Nanocristais de besifloxacino para o tratamento de conjuntivite bacteriana: preparação, caracterização físico-química e toxicidade / Besifloxacin nanocrystals for treating bacterial conjunctivitis: preparation, physicochemical characterization, and toxicity

A conjuntivite bacteriana tem significante impacto na Saúde Pública. Essa infecção representa mais de um terço das doenças oculares relatadas em âmbito global. É uma doença altamente contagiosa causada por variedade de bactérias aeróbias e anaeróbias. Diferentes antibióticos empregados no tratamento dessa doença têm apresentado elevada incidência de resistência bacteriana. Dentre os antibióticos de última geração, destaca-se o besifloxacino, antibiótico de quarta geração da classe das fluoroquinolonas, indicado exclusivamente para uso oftálmico tópico. Entretanto, esse fármaco possui baixa solubilidade em água, diminuindo sua biodisponibilidade. Tendo em vista superar esse desafio, foi proposta abordagem nanotecnológica para o desenvolvimento de nanocristais desse fármaco. A preparação de nanocristais de besifloxacino empregando moagem via úmida em escala reduzida foi promissora empregando tensoativo Povacoat®. O Diâmetro hidrodinâmico médio (DHM) da partícula foi de aproximadamente 550 nm, com índice de polidispersão (IP) menor que 0,2. Esse resultado permitiu aumentar a solubilidade de saturação em aproximadamente duas vezes em relação a matéria-prima, possibilitando aumentar a velocidade de dissolução desse fármaco e melhorar sua biodisponibilidade e segurança. Além disso, foi validado o método para quantificação do besifloxacino por CLAE, apresentando especificidade, linearidade no intervalo de 20 a 80µg/mL (r= 0,9996), precisão por repetibilidade (DPR= 1,20%, 0,84% e 0,39%), precisão intermediária (DPR= 0,94%) e exatidão 99,03%. Estudo de estabilidade acelerado (90 dias) na condição 40°C±2°C/75%UR±5%UR e estudo de estabilidade de acompanhamento (150 dias) na condição: 25°C ± 2°C / 60% UR ± 5% UR evidenciaram a estabilidade do teor no período avaliado. Ainda, a nanossuspensão de besifloxacino 0,6% m/m (nanocristais) na dose máxima (500 mg/kg) e o estabilizante Povacoat® (750 mg/kg) não apresentaram toxicidade em larvas de G. mellonella. A concentração inibitória mínima (CIM) para a formulação inovadora foi de 0,0960 µg/mL e 1,60 µg/mL frente a Staphylococcus aureus e Pseudomonas aeruginosa, respectivamente, confirmando eficácia in vitro

Bacterial conjunctivitis greatly impacts the population's health, presenting more than a third of eye diseases reported worldwide. It is an infection caused by various aerobic and anaerobic bacteria and is highly contagious. Therefore, it presents a high incidence of bacterial resistance to the antibiotics commonly used for treatment. Among the most recent antibiotics, besifloxacin is a fourth-generation fluoroquinolone antibiotic indicated exclusively for topical ophthalmic use. Due to its importance in treating bacterial conjunctivitis and its low solubility in the water, a nanotechnological approach was proposed to develop besifloxacin nanocrystals. The preparation of besifloxacin nanocrystals using small-scale wet milling was promising using Povacoat® surfactant. The particle's average hydrodynamic diameter (DHM) was approximately 550 nm, with a polydispersity index (IP) of less than 0.2. This result increased the saturation solubility approximately two times concerning the raw material, making it possible to increase the dissolution rate of this drug and improve its bioavailability and safety. In addition, the method for quantification of besifloxacin by HPLC was validated, presenting specificity, linearity in the range of 20 to 80µg/mL (r= 0.9996), precision by repeatability (DPR= 1.20%, 0.84% and 0.39%), intermediate precision (DPR= 0.94%) and accuracy 99.03%. Accelerated stability study (90 days) at 40°C±2°C/75%RH±5%RH condition and follow-up stability study (150 days) at 25°C ± 2°C / 60% RH ± condition 5% RH showed the stability of content in the evaluated period. Furthermore, the 0.6% besifloxacin nanosuspension (nanocrystals) at the maximum dose (500 mg/kg) and the Povacoat® stabilizer (750 mg/kg) did not show toxicity in G. mellonella larvae. The minimum inhibitory concentration (MIC) to innovative formulation was 0.0960 µg/mL and e 1.60 µg/mL against Staphylococcus aureus and Pseudomonas aeruginosa, respectively, confirming in vitro efficacy

Composition of semen and foreskin mucosa aerobic microbiota and its impact on sperm parameters of captive collared peccaries (Pecari tajacu).

