Influence of culture medium on in-vitro biofilm formation by Candida species

Objectives: Objective of this study was to establish an in vitro biofilm on the 96 well plates and to determine the efficacy of three different culture media on biofilm formation of Candida albicans and C. tropicalis Methods: A 96 well sterile, polystyrene plate was inoculated using 10^6 cell/ml of C. albicans and C. tropicalis suspensions and the growth rate of planktonic cells was determined by measuring the absorbance (OD492) at 2 hour intervals. Adhesion of Candidial cells to initiate the biofilm formation in the presence of three culture media (Yeast Nitrogen Base (YNB) supplemented with 100 mM glucose, Sabouraud Dextrose Broth (SDB) and RPMI1640) was quantified using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Crystal Violet (CV) assay after 90 minutes. Biofilms of C. albicans, C. tropicalis and 1:1 co-biofilms were developed and the growth rates were quantified at 24 hours’ time intervals. Scanning electron microscope (SEM) was performed to assess the architecture. Results: Planktonic cells of both C. albicans and C. tropicalis showed maximum growth with SDB. C. albicans and co-biofilm adhesion were significantly facilitated with RPMI1640 and the best medium for C. tropicalis adhesion was YNB. Biofilms showed the maximum growth rate in RPMI 1640. C. tropicalis exhibited the minimum growth with all three culture media.Conclusions: The maximum growth rate for planktonic C. albicans and C. tropicalis was achieved with SDB. However RPMI 1640 was the best medium for growth of biofilms

Development and validation of a reference marker for identification of aerobic and anaerobic bacteria associated with diabetes chronic wound ulcers using PCR denaturing gradient gel electrophoresis

Introduction: Diabetes chronic wounds consist with a diverse microbial community and unculturablespecies may be highly prevalent.Objectives: This study aimed to establish a bacterial reference marker consisting of a group ofchronic wound related bacteria, using polymerase chain reaction-denaturing gradient gelelectrophoresis (PCR-DGGE) for profiling of bacteria in diabetes chronic wound infections.Methods: DNA was extracted from the known wound bacterial strains. PCR–DGGE was performedusing eubacterial specific primers targeting V2-V3 region of 16S rDNA. DGGE was performed usinga 30-55% denaturing gradient. Migration position of each organism was detected on DGGE gel andimportant organisms were selected. Equal volume from PCR products of each selected organism wasmixed, diluted with gel loading dye in 1:1.5 ratio and used for all DGGE gels. The ladder was thensubjected to species identification of fifteen tissue debridement specimens obtained from diabeteschronic wound ulcers. The identification efficacy was tested by sequencing.Results: DNA of bacterial pathogens which showed different migration distances on the gel werecombined and used as a reference panel. This bacterial ladder consisted of eleven different bacterialspecies including Bacteroides sp., S. aureus, Acineto bacter sp., P. aeruginosa, Streptococcus Group Aand Group B sp., E. faecalis, Providencia sp., Veillonella sp., E .coli and Enterobacter sp. Accordingto the reference panel, Pseudomonas species were abundant. Further the results were confirmed bysequencing.Conclusion: Reference marker allows comparative analysis of DGGE patterns and can be used as atool for presumptive identification of polymicrobial microbiota in chronic wound infections

Impact of routine laboratory culture media on in-vitro biofilm formation of Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis

Objectives: This study was aimed to determine the efficacy of four routine laboratory culture media onbiofilm formation of Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus feacalis.Methods: A sterile flat bottom 96 well plate was inoculated using 0.5 McFarland equivalent standardcell suspension of P. aeruginosa, S. aureus and E. feacalis and the growth rate of planktonic cells wasquantified by measuring the optical density (OD492) at two hour intervals. Influence of culture mediumon adhesion of bacteria as an initial step of biofilm formation in the presence of four culture media(Nutrient broth (NB), Brain Heart Infusion (BHI) broth, Luria-Bertani (LB) broth and RPMI 1640) wasquantified using MTT (3-[4, 5- dimethylthiazole-2-yl]-2, 5- diphenyltetrazolium bromide) assay after90 minutes adhesion. Biofilms of P. aeruginosa, S. aureus, E. feacalis and their 1:1 mixed biofilmswere developed and the growth was quantified using MTT metabolic activity at 24 hour time intervals.Scanning electron microscopy (SEM) was performed to assess the ultrastructure.Results: On comparing the relative growth of the bacteria in different culture media, the maximumgrowth of all three planktonic cultures was achieved using BHI broth. All mono species and mixedspecies cultures exhibited their maximum adhesion in the presence of RPMI 1640. All biofilm exhibitedthe maximum growth in BHI broth. SEM imaging had shown the enhanced growth of ultrastructure ofthe biofilm with the presence of BHI broth.Conclusions: The maximum planktonic and biofilm growth was achieved with BHI broth. However,bacterial adhesion was enhanced in the presence of RPMI 1640

