Stress conditions in the host induce persister cells and influence biofilm formation by Staphylococcus epidermidis RP62A

Abstract INTRODUCTION: Studies have demonstrated that pathogens react to the harsh conditions in human tissues by inducing mechanisms that promote survival. METHODS: Persistence and biofilm-forming ability were evaluated during stress conditions that mimic those in the host. RESULTS: Carbon-source availability had a positive effect on Staphylococcus epidermidis RP62A adhesion during hypoxia, accompanied by a decrease in pH. In contrast, iron limitation led to decreased surface-adherent biomass, accompanied by an increase medium acidification and lactate levels. Interestingly, iron starvation and hypoxia induced persister cells in planktonic culture. CONCLUSIONS: These findings highlight the role of host stress in the virulence of S. epidermidis.

Lactoferrina bovina disminuye la invasión de Salmonella enterica en células HEp-2 / Bovine lactoferrin decreases the invasion of Salmonella enterica to HEp-2 cells

Objetivo: Evaluar el efecto de la lactoferrina bovina (Lfb) en el proceso de invasión de Salmonella enterica ser. Typhimurium en células HEp-2. Materiales y métodos: Se infectaron células HEp-2 con 10(6) unidades formadoras de colonia (UFC) de la bacteria en ausencia y presencia de 1 y 10 mg/mL de Lfb (saturada con hierro) durante 1,5 horas a 37°C. En este estudio evaluamos 2 tratamientos: pre-infección (las células HEp-2 se incubaron con Lf 1 hora, previo a la infección con Salmonella) y post-infección (la Lfb se adicionó 15 minutos después de la infección). La capacidad de invasión de Salmonella se determinó mediante la cuantificación de las UFC recuperadas desde el interior de las células HEp-2 (después del tratamiento con 100 μg/mL y 10 μg/mL de gentamicina y Triton X-100). Resultados: En el tratamiento pre-infección se observó una disminución de 23% en la invasión de Salmonella cuando las células HEp-2 fueron pre-incubadas con 1 mg/mL de Lfb (2,8x10(5) vs 2,1x10(5), p=0,04) y una disminución de 50% cuando fueron pre-incubadas con 10 mg/mL de Lfb (2,8x10(5) vs 1,4x10(5), p=0,04). Con el tratamiento post-infección no se observaron cambios en la capacidad de invasión de Salmonella. Conclusiones: Los resultados indican que Lfb reduce la capacidad de invadir de Salmonella enterica ser. Typhimurium a células HEp-2 en el tratamiento pre-infección

Objective: To assess the effect of bovine lactoferrin (bLf) on the invasion of Salmonella enterica ser. Typhimurium to HEp-2 cells. Materials and methods: HEp-2 monolayers were infected with 10(6) colony forming unit (CFU) of bacteria in the absence and presence of 1 and 10 mg/mL of bLf (iron-saturated) and incubated 1.5 hours at 37°C. Two treatments were evaluated: preinfection (HEp-2 cells were incubated with bLf one hour prior to infection with Salmonella) and post-infection (bLf was added 15 minutes after the infection). Invasiveness of Salmonella was determined throgh quantification of CFU recovered from inside the HEp-2 cells (after treatment with 100 μg/mL and 10 μg/mL of gentamicin and Triton X -100). Results: In the pre-infection treatment, we observed a decrease of 23% of Salmonella invasion when HEp-2 cells were pre incubated with 1 mg/mL of bLf (2.8x105 vs 2.1x105, p=0.04) and 50% when them were pre-incubated with 10 mg/mL of bLf (2.8x10(5) vs 1.4x10(5), p=0.04). In post-infection treatment, no changes were observed in the invasiveness of Salmonella. Conclusion: The results indicated that bLf reduces the invasiveness of Salmonella enterica ser. Typhimurium to HEp-2 cells in the pre-infection treatment

Citronellal-induced disruption of membrane homeostasis in candida albicans and attenuation of its virulence attributes