AIM: To evaluate the bacterial composition of collared peccary semen and foreskin mucosa, and to verify the sensitivity of isolates to antimicrobials used in semen conservation and to Aloe vera gel, which is an alternative external cryoprotectant. METHODS AND RESULTS: Nine foreskin mucosa and ejaculate samples from adult animals were used. Sperm characteristics and bacterial load were evaluated in fresh semen. The preputial mucosa and semen bacterial isolates were identified and tested against five concentrations of each antimicrobial (streptomycin-penicillin and gentamicin) and A. vera gel. Corynebacterium sp. and Staphylococcus sp. were isolated in greater numbers than others in both semen (64·10 and 20·51%, respectively) and the foreskin mucosa (60·60 and 24·25%, respectively), and ranged from 0·4 to 21 × 105 colony-forming units (CFU) per ml. The average load of Corynebacterium sp. was negatively correlated (P < 0·05) with the sperm membrane integrity (r = -0·73055) and curvilinear velocity (r = -0·69048). Streptomycin-penicillin and gentamicin inhibited most micro-organisms, and A. vera showed lower antimicrobial activity. CONCLUSION: Several Gram-positive bacteria are present in semen and foreskin mucosa of collared peccary, and the benefits of using primarily penicillin-streptomycin and gentamicin antimicrobials in the bacterial control of diluted semen of these animals are strongly indicated. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides insight into the reproductive microbiota of captive male-collared peccary. This work provides a theoretical basis to assist reproductive biotechnologies for ex situ conservation of the species.

Fecal cultivable aerobic microbiota of dairy cows and calves acting as reservoir of clinically relevant antimicrobial resistance genes.

Antimicrobial resistance has become a global threat to public health since multidrug-resistant (MDR) bacteria have been reported worldwide carrying different antimicrobial resistance genes (ARGs), and animals have been described as a reservoir of ARGs. The presence of antimicrobial-resistant bacteria and ARGs in the food matrix is a risk to public health. This study aimed to research the presence of clinically relevant ARGs for important antimicrobials and genetic elements in fecal samples from dairy cows and calves on a Brazilian farm. In this study, a total of 21 fecal samples were collected, and then, the DNA of cultivable aerobic bacteria was extracted. Fifty-seven ARGs and twenty-three genetic elements were researched by PCR and confirmed by sequencing. Several ARGs that confer resistance to ß-lactams, tetracyclines, fluoroquinolones, sulphonamides, phenicols, aminoglycoside, glycopeptides, and macrolides were detected. A total of 200 amplicons from 23 ARGs (blaCTX-M-Gp2, blaCMY, blaSHV, tetA, tetB, tetC, qepA, qnrB, qnrS, oqxA, oqxB, vanC1, vanC2/3, aadA, sul1, sul2, sul3, ermB, mefAE, floR, cmlA, aadA, aph(3')-Ia, aac(3')-Ia), and 145 amplicons from 12 genetic elements (IncF, IncFIA, IncFIB, IncI1, IncY, IncU, IncK, IncP, IncR, IncHI1, ColE-like, intI1) were detected. The results presented in this study call attention to the monitoring of antimicrobial resistance in dairy farms worldwide. MDR bacteria and ARGs can spread to different sources, including milk products, which are one of the most consumed products worldwide, representing a potential risk to human health.

AEROBIC MICROFLORA AND ENDOPARASITES OF FRESHLY SHOT WILD AGOUTI (DASYPROCTA LEPORINA) IN TRINIDAD, WEST INDIES.

