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

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.

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).

Acute lower respiratory tract infections in children.

Acute lower respiratory tract infection (ALRTI) is a common illness, but there have been relatively few studies of the bacterial etiology in developing countries. Nasopharyngeal aspirates of 70 children under 10 years of age with ALRTI were cultured for aerobic bacterial pathogens. Klebsiella pneumoniae was the commonest organism (32.2%) isolated followed by S. pneumoniae (10%), E. coli (10%), P. aeruginosa (5.7%), S. aureus (2.8%) and H. influenzae (1.4%). There were significantly more bacterial pathogens isolated in children <1 year of age (73.7%) than in those >1 year of age (56.2%) (P=0.03). A shift in spectrum from Gram-positive cocci to Gram-negative bacilli in ALRTI was observed in our study.

Microbial contamination of pig carcasses at a slaughterhouse in Vientiane capital, Lao PDR.

A cross-sectional study was conducted to determine microbial contamination of pig carcasses at a slaughterhouse in Vientiane, capital of Lao People Democratic Republic (Lao PDR). Between November 2004 and April 2005, 62 pig carcasses were randomly selected. From each carcass, pooled swabs (from "1" prior to and "2" after evisceration) and 25 g of tissue of mesenteric lymph nodes (MLN) were collected. The swab samples were examined for Aerobic Plate Count (APC) and Enterobacteriaceae Counts (EBC) and cultured for Salmonella. The lymph nodes were cultured for Salmonella only. Swabs1 and 2 had mean APC of 4.70 and 4.85 log10CFU/cm2, respectively. These two means were significantly (p = 0.0001) different. The means of EBC were 2.81 log10CFU/cm2 for Swab 1, and 2.98 log10CFU/cm2 for Swab 2. The difference were also statistical significant (p = 0.0001). The frequency of Salmonella isolation from Swab 1 was 46.8%, for Swab 2 was 66.1%, and from mesenteric lymphnodes was 53.2%. Eight different Salmonella serotypes were identified. The most frequent (29.1%) serotype was S. Rissen, followed by S. Anatum (26.2%), S. Derby (18.4%), and S. Elisabethville (8.7%). The other serotypes identified were S. Amsterdam (7.8%), S. Typhimurium (4.9%), S. Agona (2.9%), and S. Enteritidis (1.9%). Results of this study showed the levels of contamination with aerobic bacteria and Enterobacteriaceae were higher than recommended standards, and the carcasses were contaminated with Salmonella.

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.

Bacteriological study of paediatric and adult chronic suppurative otitis media.

Chronic suppurative otitis media (CSOM) is one of the commonest illnesses in ENT practice which requires medical attention all the more in children of poor socio-economic status having in past inadequate treatment and negligent medical care. The present study was conducted to find out the various aerobic and anaerobic microorganisms associated with CSOM in paediatric and adult cases and their current antimicrobial susceptibility pattern as a guide to therapy. Samples were collected from 109 clinically diagnosed cases of CSOM and processed according to standard protocols. Out of 74 paediatric CSOM cases, 72 (97.2%) were bacterial culture positive while out of 35 adult CSOM cases, 28 (80%) were culture positive. Bilateral CSOM was slightly more common in adults (25%) than paediatric (21.4%) age group. Polymicrobial nature of CSOM was noted in both paediatric (70.8%) and adult (71.4%) cases while number of organisms isolated per lesion was slightly higher in adults (2.5) as compared to paediatric (1.95) cases. In paediatric CSOM, Staphylococcus aureus was the commonest aerobic isolate while in adult CSOM, Pseudomonas aeruginosa was the commonest one. Among anaerobes Peptostreptococcus spp. was commonest in CSOM where as Prevotella melaninogenica in adult CSOM. Sensitivity of S. aureus to cefuroxime was 72.2% while that of gram negative bacilli was higher to cefotaxime (90 to 100%). Among anaerobes higher sensitivity was seen to metronidazole (98.6%), clindamycin (95.7%) and chloramphenicol (98.6%). In view of the polymicrobial etiology of CSOM, prompt appropriate antimicrobial therapy can effectively reverse the disease process thereby preventing longterm sequelae.

Studies on microbial aerobic flora of skin in leprosy patients.

This study reports the isolation and identification of aerobic organisms from biopsies/slit-skin smears/scrapings from 129 leprosy patients and 50 healthy controls. These include 56 paucibacillary (PB) and 73 multibacillary (MB) cases. Thirty-six isolates from the specimens from 21 patients and 15 healthy controls were grown. The non-mycobacterial isolates from clinically PB leprosy (TT/BT/I) patients were: (1) Gram-positive cocci: Staphylococcus aureus(1), Staphylococcus albus(1); (b) Gram-positive bacilli: Bacillus subtilis(1), Corynebacterium xerosis(1); (c) Gram-negative bacilli: Escherichia coli(1), Proteus mirabilis(2), Klebsiella pneumoniae(1) and Pseudomonas aeruginosa(1). The isolates from clinically MB leprosy (BB/BL/LL) patients were: (a) Gram-positive cocci: Micrococci(1), Staphylococcus aureus(1) and Staphylococcus albus(1); (b) Gram-positive bacilli: Corynebacterium xerosis(1); Corynebacterium hofmanni(1) and Bacillus cereus(1). (c) Gram-negative bacilli: Escherichia coli(2), Klebsiella pneumoniae(1) and Proteus mirabilis(2). The specimens from healthy controls yielded similar organisms. These were (a) Gram-positive cocci: Staphylococcus albus(2), Staphylococcus aureus(2) and Micrococci(2); (b) Gram-positive bacilli: Corynebacterium xerosis(1), Bacillus subtilis(2), Corynebacterium hofmanni(1) and Bacillus cereus(1); (c) Gram-negative bacilli: Escherichia coli(3), Proteus vulgaris(1) and Proteus mirabilis(1). While these results show no significant differences in the species types of non-mycobacterial aerobic organisms isolated from healthy skin and PB/MB types of leprosy, these isolates need to be characterized by immunological/molecular methods to find out subtypes if any.