Abstract: INTRODUCTION There is an increasing burden of multidrug resistance. As a result, deciphering the mechanisms of action of natural compounds with antifungal activity has gained considerable prominence. We aimed to elucidate the probable mechanism of action of citronellal, a monoterpenoid found in the essential oil extracted from Cymbopogon plants, against Candida albicans. METHODS Drug susceptibility was measured by broth microdilution and spot assays. Ergosterol levels were estimated using the alcoholic potassium hydroxide method and H+ extrusion was assessed by monitoring the glucose-induced acidification of the external medium. Virulence traits were studied by hyphal morphogenesis and biofilm formation, along with fungal cell adherence to polystyrene surface and human oral epithelial cells. RESULTS Citronellal showed anticandidal activity against C. albicans and non-albicans species of Candida at a minimum inhibitory concentration of 1 mg/ml. Citronellal interfered with membrane homeostasis, which is the major target of known antifungal drugs, by increasing the hypersensitivity of the fungi to membrane-perturbing agents, reducing ergosterol levels, and diminishing glucose-induced H+ extrusion. In addition, oxidative and genotoxic stresses were induced via an increased production of reactive oxygen species. Furthermore, citronellal inhibited the virulent attributes of yeast-to-hypha transition and biofilm formation. It also reduced cell adherence to polystyrene surface and the human oral epithelial cells. CONCLUSIONS This is the first study to propose the cell membrane, morphogenetic switching, biofilm formation, and cell adherence of Candida albicans as potential targets for the anticandidal activity of citronellal. However, clinical investigations on the therapeutic applications of citronellal are required.

Effect of plant extracts and a disinfectant on biological parameters and pathogenicity of the fungus Beauveria bassiana (Bals.) Vuill. (Ascomycota: Cordycipitaceae) / Ação de extratos vegetais e desinfetantes sobre parâmetros biológicos e patogenicidade do fungo Beauveria bassiana (Bals.) Vuill. (Ascomycota: Cordycipitaceae)

Abstract The fungus Beauveria bassiana is naturally found in poultry houses and causes high rates of mortality in Alphitobius diaperinus. Laboratory and field experiments have shown the potential of this fungus as an insect control agent. However, in poultry houses, bacteria as Salmonella, can be found and have been studied alternative control methods for this pathogen. Thus, this study aimed to evaluate the effect of plant extracts and a disinfectant on the fungus Beauveria bassiana (strain Unioeste 4). Conidial viability, colony-forming unit (CFU) counts, vegetative growth, conidia production, insecticidal activity of the fungus and compatibility were used as parameters in the evaluation of the effect of these products on the fungus. Alcoholic and aqueous extracts of jabuticaba (Myrciaria cauliflora (Mart.), guava (Psidium guajava (L.)), and jambolan (Syzygium cumini (L.), at concentrations of 10% as well as the commercial disinfectant, Peroxitane® 1512 AL, were evaluated at the recommended concentrations (RC), 1:200 (RC), 0.5 RC and 2 RC. There was a negative influence of alcoholic and aqueous extracts of jabuticaba, guava and three dilutions of Peroxitane on the viability of conidia. The CFUs and vegetative growth of the fungus were affected only by the Peroxitane (all dilutions). For conidial production, the aqueous extract of guava had a positive effect, increasing production, while the Peroxitane at the R and RC concentrations resulted in a negative influence. The mortality of A. diaperinus, caused by the fungus after exposure to these products, was 60% for the peracetic acid at 0.5 RC, and above 80% for the extracts. Thus, the results showed that all the extracts and Peroxitane at RC 0.5 are compatible with the fungus B. bassiana Unioeste 4, however only the extracts had a low impact on inoculum potential.