The agouti ( Dasyprocta leporina ) is a New World wild rodent hunted for its meat in Trinidad and other Latin American countries. Studies on agouti under captive conditions have yielded some data on health-related aspects, but relatively very little is known about their wild counterparts. The environment of the agouti can influence the microflora and parasites harbored by the animals, which may contain zoonotic pathogens. Here, the microflora found on the nasal mucosa and sections of the intestinal tract and endoparasites of freshly shot agouti from various areas of Trinidad are described. Staphylococcus epidermidis , S. intermedius , Bacillus spp., Enterobacter spp. and Escherichia coli comprised the majority of bacteria isolated from the nasal mucosa whereas Escherichia coli , Streptococcus viridans, Bacillus spp. and Klebsiella pneumoniae were predominant in all sections of the intestinal tract. The fungi Aspergillus fumigatus , Aspergillus spp., Candida spp., Penicillium spp., and Mucor spp. were only isolated from the nasal cavity but not in any section of the intestinal tract. The parasites Strongyloides spp., Ascaridia spp., a hookworm, a trematode, and Trichuris spp. were detected at variable frequencies in each of the sections of the intestines (small intestine, large intestine, caecum), whereas Eimeria spp. were found in all sections (76.9%, 10 of 13 agoutis). These wild agoutis were presumably healthy at the time of death and represent animals that hunters may encounter. Some of the detected pathogens and parasites have the potential to cause opportunistic infections or infestations, especially in immune-compromised hosts.

Exploiting the aerobic endospore-forming bacterial diversity in saline and hypersaline environments for biosurfactant production.

BACKGROUND: Biosurfactants are surface-active biomolecules with great applicability in the food, pharmaceutical and oil industries. Endospore-forming bacteria, which survive for long periods in harsh environments, are described as biosurfactant producers. Although the ubiquity of endospore-forming bacteria in saline and hypersaline environments is well known, studies on the diversity of the endospore-forming and biosurfactant-producing bacterial genera/species in these habitats are underrepresented. METHODS: In this study, the structure of endospore-forming bacterial communities in sediment/mud samples from Vermelha Lagoon, Massambaba, Dois Rios and Abraão Beaches (saline environments), as well as the Praia Seca salterns (hypersaline environments) was determined via denaturing gradient gel electrophoresis. Bacterial strains were isolated from these environmental samples and further identified using 16S rRNA gene sequencing. Strains presenting emulsification values higher than 30 % were grouped via BOX-PCR, and the culture supernatants of representative strains were subjected to high temperatures and to the presence of up to 20 % NaCl to test their emulsifying activities in these extreme conditions. Mass spectrometry analysis was used to demonstrate the presence of surfactin. RESULTS: A diverse endospore-forming bacterial community was observed in all environments. The 110 bacterial strains isolated from these environmental samples were molecularly identified as belonging to the genera Bacillus, Thalassobacillus, Halobacillus, Paenibacillus, Fictibacillus and Paenisporosarcina. Fifty-two strains showed emulsification values of at least 30%, and they were grouped into 18 BOX groups. The stability of the emulsification values varied when the culture supernatants of representative strains were subjected to high temperatures and to the presence of up to 20% NaCl. The presence of surfactin was demonstrated in one of the most promising strains. CONCLUSION: The environments studied can harbor endospore-forming bacteria capable of producing biosurfactants with biotechnological applications. Various endospore-forming bacterial genera/species are presented for the first time as biosurfactant producers.

Aerobic Microbial Respiration In Oceanic Oxygen Minimum Zones.

Oxygen minimum zones are major sites of fixed nitrogen loss in the ocean. Recent studies have highlighted the importance of anaerobic ammonium oxidation, anammox, in pelagic nitrogen removal. Sources of ammonium for the anammox reaction, however, remain controversial, as heterotrophic denitrification and alternative anaerobic pathways of organic matter remineralization cannot account for the ammonium requirements of reported anammox rates. Here, we explore the significance of microaerobic respiration as a source of ammonium during organic matter degradation in the oxygen-deficient waters off Namibia and Peru. Experiments with additions of double-labelled oxygen revealed high aerobic activity in the upper OMZs, likely controlled by surface organic matter export. Consistently observed oxygen consumption in samples retrieved throughout the lower OMZs hints at efficient exploitation of vertically and laterally advected, oxygenated waters in this zone by aerobic microorganisms. In accordance, metagenomic and metatranscriptomic analyses identified genes encoding for aerobic terminal oxidases and demonstrated their expression by diverse microbial communities, even in virtually anoxic waters. Our results suggest that microaerobic respiration is a major mode of organic matter remineralization and source of ammonium (~45-100%) in the upper oxygen minimum zones, and reconcile hitherto observed mismatches between ammonium producing and consuming processes therein.

Uncovering the cultivable microbial diversity of costa rican beetles and its ability to break down plant cell wall components.

