Citral and geraniol induce necrotic and apoptotic cell death on Saccharomyces cerevisiae.

Essential oils and their main components, monoterpenes, have been proven to be important alternatives for the control of pathogenic and spoiling microorganisms, but the mode of action of these compounds is poorly understood. This work aimed to determine the mode of action of citral and geraniol on the model yeast Saccharomyces cerevisiae using a flow cytometry approach. Exponentially growing yeast cells were treated with different concentrations of citral and geraniol for 3 h, and evaluated for cell wall susceptibility to glucanase, membrane integrity, reactive oxygen species (ROS) accumulation, mitochondrial membrane potential, and metacaspase activity. Results provide strong evidence that citral and geraniol acute fungicidal activity against Saccharomyces cells involves the loss of membrane and cell wall integrity resulting in a dose-dependent apoptotic/necrotic cell death. However, yeast cells that escape this first cell membrane disruption, particularly evident on sub-lethal concentration, die by metacaspase-mediated apoptosis induced by the accumulation of intracellular ROS. The deleted mutant on the yca1 gene showed high tolerance to citral and geraniol.

Activity of monoterpenoids on the in vitro growth of two Colletotrichum species and the mode of action on C. acutatum.

Essential oils and their main compounds, monoterpenoids, are considered as alternative control systems for phytopathogenic fungi, particularly those related to late diseases of fruits and vegetables, like anthracnose caused by Colletotrichum species. In this context, we studied the effect of twenty monoterpenoids on Colletotrichum fructicola and C. acutatum to elucidate their effectiveness and mechanisms of action. Thus, we analyzed mycelial growth and conidial inhibitory concentration, as well as the effect of selected monoterpenoids on membrane integrity and cell vitality, reactive oxygen species (ROS) accumulation, and mitochondrial membrane potential by flow cytometry. The results showed that oxygenated monoterpenoids (alcohols and aldehydes) exhibited higher antifungal activity than their corresponding hydrocarbons, esters, and cyclic counterparts. Indicating that OH- and O- radicals react with cellular components affecting fungal homeostasis. In this sense, selected monoterpenoids (citral, citronellol, geraniol, carvacrol, and thymol) inhibited conidial germination of C. acutatum in a dose-dependent manner. The inhibition of conidial germination is associated with a loss of membrane integrity, a decrease of cell metabolism, and a dose-dependent accumulation of ROS, which was non-directly associated with modifications on mitochondrial membrane potential. Membrane dysfunction and ROS accumulation may be responsible for the necrotic behavior induced by high monoterpenoids concentrations, and possible apoptotic response in sub dosages of these compounds.

Necrotic cell death induced by dithianon on Saccharomyces cerevisiae.

Dithianon is a broad-spectrum anthraquinone fungicide used to control several diseases of grapes, apples, and other fruits and vegetables. Its mode of action is described as multi-site and associated to thiol-reactivity. As other fungicides can affect non-phytopathogenic organisms as yeasts and bacteria, with impact on microbial population, diversity, and fermentation processes. In this context, we study the effect of dithianon on the model organism and fermentative yeast Saccharomyces cerevisiae in order to elucidate the mechanisms involved in yeast cell death., and explain its interference on wine fermentation kinetics. Thus for, we analyzed cellular protein and non-protein thiols, membrane and cell wall integrity, reactive oxygen species accumulation, mitochondrial membrane potential, and phosphatidylserine externalization. The results showed that when exponentially aerobic growing cells of S. cerevisiae are submitted to acute dithianon treatment they loss cell wall and membrane integrity, dying by necrosis, and this behavior is associated to a depletion of reduced proteic and non-proteic thiol groups. We also detected an important increase of cellular reactive oxygen species (ROS) associated to mitochondrial membrane potential modifications on dithianon treated cells. ROS accumulation was not associated to apoptotic cell death, but can be responsible for intracellular damages. Moreover, necrotic cell death induced by dithianon explains its effect on the kinetics of wine fermentations.

Necrotic and apoptotic cell death induced by Captan on Saccharomyces cerevisiae.

