidad, el cianuro es usado en diversos procesos industriales, y como resultado, grandes volúmenes de aguas residuales de cianuro deben ser tratados antes de su descarga. Una alternativa atractiva a los métodos químicos convencionales de descontaminación es la degradación enzimática por la enzima cianuro dihidratasa, la cual es capaz de convertir cianuro en amonio y ácido fórmico. No obstante, la inactivación de esta enzima a pH superior a 8.5 y su poca termoestabilidad han sido el principal impedimento para la implementación exitosa de esta alternativa de biorremediación. En el presente estudio, las propiedades catalíticas de células completas de Escherichia coli que sobre expresan el gen de cianuro dihidratasa de Bacillus pumilus se estudian bajo condiciones similares a las encontradas en aguas residuales industriales de cianuro y los resultados se discuten en comparación con las de la enzima purificada. Además, se determinó la capacidad de las células completas para degradar el cianuro libre en aguas residuales resultantes del proceso de extracción de oro. Las características de la enzima intracelular, relativa a la enzima purificada, incluyeron un incremento en la termoestabilidad (>60% actividad a 50°C), así como mayor tolerancia a metales pesados y en menor medida al pH (20% actividad residual a pH 9.0). Por otra parte, la degradación enzimática del 70% del cianuro libre en la muestra industrial (concentración inicial 528 mM) se logró solo después de la dilución de la muestra. Sin embargo, el incremento en la termoestabilidad observado para CynD intracelular sugiere que las células completas de E. coli que sobre expresan CynD son adecuadas para procesos que operan a temperaturas elevadas (50°C), una limitación observada para la enzima purificada.

Immobilization of E. coli expressing Bacillus pumilus CynD in three organic polymer matrices.

Cyanide is toxic to most living organisms. The toxicity of cyanide derives from its ability to inhibit the enzyme cytochrome C oxidase of the electronic transport chain. Despite its high toxicity, several industrial processes rely on the use of cyanide, and considerable amounts of industrial waste must be adequately treated before discharge. Biological treatments for the decontamination of cyanide waste include the use of microorganisms and enzymes. Regarding the use of enzymes, cyanide dihydratase (CynD), which catalyzes the conversion of cyanide into ammonia and formate, is an attractive candidate. Nevertheless, the main impediment to the effective use of this enzyme for the biodegradation of cyanide is the marked intolerance to the alkaline pH at which cyanide waste is kept. In this work, we explore the operational capabilities of whole E. coli cells overexpressing Bacillus pumilus CynD immobilized in three organic polymer matrices: chitosan, polyacrylamide, and agar. Remarkably, the immobilized cells on agar and polyacrylamide retained more than 80% activity even at pH 10 and displayed high reusability. Conversely, the cells immobilized on chitosan were not active. Finally, the suitability of the active complexes for the degradation of free cyanide from a solution derived from the gold processing industry was demonstrated.