Resumo O fungo Beauveria bassiana é encontrado naturalmente em aviários de frango de corte, tendo sua eficácia como agente controlador do Alphitobius diaperinus, em condições de laboratório e campo. No entanto, nos aviários encontram-se também bactérias, como a Salmonella, para a qual vêm sendo pesquisadas alternativas de controle. Sendo assim, o objetivo do trabalho foi avaliar o efeito de extratos vegetais e um desinfetante com potencial de uso contra Salmonella spp., sobre os parâmetros biológicos do fungo B. bassiana isolado Unioeste 4. Foram avaliados extratos alcoólicos e aquosos de folhas de jabuticabeira (Myrciaria cauliflora (Mart.)), goiabeira (Psidium guajava (L.)), jamboleiro (Syzygium cumini (L.)), na concentração de 10% e também o desinfetante comercial Peroxitane®1512 AL na concentração recomendada – 1:200 (CR), 0,5 CR e 2CR. Foram avaliados a: germinação dos conídios, unidades formadoras de colônias (UFC), crescimento vegetativo, produção de conídios e efeito sobre a atividade inseticida do fungo contra adultos de A. diaperinus, bem como a compatibilidade entre produtos e o fungo. Verificou-se influência negativa dos extratos alcoólico e aquoso de jabuticabeira, goiabeira e das três diluições de Peroxitane sobre a viabilidade dos conídios. Já, a UFC e o crescimento vegetativo foram afetados apenas com Peroxitane (em todas as diluições). Para produção de conídios, o extrato aquoso de goiabeira teve efeito positivo, elevando a produção, enquanto as diluições recomendada e o dobro de Peroxitane mostraram influência negativa. Observou-se ainda que a mortalidade de A. diaperinus causada pelo fungo após a exposição aos produtos foi de 60% para o ácido peracético na 0,5 CR, já para os extratos foi acima de 80%. Assim, os resultados demonstraram que todos os extratos e o Peroxitane na 0,5 CR são compatíveis com o fungo B. bassiana Unioeste 4, porém apenas os extratos tiveram baixo impacto sobre o potencial de inóculo do fungo.

Carum copticum and Thymus vulgaris oils inhibit virulence in Trichophyton rubrum and Aspergillus spp

Emergence of drug-resistant strains has demanded for alternative means of combating fungal infections. Oils of Carum copticum and Thymus vulgaris have long been used in ethnomedicine for ailments of various fungal infections. Since their activity has not been reported in particular against drug-resistant fungi, this study was aimed to evaluate the effects of oils of C. copticum and T. vulgaris on the growth and virulence of drug-resistant strains of Aspergillus spp. and Trichophyton rubrum. The gas chromatography-mass spectrometry analysis revealed thymol constituting 44.71% and 22.82% of T. vulgaris and C. copticum, respectively. Inhibition of mycelial growth by essential oils was recorded in the order of thymol > T. vulgaris > C. copticum against the tested strains. RBC lysis assay showed no tested oils to be toxic even up to concentration two folds higher than their respective MFCs. Thymol exhibited highest synergy in combination with fluconazole against Aspergillus fumigatus MTCC2550 (FICI value 0.187) and T. rubrum IOA9 (0.156) as determined by checkerboard method. Thymol and T. vulgaris essential oil were equally effective against both the macro and arthroconidia growth (MIC 72 µg/mL). A > 80% reduction in elastase activity was recorded for A. fumigatus MTCC2550 by C. copticum, T. vulgaris oils and thymol. The effectiveness of these oils against arthroconidia and synergistic interaction of thymol and T. vulgaris with fluconazole can be exploited to potentiate the antifungal effects of fluconazole against drug-resistant strains of T. rubrum and Aspergillus spp.

Molecular characterization of Listeria species isolated from frozen fish

The objective of this study was to investigate the molecular characterization of Listeria species isolated from frozen raw fish. A total of 219 samples consisting of 104 mackerel, 52 horse mackerel, 51 catfish and 12 herring were collected and analyzed by bacteriological, serological, antimicrobial and molecular methods. Overall, 29[56.9%] and 1[0.96%] of catfish samples and mackerel samples respectively were positive for Listeria spp. No Listeria was detected in herring and horse mackerel. In catfish, L. welshimeri [13.7%] was the most commonly isolated species followed by L. monocytogenes [11.8%], L. innocua [9.8%], L. grayi subsp. murrayi [9.8%], L. grayi subsp. grayi [7.8%], and L. ivanovii [3.9%]. In mackerel, only L. monocytogenes was detected in one sample.L. monocytogenes isolates serotyped as type 1 and type 4 [3 isolates each] and one non-typeable.Antimicrobial resistance profiling showed all L. monocytogenes isolates were resistant to ampicillin and tetracycline. Two were resistant to erythromycin. However, they were susceptible to rifampicin, vancomycin, chloramphenicol and streptomycin. Four virulence-associated genes [prfA, hlyA, actA and inlA] in addition to the genus gene [prs] were investigated using multiplex PCR. All the isolates were positive for prs gene but, onlyL. monocytogenes isolates were positive for all tested virulence genes. Our study indicates that imported raw catfish can represent a significant source of L. monocytogenes and potential health risk for listeriosis