Coleopterans are the most diverse insect order described to date. These organisms have acquired an array of survival mechanisms through their evolution, including highly efficient digestive systems. Therefore, the coleopteran intestinal microbiota constitutes an important source of novel plant cell wall-degrading enzymes with potential biotechnological applications. We isolated and described the cultivable fungi, actinomycetes and aerobic eubacteria associated with the gut of larvae and adults from six different beetle families colonizing decomposing logs in protected Costa Rican ecosystems. We obtained 611 isolates and performed phylogenetic analyses using the ITS region (fungi) and 16S rDNA (bacteria). The majority of fungal isolates belonged to the order Hypocreales (26% of 169 total), while the majority of actinomycetes belonged to the genus Streptomyces (86% of 241 total). Finally, we isolated 201 bacteria spanning 19 different families belonging into four phyla: Firmicutes, α, ß and γ-proteobacteria. Subsequently, we focused on microbes isolated from Passalid beetles to test their ability to degrade plant cell wall polymers. Highest scores in these assays were achieved by a fungal isolate (Anthostomella sp.), two Streptomyces and one Bacillus bacterial isolates. Our study demonstrates that Costa Rican beetles harbor several types of cultivable microbes, some of which may be involved in symbiotic relationships that enable the insect to digest complex polymers such as lignocellulose.

Accuracy of the cytopathology, bacterioscopy, and vaginal flora culture.

INTRODUCTION: An over-population of vaginal microorganisms causing inflammatory processes renders it difficult to properly assess the cytopathological exam that aims to screen precedent cervical lesions. On the contrary, the occurrence of the microbial flora saprophyte does not influence correct cythodiagnosis. OBJECTIVE: To assess the composition of vaginal tract aerobic microorganisms of asymptomatic women in menacme and post-menopause, and to analyze the accuracy of cytopathologic, bacterioscopic exams, and culturing of the flora. METHODS: The women were first submitted to a focused anamnestic interrogatory and then submitted to gynecological exam. A sample of the vaginal fluid was collected with a culture swab and a smear was made on two glass slides for stained bacterioscopic exam (GRAM). The collection of material was then compiled in a cytopathologic smear analysis. All women signed the free and informed consent letter and the project was approved by the Ethics Research Board of Hospital São Paulo - UNIFESP. RESULTS: Bacterioscopy and culture proved to be better than the cytopathologic exam in featuring the bacilli and cocci. The bacterioscopy provided a better detection of the presence of bacilli (p < 0.001); no statistical difference was seen between both exams with respect to the detected cocci. The beta-hemolytic Streptococcus group was of significance in post-menopausal women (p < 0.05). CONCLUSION: In this study, the bacterioscopic and culture exams of the vaginal fluid were more effective in assessing the vaginal flora and in the detection of bacilli, compared to the cytopathological exam.

[Impact of mass spectrometry by MALDI-TOF MS for the rapid identification of aerobic and anaerobic bacteria of clinical importance]. / Impacto de la espectrometría de masas por MALDI-TOF MS en la identificación rápida de bacterias aeróbicas y anaeróbicas de importancia clínica.

BACKGROUND: MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization -Time of Flight Mass Spectrometry) technology, recently introduced in the microbiology laboratory has proven to be a precise and rapid method for bacterial identification. OBJECTIVE: To evaluate the performance, costs associated and turnaround time of MALDI-TOF in a routine laboratory. MATERIAL AND METHOD: Five hundred and sixty one clinical isolates (281 aerobes and 280 anaerobes) previously identified by conventional methods were evaluated. Discordances were resolved by means of 16S rRNA sequencing. RESULTS: MALDI-TOF identified 95, 7% of the aerobes isolates and 86, 4% of the anaerobes. The groups with better performance were the enterobacteriacea and Bacteroides spp with 95% and 100% identification at the species level. The error rate of MALDI-TOF and conventional methods compared to sequencing was 0, 39% and 9, 4% respectively. The costs associated were 8 times lower with a turnaround time of 6 hours. CONCLUSION: MALDI-TOF proved to be simple, precise and less expensive technology compared to the traditional methods.

Impacto de la espectrometría de masas por MALDI-TOF MS en la identificación rápida de bacterias aeróbicas y anaeróbicas de importancia clínica / Impact of mass spectrometry by MALDI-TOF MS for the rapid identification of aerobic and anaerobic bacteria of clinical importance

Background: MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization -Time of Flight Mass Spectrometry) technology, recently introduced in the microbiology laboratory has proven to be a precise and rapid method for bacterial identification. Objective: To evaluate the performance, costs associated and turnaround time of MALDI-TOF in a routine laboratory. Material and Method: Five hundred and sixty one clinical isolates (281 aerobes and 280 anaerobes) previously identified by conventional methods were evaluated. Discordances were resolved by means of 16S rRNA sequencing. Results: MALDI-TOF identified 95, 7% of the aerobes isolates and 86, 4% of the anaerobes. The groups with better performance were the enterobacteriacea and Bacteroides spp with 95% and 100% identification at the species level. The error rate of MALDI-TOF and conventional methods compared to sequencing was 0, 39% and 9, 4% respectively. The costs associated were 8 times lower with a turnaround time of 6 hours. Conclusion: MALDI-TOF proved to be simple, precise and less expensive technology compared to the traditional methods.