Captan is one of the most widely used broad-spectrum fungicide applied to control several early and late diseases of grapes, apples, and other fruits and vegetables, and as other phthalimide fungicides is defined as a multisite compound with thiol-reactivity. Captan can affect non-target organisms as yeasts, modifying microbial populations and fermentation processes. In this study, we asked whether Captan thiol-reactivity and other mechanisms are involved in acute Captan-induced cell death on aerobic growing Saccharomyces cerevisiae. Thus for, we analyze cellular protein and non-protein thiols, cell membrane integrity, reactive oxygen species accumulation, phosphatidylserine externalization, and apoptotic mutants behavior. The results showed that when submitted to acute Captan treatment most cells lost their membrane integrity and died by necrosis due to Captan reaction with thiols. However, part of the cells, even maintaining their membrane integrity, lost their culture ability. These cells showed an apoptotic behavior that may be the result of non-protein thiol depletion and consequent increase of reactive oxygen species (ROS). ROS accumulation triggers a metacaspase-dependent apoptotic cascade, as shown by the higher viability of the yca1-deleted mutant. Together, necrosis and apoptosis are responsible for the high mortality detected after acute Captan treatment of aerobically growing cells of S. cerevisiae.

Nosocomial and community infections due to class A extended-spectrum ß-lactamase (ESBLA)-producing Escherichia coli and Klebsiella spp. in southern Brazil.

OBJECTIVES: To determine the prevalence of class A extended spectrum ß-lactamases (ESBL)-producing Escherichia coli and Klebsiella spp., and to investigate clonality among ESBL-producing isolates of nosocomial and community infections. METHODS: The study involved 354 nosocomial infections samples and 992 community infections samples, obtained between 2003 and 2006 at Caxias do Sul, RS. The detection of ESBL was performed by the disk-diffusion test. Presence of blaCTX-M, blaSHV and blaTEM ß-lactamase genes was evaluated by PCR, and genomic typing was determined by pulsed-field gel electrophoresis analysis. RESULTS: Higher frequency of ESBL-producing isolates were detected among nosocomial samples of E. coli (6.7%) and Klebsiella (43.7%), than those obtained from community infections (0.4% and 2.6%). blaTEM and blaCTX were the most prevalent ESBL gene families in both E. coli and Klebsiella isolates. Different pulsotypes were obtained among ESBL-producing E. coli and 11 clones for Klebsiella spp., which occurred over the years and in different hospital wards. Among ESBL-producing K. pneumoniae, 74.3% transferred ESBL genes by conjugation and exhibited concomitant decreased aminoglycosides susceptibility. CONCLUSION: ESBL-producing E. coli, and especially K. pneumoniae are essentially a nosocomial problem, and their dissemination to the community is relatively limited. The great genetic variability observed among ESBL-producing bacteria indicates polyclonal spread and high transference of ESBL genes between bacteria in the hospital environment. This information is of paramount importance for nosocomial infection control.

Nosocomial and community infections due to class A extended-spectrum β-lactamase (ESBLA)-producing Escherichia coli and Klebsiella spp. in southern Brazil

OBJECTIVES: To determine the prevalence of class A extended spectrum β-lactamases (ESBL)-producing Escherichia coli and Klebsiella spp., and to investigate clonality among ESBL-producing isolates of nosocomial and community infections. METHODS: The study involved 354 nosocomial infections samples and 992 community infections samples, obtained between 2003 and 2006 at Caxias do Sul, RS. The detection of ESBL was performed by the disk-diffusion test. Presence of blaCTX-M, blaSHV and blaTEM β-lactamase genes was evaluated by PCR, and genomic typing was determined by pulsed-field gel electrophoresis analysis. RESULTS: Higher frequency of ESBL-producing isolates were detected among nosocomial samples of E. coli (6.7 percent) and Klebsiella (43.7 percent), than those obtained from community infections (0.4 percent and 2.6 percent). blaTEM and blaCTX were the most prevalent ESBL gene families in both E. coli and Klebsiella isolates. Different pulsotypes were obtained among ESBL-producing E. coli and 11 clones for Klebsiella spp., which occurred over the years and in different hospital wards. Among ESBL-producing K. pneumoniae, 74.3 percent transferred ESBL genes by conjugation and exhibited concomitant decreased aminoglycosides susceptibility. CONCLUSION: ESBL-producing E. coli, and especially K. pneumoniae are essentially a nosocomial problem, and their dissemination to the community is relatively limited. The great genetic variability observed among ESBL-producing bacteria indicates polyclonal spread and high transference of ESBL genes between bacteria in the hospital environment. This information is of paramount importance for nosocomial infection control.

