Recovery of Candida spp. from a denture rat model

Denture stomatitis is a common infection in denture wearers. This study evaluated the recovery of Candida spp. from the palate of Wistar rats after using an acrylic device with single and mixed-species of Candida spp. After approval of the Ethics Committee, 84 male and female Wistar rats were used. Custom-made acrylic devices were fabricated for each animal and sterilized by microwave irradiation. Single and mixed species biofilms of C. albicans (Ca), C. glabrata (Cg), and C. tropicalis (Ct) were grown on the devices for 48 h at 37°C. Rats were anesthetized and the devices were cemented on the molar teeth (n=5 for each sex and Candida spp.). Rats received a carbohydrate-rich diet. Single and mixed species were inoculated in the oral cavity thrice after three-day intervals. Controls received only dentures without Candida spp. After 4 weeks, the devices were removed, the palates were swabbed, and diluted samples were plated on Agar Sabouraud Dextrose and CHROMAgar Candida for colony counting and presumptive identification, respectively, after 48 h. Data were analyzed by 3way ANOVA (α=5%). There was a significant interaction (p=0.003) between sex and species. For females, all groups recovered significant values (p≤0.027) compared with controls. For males, groups with Ct as single and dual-species showed the lowest values without difference (p≥0.183) with the control. The groups with triple-species showed the highest values but without difference (p≥0.071) with the groups with single and dual-species, except males with Ct. Ct alone showed reduced recovery from palate of male rats.

A rapid and non-destructive method for spatial–temporal quantification of colonization by Pseudomonas syringae pv. tomato DC3000 in Arabidopsis and tomato

Background The bacterial leaf pathogen Pseudomonas syringae pv tomato (Pst) is the most popular model pathogen for plant pathology research. Previous methods to study the plant-Pst interactions rely on destructive quantification of Pst colonisation, which can be labour- and time-consuming and does not allow for spatial–temporal monitoring of the bacterial colonisation. Here, we describe a rapid and non-destructive method to quantify and visualise spatial–temporal colonisation by Pst in intact leaves of Arabidopsis and tomato. Results The method presented here uses a bioluminescent Pst DC3000 strain that constitutively expresses the luxCDABE operon from Photorhabdus luminescens (Pst::LUX) and requires a common gel documentation (Gel Doc) system with a sensitive CCD/CMOS camera and imaging software (Photoshop or Image J). By capturing bright field and bioluminescence images from Pst::LUX-infected leaves, we imaged the spatiotemporal dynamics of Pst infection. Analysis of bioluminescence from live Pst bacteria over a 5-day time course after spray inoculation of Arabidopsis revealed transition of the bacterial presence from the older leaves to the younger leaves and apical meristem. Colonisation by Pst:LUX bioluminescence was obtained from digital photos by calculating relative bioluminescence values, which is adjusted for bioluminescence intensity and normalised by leaf surface. This method detected statistically significant differences in Pst::LUX colonisation between Arabidopsis genotypes varying in basal resistance, as well as statistically significant reductions in Pst::LUX colonisation by resistance-inducing treatments in both Arabidopsis and tomato. Comparison of relative bioluminescence values to conventional colony counting on selective agar medium revealed a statistically significant correlation, which was reproducible between different Gel Doc systems. Conclusions We present a non-destructive method to quantify colonisation by bioluminescent Pst::LUX in plants. Using a common Gel Doc system and imaging software, our method requires less time and labour than conventional methods that are based on destructive sampling of infected leaf material. Furthermore, in contrast to conventional strategies, our method provides additional information about the spatial–temporal patterns of Pst colonisation.

Targeting bacteria causing otitis media using nanosystems containing nonspherical gold nanoparticles and ceragenins

Aim: To evaluate the antibacterial and antibiofilm activity of ceragenin-conjugated nonspherical gold nanoparticles against the most common agents of otitis media. Methods: Minimal inhibitory and bactericidal concentrations and colony-counting assays, as well as colorimetric and fluorimetric methods, were used to estimate the antibacterial activity of compounds in phosphate-buffered saline and human cerumen. The nanosystems’ biocompatibility and ability to decrease IL-8 release was tested using keratinocyte cells. Results: The tested compounds demonstrated strong antimicrobial activity against planktonic and biofilm cultures at nontoxic doses due to the induction of oxidative stress followed by the damage of bacterial membranes. Conclusions: This study indicates that ceragenin-conjugated nonspherical gold nanoparticles have potential as new treatment methods for eradicating biofilm-forming pathogens associated with otitis media.

Identity and Antibiogram of Bacterial Isolates from Owerri Modern Abattoir, Imo State, Nigeria

Aim: The identity and antibiogram of bacterial isolates from Owerri modern abattoir in Imo State, Nigeria, was investigated with the aim of determining the bacterial profile of the abattoir and their susceptibility pattern to commonly used antibiotics. Study Design: Cross sectional study Place and Duration of Study: This study was carried out in Owerri Modern Abattoir located within Owerri metropolis from June to November, 2020. Methodology: Questionnaires were used to obtain participants’ consent, demographic data and sanitary practices in the abattoir. Samples were taken and bacteriological analysis of the samples done using pour plate method. Disc diffusion antibiotic susceptibility testing and minimum inhibitory concentration were performed after colony counting, identification and characterization of the isolates using standard microbiological and biochemical techniques. Results: Mean viable bacterial counts were generally high with highest counts from contaminated soil (6.13x106CFU/ml) and least from workers hands (1.17x106CFU/ml). Escherichia coli had the highest prevalence (18.0%), with the highest counts from soil (3.10%). Vibrio cholerae recorded the least prevalence (0.62%), and was isolated only from washing water. High resistance to antibiotics was observed. Conclusion: Government authority and the general public are advised to ensure adequate environmental sanitation and proper cooking of meat before consumption in order to mitigate the incidence of infection and antibiotic resistance.