Introducción: La tecnología MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry) incorporada recientemente en el laboratorio de microbiología ha demostrando ser un método rápido y preciso para la identificación bacteriana. Objetivo: Evaluar el desempeño de MALDI-TOF para la identificación de aislados clínicos, comparar los costos asociados y el tiempo en la entrega de resultados en un laboratorio de rutina. Material y Método: Se evaluaron un total de 561 aislados de pacientes (281 aeróbicos y 280 anaeróbicos estrictos) identificados previamente por métodos convencionales, los que fueron identificados por MALDI-TOF. Las discordancias fueron resueltas mediante secuenciación del 16S ARNr. Resultados: MALDI-TOF identificó adecuadamente a 95,7% de los aislados aeróbi-cos y 86,4% de los anaeróbicos estrictos, observándose el mayor porcentajes de identificación a nivel de especie en los grupos de enterobacterias y Bacteroides spp (95 y 100% respectivamente). La tasa de error de MALDI-TOF y métodos convencionales vs secuenciación fue de 0,39 y 9,4%, respectivamente. El costo asociado por identificación fue ocho veces menor que el de los métodos tradicionales con una demora promedio de seis horas en la entrega de resultados. Conclusión: MALDI-TOF mostró ser una tecnología simple, precisa y de menor costo que los métodos tradicionales.

Sialidase activity in aerobic vaginitis is equal to levels during bacterial vaginosis.

OBJECTIVE: To evaluate levels of proinflammatory cytokines and sialidase activity in aerobic vaginitis (AV) in relation to normal vaginal flora and bacterial vaginosis (BV). STUDY DESIGN: In this cross-sectional study, a total of 682 consecutive non-pregnant women attending the gynecology service were assessed and 408 women were included. Vaginal rinsing samples were collected from 223 women with microscopic finding of BV (n=98), aerobic vaginitis (n=25) and normal flora (n=100). Samples were tested for interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor (TNF)-α, and sialidase activity. RESULTS: Compared to women with normal flora, vaginal levels of IL-1ß were highly increased in both BV and AV (p<0.0001). Significantly higher vaginal IL-6 was detected in AV (p<0.0001) but not in BV, in relation to normal flora. Women with AV also presented increased IL-8 levels (p<0.001), while those with BV presented levels similar to normal flora. Sialidase was increased in BV and AV compared with the normal group (p<0.0001) but no difference in sialidase activity was observed between BV and AV. CONCLUSION: A more intense inflammatory host response occurs for AV than for BV when compared with normal flora. Furthermore, the increased sialidase activity in AV and BV indicates that both abnormal vaginal flora types can be harmful to the maintenance of a healthy vaginal environment.

Aerobic bacteria from mucous membranes, ear canals, and skin wounds of feral cats in Grenada, and the antimicrobial drug susceptibility of major isolates.

In a 2-year period 54 feral cats were captured in Grenada, West Indies, and a total of 383 samples consisting of swabs from rectum, vagina, ears, eyes, mouth, nose and wounds/abscesses, were cultured for aerobic bacteria and campylobacters. A total of 251 bacterial isolates were obtained, of which 205 were identified to species level and 46 to genus level. A commercial bacterial identification system (API/Biomerieux), was used for this purpose. The most common species was Escherichia coli (N=60), followed by Staphylococcus felis/simulans (40), S. hominis (16), S. haemolyticus (12), Streptococcus canis (9), Proteus mirabilis (8), Pasteurella multocida (7), Streptococcus mitis (7), Staphylococcus xylosus (7), S. capitis (6), S. chromogenes (4), S. sciuri (3), S. auricularis (2), S. lentus (2), S. hyicus (2), Streptococcus suis (2) and Pseudomonas argentinensis (2). Sixteen other isolates were identified to species level. A molecular method using 16S rRNA sequencing was used to confirm/identify 22 isolates. Salmonella or campylobacters were not isolated from rectal swabs. E. coli and S. felis/simulans together constituted 50% of isolates from vagina. S. felis/simulans was the most common species from culture positive ear and eye samples. P. multocida was isolated from 15% of mouth samples. Coagulase-negative staphylococci were the most common isolates from nose and wound swabs. Staphylococcus aureus, or S. intemedius/S. pseudintermedius were not isolated from any sample. Antimicrobial drug resistance was minimal, most isolates being susceptible to all drugs tested against, including tetracycline.