Infection in a rat model reactivates attenuated virulence after long-term axenic culture of Acanthamoeba spp

Prolonged culturing of many microorganisms leads to the loss of virulence and a reduction of their infective capacity. However, little is known about the changes in the pathogenic strains of Acanthamoeba after long culture periods. Our study evaluated the effect of prolonged culturing on the invasiveness of different isolates of Acanthamoeba in an in vivo rat model. ATCC strains of Acanthamoeba, isolates from the environment and clinical cases were evaluated. The in vivo model was effective in establishing the infection and differentiating the pathogenicity of the isolates and re-isolates. The amoebae cultured in the laboratory for long periods were less virulent than those that were recently isolated, confirming the importance of passing Acanthamoeba strains in animal models.

Effect of exposure to hydrogen peroxide on the virulence of Escherichia coli.

PURPOSE: To eliminate pathogenic bacteria, the host presents conditions that are stressful for bacteria. Oxidative stress arises when the concentration of pro-oxidants like hydrogen peroxide (H2O2 ) and superoxide anion increases to a level over the basal defence capacity of the cell. In the present study, we studied the effect of oxidative stress on the production of certain virulence factors by Escherichia coli . METHODS: E. coli was exposed to oxidative stress by growing in the presence of different concentrations of H2O2 . The effect of oxidative stress on the expression of surface hydrophobicity, adherence, haemolysin production, serum resistance and phagocytosis was studied. RESULTS: Oxidative stress caused a significant decrease in the expression of all the virulence factors of E. coli . CONCLUSIONS: Synthesis of virulence factors can be significantly altered by oxidative stress and such changes may affect the pathogenicity of E. coli.

Low-density lipoprotein protects Vibrio vulnificus-induced lethality through blocking lipopolysaccharide action

Lipoprotein plays a role in the host defense against bacterial infection, and its serum level has been demonstrated to be an important prognosis factor of survival. We have previously demonstrated that LDL directly inactivates the hemolytic activity of Vibrio vulnificus cytolysin (VVC) in vitro. The object of this study was therefore to examine whether the LDL-mediated inactivation of VVC leads to protection against lethal infection of V. vulnificus in vivo, using wild and VVC-deficient V. vulnificus strains. Unexpectedly, we found that LDL protects mouse lethality induced by VVC-deficient as well as wild V. vulnificus strain. We also demonstrated that LDL blocks V. vulnificus LPS-induced lethality in mice. These results suggest that LDL preferentially act on endotoxin rather than exotoxin in the protection against V. vulnificus-induced mice lethality.

Adherencia de acinetobacter baumannii al tejido de tráquea de la rata / Adherence of acinetobacter baumannii to rat trachial tissue

Background: Acinetobacter baumannii is an important nosocomial pathogen whose virulence factors have not been fully elucidated. Aim: To study the adherence and hemagglutinating capacity of several biotypes of Acinetobacter baumannii. Material and methods: Thirty nine strains of Acinetobacter baumannii isolated from hospitalized patients were studied. The adherence of these strains to small pieces of rat tracheal tissue was studied. Additionally, their ability to hemagglutinate human erythrocytes and the effect of D-mannose and D-galactose on the adherence and hemagglutinating capacity was assessed. Transmission electron microscopy of strains was performed looking for the presence of fimbriae. Results: All strains exhibited adherence to tissues. All strains had also D-mannose and D-galactose resistant hemagglutinating ability. Fimbriae were found in Acinetobacter baumannii and E coil cells. Conclusions: Adherence of Acinetobacter baumannii to rat tracheal tissue, apparently not related to the presence of fimbriae, may be a virulence mechanism of this bacterium

Effect of bacterial association on virulence of Entamoeba histolytica to baby hamster kidney cell monolayers.

Axenic E. histolytica trophozoite strain NIH:200 and HMI:IMSS when co-associated with aerobic bacteria Escherichia coli strain K12 and serotype 056 showed marked increase in virulence as observed by destruction of baby hamster kidney (BHK) monolayers. However, when incubated with anaerobic bacterial strains Clostridium perfringens and Bacteroides fragilis virulence remained unaltered. Further, adherence of E. histolytica to BHK monolayer was found to be mediated by N-acetyl-D-galactosamine.

