Evaluation of different detection methods of biofilm formation in the clinical isolates

BACKGROUND: Microorganisms growing in a biofilm are associated with chronic and recurrent human infections and are highly resistant to antimicrobial agents. There are various methods to detect biofilm production like Tissue Culture Plate (TCP), Tube method (TM), Congo Red Agar method (CRA), bioluminescent assay, piezoelectric sensors, and fluorescent microscopic examination. OBJECTIVE: This study was conducted to compare three methods for the detection of biofilms. METHOD: The study was carried out at the Department of Microbiology, Army Medical College, National University of Sciences and Technology, Pakistan, from January 2010 to June 2010. A total of 110 clinical isolates were subjected to biofilm detection methods. Isolates were identified by standard microbiological procedures. Biofilm detection was tested by TCP, TM and CRA. Antibiotic susceptibility test of biofilm producing bacteria was performed by using the Kirby-Bauer disc diffusion technique according to CLSI guidelines. RESULTS: The TCP method was considered to be superior to TM and CRA. From the total of 110 clinical isolates, TCP method detected 22.7 percent as high, 41 percent moderate and 36.3 percent as weak or non-biofilm producers. We have observed higher antibiotic resistance in biofilm producing bacteria than non-biofilm producers. CONCLUSION: We can conclude from our study that the TCP method is a more quantitative and reliable method for the detection of biofilm forming microorganisms as compared to TM and CRA methods, and it can be recommended as a general screening method for detection of biofilm producing bacteria in laboratories.

Clinical status and detection of periodontopathogens and Streptococcus mutans in children with high levels of supragingival biofilm

Knowledge about the presence of some important oral pathogens is an important step in better identifying children at risk for periodontal and/or caries diseases in later life. The purpose of this study was to detect the presence of Streptococcus mutans (Sm), Aggregatibacter actinomycetemcomitans (Aa), Campylobacter rectus (Cr), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), and Tannerella forsythia (Tf) in gingival biofilm samples from 196 children, and to assess whether any of these pathogens are more associated with gingival inflammation extension and the Decayed/Missing/Filled teeth (DMFT/dmft) index. The subjects presented plaque index greater than 80 percent and were divided in 3 groups according to the bleeding index (BI): I) Low bleeding (< 30 percent), II) Medium bleeding (31 - 59 percent) and III) High bleeding (> 60 percent). The presence of each pathogen was determined by PCR. The prevalence of Sm was 71.9 percent and the mean dmft/DMFT was 6.68. The prevalence in low, medium and high bleeding groups was 43.5 percent, 34.5 percent and 46.7 percent for Aa; 43.5 percent, 37.9 percent, and 36.7 percent for Cr; 99.1 percent, 100 percent, and 96.7 percent for Pg; 56.5 percent, 56.9 percent, and 66.7 percent for Pi; and 58.3 percent, 60.3 percent, and 56.7 percent for Tf, respectively. Pg (99.0 percent) was the most prevalent periodontal pathogen detected followed by Tf (58.7 percent), Pi (58.2 percent), Aa (41.3 percent) and Cr (40.8 percent). Our study indicated that in this high plaque index population studied, a high prevalence of Sm and high mean DMFT were observed. In addition, the presence of Pi was associated with the presence of inflammation (P < 0.05) whereas Cr was associated with periodontal health (P < 0.05).

Survival and surface adherence ability of bacterial pathogens in oral liquid pharmaceuticals and their containers

The survival and surface adherence ability of Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa in nutrient broth and in five oral liquid pharmaceuticals (nivaquine syrup, cough mixture, paracetamol elixir, cotrimoxazole and vitamin C) were investigated The bacteria grew more in nutrient broth than in the pharmaceuticals (p < 0. 001) and the recovery of stressed cells was enhanced when 3 Tween 80 was used as the recovery medium as against the use of normal saline (p < 0.01). The Gram-negative bacteria were more adapted to the pharmaceuticals than their Gram-positive counterparts. Klebsiella pneumoniae and Ps. aeruginosa were recovered in large numbers from nivaquine and cotrimoxazole suspensions that did not support the growth of the other bacteria. The effect of bacterial growth on the physico-chemical properties of the pharmaceuticals was also evaluated The properties were not altered significantly except for pH shifts of 0.3 to 1.1 caused by E. coli and S. aureus in paracetamol and vitamin C. Adherence capability was found to correlate with the survival ability of the bacteria. Populations on coupons were significantly higher when nutrient broth was used as the suspending medium compared with any of the pharmaceuticals (p < 0.01). Rubber and plastic coupons were significantly more accessible to the bacteria than glass coupon as revealed by the high population of bacteria recovered from their surfaces

Inhibition of the growth of Ascosphaera apis by Bacillus and Paenibacillus strains isolated from honey / Inhibición del crecimiento de Ascosphaera apis mediante cepas de Bacillus y Paenibacillus aisladas de miel

The fungus Ascosphaera apis, the causative agent of chalkbrood disease in honeybee larvae, occurs throughout the world and is found in many beekeeping areas of Argentina. The potential as biocontrol agents of 249 aerobic spore-forming bacterial antagonists isolated from honey samples was evaluated. Each isolate was screened against A. apis by a central disk test assay. Ten bacterial strains that showed the best antagonistic effect to A. apis were selected for further study and identified as Bacillus cereus (m363, mv86, mv81, mv75), Bacillus circulans (Fr231, m448b), Bacillus megaterium (m435), Bacillus pumilus (m354), Bacillus subtilis (m329), and Paenibacillus alvei (m321). For testing the efficiency of the selected strains, a paired culture test was used with 5 replicates of each combination bacterial antagonist / A. apis strain, and 5 replications for each control on 4 different culture media. The analysis of variance and posterior comparison of means according to LSD method showed that the best antagonists when using YGPSA medium were B. subtilis (m329) and B. megaterium (m435), and in the case of MYPGP medium the most efficient were B. circulans strains Fr 231 and m448b.

La cría yesificada es una micosis invasiva ocasionada por el hongo heterotálico Ascosphaera apis que afecta exclusivamente a las larvas de las abejas. La enfermedad tiene difusión mundial y en la Argentina se halla diseminada en todas las áreas donde se realiza apicultura. Se estudió la potencialidad de 249 cepas de bacterias esporuladas aeróbicas aisladas de miel como agentes biocontroladores del hongo mediante un ensayo en disco central en condiciones de laboratorio. Se seleccionaron como mejores antagonistas 10 cepas bacterianas identificadas como Bacillus cereus (m363, mv86, mv81, mv75), Bacillus circulans (Fr231, m448b), Bacillus megaterium (m435), Bacillus pumilus (m354), Bacillus subtilis (m329), y Paenibacillus alvei (m321). Para probar la eficiencia de las cepas seleccionadas, se empleó la técnica de cultivo dual con 5 repeticiones de cada combinación antagonista bacteriano / cepa de A. apis, 5 para cada control y 4 medios de cultivo diferentes empleando 10 cepas del hongo de distintos orígenes geográficos. El análisis de la variancia y posterior comparación de medias LSD (Least Square Dfferences) mostró que los mejores antagonistas fueron B. subtilis (m329) y B. megaterium (m435) para el caso del medio YGPSA, mientras que para MYPGP las más eficientes fueron las cepas de B. circulans Fr 231 y m448b.