Normal bacterial flora from vaginas of Criollo Limonero cows.

In order to describe the normal bacterial flora in vaginas of Criollo Limonero cows, 51 healthy multiparous cows, at least 90-day postpartum, were selected. Duplicated swabs (N = 102) were taken from the vaginal fornix of cows to perform aerobic and anaerobic cultures as well as conventional biochemical tests. Out of 102 swabs, bacterial growth was obtained in 55 (53.9%) while the remaining 47 (46.1%) did not exhibited any bacterial growth. Of the 55 bacterial growths, 23 (41.8%) were aerobic whereas 32 (58.1%) were anaerobic. Likewise, 29 (52.72%) of bacterial growths were pure and 26 (47.27%) were mixed. Under both aerobic and anaerobic conditions, Gram positive bacteria were predominant (81.82% and 73.08%, respectively) over Gram negative bacteria (18.18% and 26.92%, respectively). Isolated bacteria were Arcanobacterium pyogenes (22.92%), Staphylococcus aureus (15.63%), Staphylococcus coagulase negative (17.71%), Erysipelothrix rhusiopathiae (6.25%), Bacteroides spp. (13.54%), and Peptostreptococcus spp. (7.29%). In conclusion, normal vaginal bacterial flora of Criollo Limonero cows was predominantly Gram positive and included A. pyogenes, S. aureus, coagulase negative Staphylococcus, E. rhusiopathiae, Bacteroides spp., and Peptostreptococcus spp. In Criollo Limonero cattle, adaptive aspects such as development of humoral and physical mechanisms for defense, and bacterial adaptation to host deserve research attention.

Production of fermented cheese whey-based beverage using kefir grains as starter culture: evaluation of morphological and microbial variations.

Whey valorization concerns have led to recent interest on the production of whey beverage simulating kefir. In this study, the structure and microbiota of Brazilian kefir grains and beverages obtained from milk and whole/deproteinised whey was characterized using microscopy and molecular techniques. The aim was to evaluate its stability and possible shift of probiotic bacteria to the beverages. Fluorescence staining in combination with Confocal Laser Scanning Microscopy showed distribution of yeasts in macro-clusters among the grain's matrix essentially composed of polysaccharides (kefiran) and bacteria. Denaturing gradient gel electrophoresis displayed communities included yeast affiliated to Kluyveromyces marxianus, Saccharomyces cerevisiae, Kazachatania unispora, bacteria affiliated to Lactobacillus kefiranofaciens subsp. Kefirgranum, Lactobacillus kefiranofaciens subsp. Kefiranofaciens and an uncultured bacterium also related to the genus Lactobacillus. A steady structure and dominant microbiota, including probiotic bacteria, was detected in the analyzed kefir beverages and grains. This robustness is determinant for future implementation of whey-based kefir beverages.

Isolation of a lipase-secreting yeast for enzyme production in a pilot-plant scale batch fermentation.

The production of lipase by twenty-nine yeasts isolated from the phylloplane of Hibiscus rosa-sinensis was evaluated. The highest lipase producers were Pseudozyma hubeiensis HB85A, Debaryomyces occidentalis-like HB83 and Cryptococcus sp. HB80. P. hubeiensis HB85A batch fermentations were carried out in a bioreactor and lipase production improved 3.2-fold as compared to flask submerged cultures. The production process was significantly reduced from 48 h (in flasks) to 18 h (in the bioreactor). The better hydrolytic activity was achieved with C16 p-nitrophenyl ester. Maximal activity was observed at pH 7.0, the optimum temperature was 50 degrees C at pH 7.0 and the enzyme was stable at 30 and 40 degrees C. The lipolytic activity was stimulated by Mg(2+), K(+) and Ba(2+) salts and EDTA and slightly inhibited by Ca(2+) salts. Non-ionic detergents such as Triton X-100, Tween 80 and Tween 20 strongly stimulated lipase activity, whereas SDS inhibited it. The lipase was stable in iso-octane and hexane at 80%.