Trypanosoma cruzi: metacyclogenesis in vitro - I. Changes in the properties of metacyclic trypomastigotes maintained in the laboratory by different methods

In this work we have studied the modifications in the biological properties of Trypanosoma cruzi when the parasite is maintained for a long time in axenic culture. The studies were done with a clone from an avirulent strain (Dm30L) and a non-cloned virulent strain (EP) of T. cruzi. Both parasiteswere maintained, for at least three years, by successive triatomine/mouse alternate passage (control condition), or by serial passage in axenic medium (culture condition), or only in the mouse (mouse condition). The comparison between parasites of culture and control condition showed that metacyclogenesis capacity was reduced in the former and that the resulting metacyclics displayed an attenuatedvirulence. In order to compare the virulence of metacyclics from the urine of the insect vector, Rhodnius prolixus were infected by artificial feeding with parasites of the control or culture condition. After three triatomine/triatomine passages, there was observed an almost identical biological behavior for these parasites, hence indicating that the maintenance of T. cruzi for a long time in axenic culture affects the differentiation capacity and the virulence of the parasite. Additionally, it was demonstrated that it is possible to maintain T. cruzi exclusively through passages in the invertebrate host

Mycological study of seborrheic dermatitis

The relationship between the Pityrosporum yeast and seborrheic dermatitis was studied. Seborrheic dermatitis is a chronic disease as 72.6% of cases had duration more than 10 years. The familial tendency and obesity were significantly important predisposing factors with more severity in winter. Direct microscopy was not a predictive method for fungal diagnosis as it gave only 13.6% positive smears. Dixon's modified medium was a good culture for rapid recovery of P. ovale, while SDCA medium was effective when the period of cultivation was prolonged. A trial to suppress P. ovale was performed with an antimycotic shampoo

Aeromonas spp en niños con síndrome diarreico agudo / Aeromonas spp in children with acute diarrheal disease

El interés por el género Aeromonas es creciente, tanto como causante de enfermedades diarreicas como extraintestinales. Produce cuadros intestinales a través de acción citotóxica y enterotóxica. Empleando agar sangre con 30 mg/l de ampicilina (ASA) se aislaron Aeromonas spp en 8 de 400 niños diarreicos y en ninguno de 230 controles no diarreicos. El aislamiento fue muy inferior en los medios tradicionales del coprocultivo, agar lactosa y agar Shigella-Salmonella, así como agar sangre sin ampicilina, en el que hubo sobredesarrollo de flora entérica. Cinco cepas aisladas fueron A. hydrophila, 2 A. caviae y 1 A. veroni biotipo sobria. Cuatro de cinco cepas probadas resultaron enterotoxigénicas en asa de conejo. Todas las cepas fueron hemolíticas; 6 fueron Voges Proskauer (+), lisina decarboxilasa (+) y glucosa con gas (+), características que han sido relacionadas con la virulencia. Todos los aislados resultaron resistentes a las aminopenicilinas con o sin sulbactama y sensibles a cloranfenicol, colistin, gentamicina, neomicina y norfloxacina. Dos cepas resultaron resistentes a sulfametoxazol-trimetoprima

Acción del Agua del Volcán Copahue (Neuquén, Argentina) sobre la morfología de las microbacterias / Action of the Copahue Volcano (Neuquén, Argentine) water of mycobateria morphology

Se efectuó un estudio de la acción del Agua del Volcán Copahue (AVC), Neuquén, Argentina, sobre 15 cepas de microbacterias Mycobacterium tuberculosis: M. bovis y los mycobacterum no tuberculosos ("Atipicos"), poniendo especial interés en los que forman "cuerdas". Se utilizó AVC con su pH l,3 y se la llevó a pH 6,5. En los bacilos que resistieron la acción del agua hasta el momento de la coloración, se advirtieron elementos más o menos alterados. Al aumentar el tiempo de contacto se llegó a la destrucción total, observándose en algunos casos muy pocos bacilos aislados, material deteriorado y formas granulares. Estas alteraciones fueron mucho más marcadas en las suspensiones que en el líquido del sedimento con el AVC; en las primeras directamente no se llegó a reparar la formación de cuerdas en ningun momento aun en bacilos que deberían formarla. Su acción no estaría asociada a una reacción bioquímica responsable en la síntesis de la pared celular, como la transpeptidación. El AVC actuaría sobre la síntesis del ácido micólico y se trataría de un agente bacteriolítico. Además se realizaron estudios en el "Laboratorio de tratamiento de imágenes", INEUCI (Instituto de Neurociencia), CONICET

Role of iron in the virulence of Vibrio vulnificus isolated from Cuddalore coastal waters (India).