Utilisation of an operative difficulty grading scale for laparoscopic cholecystectomy

Background A reliable system for grading operative difficulty of laparoscopic cholecystectomy would standardise description of findings and reporting of outcomes. The aim of this study was to validate a difficulty grading system (Nassar scale), testing its applicability and consistency in two large prospective datasets. Methods Patient and disease-related variables and 30-day outcomes were identified in two prospective cholecystectomy databases: the multi-centre prospective cohort of 8820 patients from the recent CholeS Study and the single-surgeon series containing 4089 patients. Operative data and patient outcomes were correlated with Nassar operative difficultly scale, using Kendall’s tau for dichotomous variables, or Jonckheere–Terpstra tests for continuous variables. A ROC curve analysis was performed, to quantify the predictive accuracy of the scale for each outcome, with continuous outcomes dichotomised, prior to analysis. Results A higher operative difficulty grade was consistently associated with worse outcomes for the patients in both the reference and CholeS cohorts. The median length of stay increased from 0 to 4 days, and the 30-day complication rate from 7.6 to 24.4% as the difficulty grade increased from 1 to 4/5 (both p < 0.001). In the CholeS cohort, a higher difficulty grade was found to be most strongly associated with conversion to open and 30-day mortality (AUROC = 0.903, 0.822, respectively). On multivariable analysis, the Nassar operative difficultly scale was found to be a significant independent predictor of operative duration, conversion to open surgery, 30-day complications and 30-day reintervention (all p < 0.001). Conclusion We have shown that an operative difficulty scale can standardise the description of operative findings by multiple grades of surgeons to facilitate audit, training assessment and research. It provides a tool for reporting operative findings, disease severity and technical difficulty and can be utilised in future research to reliably compare outcomes according to case mix and intra-operative difficulty

Global Retinoblastoma Presentation and Analysis by National Income Level

Importance: Early diagnosis of retinoblastoma, the most common intraocular cancer, can save both a child's life and vision. However, anecdotal evidence suggests that many children across the world are diagnosed late. To our knowledge, the clinical presentation of retinoblastoma has never been assessed on a global scale. Objectives: To report the retinoblastoma stage at diagnosis in patients across the world during a single year, to investigate associations between clinical variables and national income level, and to investigate risk factors for advanced disease at diagnosis. Design, Setting, and Participants: A total of 278 retinoblastoma treatment centers were recruited from June 2017 through December 2018 to participate in a cross-sectional analysis of treatment-naive patients with retinoblastoma who were diagnosed in 2017. Main Outcomes and Measures: Age at presentation, proportion of familial history of retinoblastoma, and tumor stage and metastasis. Results: The cohort included 4351 new patients from 153 countries; the median age at diagnosis was 30.5 (interquartile range, 18.3-45.9) months, and 1976 patients (45.4%) were female. Most patients (n = 3685 [84.7%]) were from low- A nd middle-income countries (LMICs). Globally, the most common indication for referral was leukocoria (n = 2638 [62.8%]), followed by strabismus (n = 429 [10.2%]) and proptosis (n = 309 [7.4%]). Patients from high-income countries (HICs) were diagnosed at a median age of 14.1 months, with 656 of 666 (98.5%) patients having intraocular retinoblastoma and 2 (0.3%) having metastasis. Patients from low-income countries were diagnosed at a median age of 30.5 months, with 256 of 521 (49.1%) having extraocular retinoblastoma and 94 of 498 (18.9%) having metastasis. Lower national income level was associated with older presentation age, higher proportion of locally advanced disease and distant metastasis, and smaller proportion of familial history of retinoblastoma. Advanced disease at diagnosis was more common in LMICs even after adjusting for age (odds ratio for low-income countries vs upper-middle-income countries and HICs, 17.92 [95% CI, 12.94-24.80], and for lower-middle-income countries vs upper-middle-income countries and HICs, 5.74 [95% CI, 4.30-7.68]). Conclusions and Relevance: This study is estimated to have included more than half of all new retinoblastoma cases worldwide in 2017. Children from LMICs, where the main global retinoblastoma burden lies, presented at an older age with more advanced disease and demonstrated a smaller proportion of familial history of retinoblastoma, likely because many do not reach a childbearing age. Given that retinoblastoma is curable, these data are concerning and mandate intervention at national and international levels. Further studies are needed to investigate factors, other than age at presentation, that may be associated with advanced disease in LMICs