Distribución de bacterias potencialmente fijadoras de nitrógeno y su relación con parámetros fisicoquímicos en suelos con tres coberturas vegetales en el sur de la Amazonia colombiana

Distribution of potentially nitrogen-fixing bacteria and its relationship with physicochemical parameters in soils with three vegetation types in the southern Colombian Amazon region. Potentially nitrogen-fixing microaerobic and aerobic bacteria were isolated from several Colombian Amazon soils (forest, pastures and chagras) and two landscapes (floodable and non floodable areas). The abundance and distribution of bacteria were evaluated, as well as their relationship with soil physical and chemical characteristics. Landscape had a direct influence on the abundance of the microaerobic bacteria, with higher numbers in forest and pasture soils in non- floodable zones. The aerobic isolates (N=51) were grouped into 19 morphologies, with the highest numbers found in forest soil in floodable zones. A higher number of aerobic morphologies was shared among forest sites (Nonmetric Multidimensional Scaling and Analysis of Similarity p<0.05), and 40% of the distribution was explained by lime percentage and Al concentration. Rev. Biol. Trop. 57 (4): 915- 927. Epub 2009 December 01.

Se evaluó la abundancia y distribución de bacterias microaerófilas y aerobias potencialmente fijadoras de nitrógeno aisladas a partir de suelos bajo coberturas de bosque, pastizal y chagra en dos paisajes, terraza y llanura inundable, en el sur de trapecio amazónico. Se relacionó el recuento en placa en el medio Ashby de estas bacterias aerobias con las características físicas y químicas del suelo. Se encontró mayor abundancia de bacterias aerobias y microaerofilas en suelos bajo cobertura de pastizal. Se observó influencia directa del paisaje sobre la abundancia de bacterias microaerófilas registrando mayores recuentos los suelos bajo terraza en las coberturas de bosque y pastizal. Los aislamientos aerobios obtenidos (51) se agruparon en 19 morfologías, de las cuales se obtuvo mayor número en suelos bajo bosque y en el paisaje de llanura inundable. A través del análisis multidimensional no métrico (NMDS) y análisis de similaridades (ANOSIM) (p<0.05) se comprobó que entre los sitios de muestreo bajo la cobertura de bosque se comparten mayor número de morfologías de bacterias aerobias que bajo las demás coberturas. El programa BIOENV indicó que esta distribución fue explicada en un 40% por las variables % limo y Al (meq/100 g).

[Distribution of potentially nitrogen-fixing bacteria and its relationship with physicochemical parameters in soils with three vegetation types in the southern Colombian Amazon region]. / Distribución de bacterias potencialmente fijadoras de nitrógeno y su relación con parámetros fisicoquímicos en suelos con tres coberturas vegetales en el sur de la Amazonia colombiana.

Potentially nitrogen-fixing microaerobic and aerobic bacteria were isolated from several Colombian Amazon soils (forest, pastures and chagras) and two landscapes (floodable and non floodable areas). The abundance and distribution of bacteria were evaluated, as well as their relationship with soil physical and chemical characteristics. Landscape had a direct influence on the abundance of the microaerobic bacteria, with higher numbers in forest and pasture soils in non-floodable zones. The aerobic isolates (N=51) were grouped into 19 morphologies, with the highest numbers found in forest soil in floodable zones. A higher number of aerobic morphologies was shared among forest sites (Nonmetric Multidimensional Scaling and Analysis of Similarity p<0.05), and 40% of the distribution was explained by lime percentage and Al concentration.

Effect of root canal procedures on endotoxins and endodontic pathogens.

BACKGROUND/AIMS: The purpose of this study was to determine the amount of endotoxin (lipopolysaccharide) and cultivable bacteria in human necrotic root canals before (S1) and after chemo-mechanical preparation using chlorhexidine (CHX) gel as auxiliary chemical substance (S2), and after 7 days of intracanal dressing (S3) in order to evaluate the anti-endotoxin and antimicrobial effects of endodontic procedures. METHOD: Twenty-four teeth were selected for the present study. Chemo-mechanical preparation was performed using 2% CHX gel and three different intracanal medicaments [CaOH2 paste; 2% CHX gel; and CaOH2 + 2% CHX gel]. A quantitative chromogenic Limulus amoebocyte lysate assay was used to measure the amount of endotoxin. Aerobic and anaerobic techniques were used to isolate and identify bacteria, and to determine the bacterial reduction by counting colony-forming units (CFU). RESULTS: Endotoxins and bacteria were present in 100% of the initial samples, with endotoxin concentration ranging from 62.93 to 214.56 UE/ml and CFU ranging from 4 x 10(5) to 2.6 x 10(6). After chemo-mechanical preparation a mean endotoxin reduction of 44.4% was found. Eight (33.3%) root canals were still positive by culture analysis with a mean reduction of bacteria (CFU) of 99.96%. After 7 days of intracanal dressing, endotoxin concentration decreased by only 1.4% compared with S2, and residual bacteria were recovered by culture analysis in 13 cases (54.1%). No significant difference was found among different intracanal medicaments. CONCLUSION: Relatively high values of endotoxin were still present in the root canal after chemo-mechanical preparation although the majority of bacteria were eliminated. No improvement was achieved by 7 days of intracanal dressing.