V. vulnificus strains isolated from different sources of Cuddalore coastal waters were tested for their virulence activity through their LD50 values in mice. As infections of V. vulnificus have been correlated with pre-existing liver disease and hemochromatosis, the role of iron on virulence was determined using. iron overloaded mice. The LD50 was in the range of 10(4)-10(7) cells in normal mice but 10(1)-10(2) cells in iron-injected mice, thus providing evidence that iron may play a major role in the pathogenesis of V. vulnificus.

Mechanisms of evasion of Schistosoma mansoni schistosomula to the lethal activity of complement

Schistosomula of Schistosoma mansoni became resistant to antibody-dependent complement damage in vitro after pre-incubation with normal human erythrocytes (NHuE) whatever the ABO or Rh blood group. Resistant parasites were shown to acquire host decay accelerating factor (DAF) , a 70 kDa glycoprotein attached to the membrane of NHue by a GPI anchor. IgG2a mAb anti-human DAF (IA10) immunoprecipitated a 70 kDa molecule from 125I-labeled schistosomula pre-incubated with NHuE and inhibited their resistance to complement-dependent killing in vtro. Incubationof schistosomula with erytrocytes from patients with paroxsimal nocturnal hemoglobinuria (PNHE) or SRBC, wich are DAF-deficient, did not protect the parasites from complement lesion. Supernatant of 100,000 x g collected from NHuE incubated for 24 h in defined medium was shown to contain a soluble form of DAF and to protect schistosomula from complement killing. Schistosomula treated with trypsin before incubation with NHuE ghosts did not become resistant to complement damage. On the other hand, pre-treatment with chymotrypsin did not interfere with the acquisition of resistance by the schistosomula. These results indicate that, in vitro, NHuE DAF can be transferred to schistosomula in a soluble form and that the binding of this molecule to the parasite surface is dependent upon trypsin-sensitive chymotrypsin-insensitive polipeptide(s) present on the surface of the worm

Compuestos triazólicos en el tratamiento de la histoplasmosis diseminada experimental del hamster / Triazoles compound in the treatment of disseminated experimental histoplasmosis in hamsters

Se llevó a cabo un estudio comparativo de la eficacia de cuatro compuestos triazólicos en el tratamiento de la histoplasmosis experimental del hamster. Fueron utilizados en total 110 hamsters, de ambos sexos. Estos animales se inocularon por vía intracardíaca con una suspensión de la fase levaduriforme del Histoplasma capsulatum. Los tratamientos comenzaron 1 semana después de la infección y se prolongaron por 3 semanas. Todas las drogas fueron administradas por gastroclisis una vez por día, a las dosis de 8 y 16 mg/kg de peso. Tres grupos de hamsters fueron empleados como control y recibieron los solventes de los antifúngicos estudiados. Fueron administrados los siguientes triazoles: itraconazol, fluconazol, Sch 39.304 y Bay r 3783. Todos los animales se sacrificaron una semana después de terminados los tratamientos. La evaluación de los resultados se realizó en base a los siguientes criterios: 1)Aspecto macroscópico de higado y bazo, 2)examen microscópico de frotis teñidos con Giemsa de los mismos órganos, 3)cortes histológicos de bazo teñidos por el método de P.A.S.y 4)cultivos de bazo (cultivos masivos de un homogeneizado del órgano y determinación de U.F.C/g). El itraconazol y el Sch 39.304 redujeron en forma muy marcada las alteraciones macroscópicas, la positividad de los exámenes microscópicos y los cultivos. Los animales tratados con fluconazol y con Bay r 3783 a la dosis de 16 mg/kg/día, sólo mostraron escasa reducción del número de U.F.C/g de bazo, en relación a los controles. El itraconazol es una droga de probada eficacia en la histoplasmosis humana. El Sch 39.304 parece ser un antifúngico interesante en el tratamiento de esta enfermedad, en especial si se tienen en cuenta sus propiedades fármaco-cinéticas distintas de las itraconazol lo que facilitaría su aplicación en otras localizaciones de la histoplasmosis diseminada