Aeromonas associated diarrhoeal disease in south Brazil: prevalence, virulence factors and antimicrobial resistance

Aeromonas were isolated from 27 (6.6 percent) of 408 patients admitted with acute gastroenteritis in two hospitals at Rio Grande do Sul, Brazil. Isolates were classified as A. hydrophila (51.8 percent), A. caviae (40.8 percent), and A. veronii biotype sobria (7.4 percent). The highest prevalence of Aeromonas associated infections occurred in lactants and children. Virulence genes (aerA -aerolysin/hemolysin, ahpA -serine-protease, satA - glycerophospholipid-cholesterol acyltransferase, lipA -lipase, and ahyB -elastase) and virulence factors (hemolytic, proteolitic, lipolitic activities, and biofilm formation) were identified in most A. hydrophila and A. veronii biotype sobria isolates, with lower frequencies on A. caviae. All Aeromonas isolates were resistant to ampicillin, ticarcillin/clavulanic acid, cephalotin, and cephazolin, and most of them (>70 percent) exhibited resistance to imipenem, carbenicillin, amoxillin/sulbactan, and piperacillin. Multiple-resistance, more than four antibiotics, was evidenced in 29.6 percent of the isolates. The most efficient antibiotics were the quinolones (ciprofloxacin and norfloxacin), and the aminoglycosides (amikacin and netilmicin).

Aeromonas foram isoladas de 27 (6.6 por cento) dos 408 pacientes admitidos com gastroenterite aguda em dois hospitais do Rio Grande do Sul, Brasil. Os isolados foram classificados com A. hydrophila (51.8 por cento), A. caviae (40.8 por cento), e A. veronii biotype sobria (7.4 por cento). A maior prevalência de Aeromonas ocorreu em lactantes e crianças. Genes (aerA -aerolisina/hemolisina, ahpA -serina-protease, satA - glicerofosfolipidio-colesterol aciltransferase, lipA -lipase, e ahyB -elastase) e factores (atividade hemolítica, proteolítica, lipolítica, e formação de biofilme) de virulência foram identificados na maioria dos isolados de A. hydrophila e A. veronii biotype sobria, com freqüências menores em A. caviae. Todos os isolados de Aeromonas apresentaram resistência a ampicilina, ticarcilina/ácido clavulânico, cefalotina e cefazolina, e a maior parte (>70 por cento) exibiram resistência a imipenem, carbenicilina, amoxacilina/sulbactam e piperacilina. Resistência múltipla foi evidenciada em 29,6 por cento dos isolados. Os antibióticos mais eficientes foram as quinolonas (ciprofloxacina e norfloxacina) e os aminoglicosídicos (amicacina e netilmicina).

The effect of organic osmoprotectors on Aeromonas trota and A. hydrophila grown under high sodium chloride concentrations

O efeito de solutos orgânicos no crescimento de Aeromonas trota e A. hydrophila foi avaliado. A prolina e o ácido glutâmico não apresentaram efeito osmoprotetor, mas a betaina exerceu osmoproteção permitindo o crescimento de ambas bactérias em concentrações inibitórias de NaCl. A cinética do crescimento sugere que a diferença em halotolerância entre as linhagens está associada à síntese de osmolitos, mais do que à capacidade de acúmulo de betaina.

The effect of organic osmoprotectors on Aeromonas trota and A. hydrophila grown under high sodium chloride concentrations

The effect of organic solutes on the growth of Aeromonas trota and A. hydrophila was evaluated. Proline and glutamic acid were not effective as osmoprotectors, but betaine exerts osmoprotection allowing the growth of both strains in inhibitory concentrations of NaCl. Growth kinetics suggests that the halotolerance difference between the strains is associated with the synthesis of osmolytes rather than betaine uptake.

O efeito de solutos orgânicos no crescimento de Aeromonas trota e A. hydrophila foi avaliado. A prolina e o ácido glutâmico não apresentaram efeito osmoprotetor, mas a betaina exerceu osmoproteção permitindo o crescimento de ambas bactérias em concentrações inibitórias de NaCl. A cinética do crescimento sugere que a diferença em halotolerância entre as linhagens está associada à síntese de osmolitos, mais do que à capacidade de acúmulo de betaina.