Feasibility study of fluorescence quantitative PCR for the detection of microecological dynamics in fermented maize stover feeds

During the fermentation of corn stalk bio-feed, the quantity of bacteria in corn stalk bio-feed was counted by fluorescence quantitative PCR and plate colony counting respectively. The comparative analysis of these studies was used to explore the feasibility of fluorescence quantification methods and changes in microbiota during fermentation. The results showed that the standard deviation of fluorescence quantitative method was smaller than that of plate method, but the trend was similar. The biomass of Bacillus subtilis, Lactobacillus plantarum and Saccharomyces cerevisiae reached their maximum on the third, fifth and fifth day respectively, and then decreased gradually and maintained at a certain level. The experiment showed that the fluorescence quantitative PCR method can accurately quantify the number of bacteria in corn stalk bio-feed, and it is a better method to quantitatively detect the dynamic changes of different kinds of bacteria in corn stalk bio-feed.

Rapid evaluation of the resistance of citrus germplasms against Xanthomonas citri subsp. citri

Xanthomonas citri subsp. citri (Xcc) is the causal agent of citrus bacterial canker (CBC), one of the most devastating citrus diseases. Most commercial citrus varieties are susceptible to CBC. However, some citrus varieties and wild citrus germplasms are CBC-resistant and are promising in genetic improvements of citrus resistance against CBC. We aimed to evaluate citrus germplasms for resistance against CBC. First, we developed a rapid evaluation method based on enhanced yellow fluorescent protein (eYFP)-labeled Xcc. The results demonstrated that eYFP does not affect the growth and virulence of Xcc. Xcc-eYFP allows measuring of bacterial titers, but is more efficient and rapid than the plate colony counting method. Next, we evaluated citrus germplasms collected in China. Based on symptoms and bacterial titers, we identified that two citrus germplasms (‘Ichang’ papeda, and ‘Huapi’ kumquat) are resistant, whereas eight citrus germplasms (‘Chongyi’ wild mandarin, ‘Mangshan’ wild mandarin, ‘Ledong’ kumquat, ‘Dali’ citron, ‘Yiliang’ citron, ‘Longyan’ kumquat, ‘Bawang’ kumquat and ‘Daoxian’ wild mandarin) are tolerant. In summary, we have developed a rapid evaluation method to test the resistance of citrus plants against CBC. This method was successfully used to identify two highly canker-resistant citrus germplasms and eight citrus germplasms with canker tolerance. These results could be leveraged in traditional breeding contexts or be used to identify canker resistance genes to improve the disease resistance of commercial citrus varieties via biotechnological approaches.

Application of the Resazurin Cell Viability Assay to Monitor Escherichia coli and Salmonella Typhimurium Inactivation Mediated by Phages

Bacterial inactivation using bacteriophages (or phages) has emerged as an effective solution for bacterial infections, but the screening methods used to evaluate the effectiveness of the phages to inactivate bacteria are not fast, reliable or precise enough. The efficiency of bacterial inactivation by phages has been evaluated by monitoring bacterial concentration either by counting colony-forming units (CFU), a laborious and time-consuming method, or by monitoring the optical density (OD), a less sensitive method. In this study, the resazurin cell viability assay was used to monitor the viability of bacteria from different genera during the inactivation by different phages, and the results were compared with the standard methods used to assess bacterial inactivation. The results showed that the resazurin colorimetric cell viability assay produces similar results to the standard method of colony-counting and giving, and also more sensitive results than the OD method. The resazurin assay can be used to quickly obtain the results of the cell viability effect profile using two different bacterial strains and several different phages at the same time, which is extremely valuable in screening studies. Moreover, this methodology is established as an effective, accurate and rapid method when compared to the ones widely used to monitor bacterial inactivation mediated by phages.

A 3×4 drop-plating protocol for estimation of Antimicrobial-resistant bacteria, taking Extended-spectrum beta-lactamases producing Escherichia coli as an example.

The estimation of antimicrobial-resistant (AMR) bacteria plays an important role in risk assessment and surveillance. To test the concentration of resistant bacteria with colony count is a fast and straightforward way to perform. Here we describe an optimized drop-plating method for colony counting of resistant bacteria. We took the ESBL-producing E. coli in freshwater samples as an example. The optimized methods can successfully quantify ESBL-producing E. coli of water samples in a concentration range of 104 CFU/L to 106 CFU/L. We have shown that this drop-plating method is comparable to the direct spreading method by testing with both methods on a series of simulated samples, which were constructed using raw surface water spiked with different concentrations of ESBL-producing E. coli. The ESBL-producing phenotype has been further confirmed with the double-disc synergy test. Compared to direct spread methods, our methods can save consumables and operate with smaller sample sizes. Therefore, this method could be more sustainable in AMR surveillance and risk assessment.