Denitrification likely catalyzed by endobionts in an allogromiid foraminifer

Author Posting. © The Author(s), 2011. This is the author's version of the work. It is posted here by permission of Nature Publishing Group for personal use, not for redistribution. The definitive version was published in The ISME Journal 6 (2012): 951–960, doi:10.1038/ismej.2011.171.Nitrogen can be a limiting macronutrient for carbon uptake by the marine biosphere. The process of denitrification (conversion of nitrate to gaseous compounds, including N2) removes bioavailable nitrogen, particularly in marine sediments, making it a key factor in the marine nitrogen budget. Benthic foraminifera reportedly perform complete denitrification, a process previously considered nearly exclusively performed by bacteria and archaea. If the ability to denitrify is widespread among these diverse and abundant protists, a paradigm shift is required for biogeochemistry and marine microbial ecology. However, to date, the mechanisms of foraminiferal denitrification are unclear and it is possible that the ability to perform complete denitrification is due to symbiont metabolism in some foraminiferal species. Using sequence analysis and GeneFISH, we show that for a symbiont-bearing foraminifer, the potential for denitrification resides in the endobionts. Results also identify the endobionts as denitrifying pseudomonads and show that the allogromiid accumulates nitrate intracellularly, presumably for use in denitrification. Endobionts have been observed within many foraminiferal species, and in the case of associations with denitrifying bacteria, may provide fitness for survival in anoxic conditions. These associations may have been a driving force for early foraminiferal diversification, which is thought to have occurred in the Neoproterozoic when anoxia was widespread.This research was supported by NSF grant EF-0702491 to JMB, KLC and VPE; some ship support was provided by NSF MCB-0604084 to VPE and JMB.2012-06-0

Identification and Genome-Wide Prediction of DNA Binding Specificities for the ApiAP2 Family of Regulators from the Malaria Parasite

The molecular mechanisms underlying transcriptional regulation in apicomplexan parasites remain poorly understood. Recently, the Apicomplexan AP2 (ApiAP2) family of DNA binding proteins was identified as a major class of transcriptional regulators that are found across all Apicomplexa. To gain insight into the regulatory role of these proteins in the malaria parasite, we have comprehensively surveyed the DNA-binding specificities of all 27 members of the ApiAP2 protein family from Plasmodium falciparum revealing unique binding preferences for the majority of these DNA binding proteins. In addition to high affinity primary motif interactions, we also observe interactions with secondary motifs. The ability of a number of ApiAP2 proteins to bind multiple, distinct motifs significantly increases the potential complexity of the transcriptional regulatory networks governed by the ApiAP2 family. Using these newly identified sequence motifs, we infer the trans-factors associated with previously reported plasmodial cis-elements and provide evidence that ApiAP2 proteins modulate key regulatory decisions at all stages of parasite development. Our results offer a detailed view of ApiAP2 DNA binding specificity and take the first step toward inferring comprehensive gene regulatory networks for P. falciparum

Histone Deacetylases Play a Major Role in the Transcriptional Regulation of the Plasmodium falciparum Life Cycle

The apparent paucity of molecular factors of transcriptional control in the genomes of Plasmodium parasites raises many questions about the mechanisms of life cycle regulation in these malaria parasites. Epigenetic regulation has been suggested to play a major role in the stage specific gene expression during the Plasmodium life cycle. To address some of these questions, we analyzed global transcriptional responses of Plasmodium falciparum to a potent inhibitor of histone deacetylase activities (HDAC). The inhibitor apicidin induced profound transcriptional changes in multiple stages of the P. falciparum intraerythrocytic developmental cycle (IDC) that were characterized by rapid activation and repression of a large percentage of the genome. A major component of this response was induction of genes that are otherwise suppressed during that particular stage of the IDC or specific for the exo-erythrocytic stages. In the schizont stage, apicidin induced hyperacetylation of histone lysine residues H3K9, H4K8 and the tetra-acetyl H4 (H4Ac4) and demethylation of H3K4me3. Interestingly, we observed overlapping patterns of chromosomal distributions between H4K8Ac and H3K4me3 and between H3K9Ac and H4Ac4. There was a significant but partial association between the apicidin-induced gene expression and histone modifications, which included a number of stage specific transcription factors. Taken together, inhibition of HDAC activities leads to dramatic de-regulation of the IDC transcriptional cascade, which is a result of both disruption of histone modifications and up-regulation of stage specific transcription factors. These findings suggest an important role of histone modification and chromatin remodeling in transcriptional regulation of the Plasmodium life cycle. This also emphasizes the potential of P. falciparum HDACs as drug targets for malaria chemotherapy