Flora bacteriana aeróbica del tracto digestive del vampiro común, Desmodus rotundus (Chiroptera: Phyllostomidae) / Aerobic bacterial flora from the digestive tract of the common vampire bat, Desmodus rotundus (Chiroptera: Phyllostomidae)

This study addresses the composition of microbial flora in the vampire bat (Desmodus rotundus) primarily because all available data are outdated, and because of the economical significance of this bat species. Twenty-one bats were collected and their aerobic bacteria documented separately for stomach and intestine. Bacteria were identified through the Analytical Profile Index (API), and results analyzed with the APILAB software. A total of thirty bacterial species were isolated from sixteen females and five males. The most common species were Escherichia coli and Staphylococcus aureus, although other bacteria, such as Acinetobacterjohnsonii, Enterobacter sakazakii, Staphylococcus chromogenes, S. hyicus and S. xylosus were also common. The number of species found in the stomach and intestine was significantly different, and the intestine presented a higher diversity compared to the stomach. This has previously been found in other mammals and it is attributed to a reduction of acidity. Most of the species found in this study are considered normal components of the digestive tract of mammals, although other bacteria common in the skin of mammals and from aquatic environments were found. Bacteria from the skin may invade the vampire's stomach and/or intestine when the bat has contact with its prey, and may suggest that the vampire's feeding habit facilitates the invasion of other microbes not common in its digestive tract. The fact that bacteria from aquatic environments were also found suggests that D. rotundus, as previously found by other researchers, drinks free water when available, and water may be another source of microbial invasion.

El objetivo de esta investigación fue determinar la flora bacteriana del vampiro común (Desmodus rotundus) primordialmente debido a que los datos al respecto están desactualizados, y además por la gran importancia económica de esta especie de murciélago. Veintiún murciélagos fueron recolectados y su flora bacteriana identificada separadamente a nivel de estómago e intestino. Las bacterias fueron identificadas con el Analytical Profile Index (API), y los resultados analizados con el paquete APILAB. Un total de treinta especies fueron aisladas en 16 hembras y cinco machos. Las especies más comunes fueron Escherichia coli y Staphylococcus aureus, aunque otras especies, como Acinetobacter johnsonii, Enterobacter sakazakii, Staphylococcus chromogenes, S. hyicus y S. xylosus también se aislaron con frecuencia. El número de especies identificadas en el estómago y el intestino fue significativamente diferente, siendo el intestino más diverso. Esto ha sido encontrado anteriormente en otros mamíferos, y se atribuye probablemente a la reducción en acidez. Asimismo, la mayoría de las especies identificadas en este estudio forman parte de la flora bacteriana normal del tracto digestivo de mamíferos, aunque también se encontraron otras bacterias comunes en la piel de mamíferos y en ambientes acuáticos Las bacterias de la piel podrían estar colonizando el estómago y/o intestino del vampiro cuando éste tiene contacto con sus presas, lo que sugiere que el hábito alimentario de esta especie facilita su colonización por microorganismos que no se encontrarían comúnmente en su tracto digestivo. Ya que también se identificaron bacterias comunes en ambientes acuáticos, es probable que D. rotundus consuma agua cuando esté disponible, lo que respalda los resultados de otros investigadores, y sugiere que esta podría ser una fuente adicional de invasión microbiana.

Microbial hydrolysis of acetylated nucleosides.

Enzymatic hydrolysis of acetylated nucleosides using microbial whole cells has been carried out for the first time. Unlike Candida antarctica B lipase-catalysed alcoholysis, none of the tested microorganisms displayed a common deacetylation profile. Depending on the substrate and the biocatalyst used, 5'-selective deprotection or mixtures of mono O-acetylated products were obtained.