Developing a Novel Murine Meningococcal Meningitis Model Using a Capsule-Null Bacterial Strain.

BACKGROUND: Neisseria meningitidis (meningococcus) is a Gram-negative bacterium that colonises the nasopharynx of about 10% of the healthy human population. Under certain conditions, it spreads into the body, causing infections with high morbidity and mortality rates. Although the capsule is the key virulence factor, unencapsulated strains have proved to possess significant clinical implications as well. Meningococcal meningitis is a primarily human infection, with limited animal models that are dependent on a variety of parameters such as bacterial virulence and mouse strain. In this study, we aimed to develop a murine Neisseria meningitidis meningitis model to be used in the study of various antimicrobial compounds. METHOD: We used a capsule-deficient Neisseria meningitidis strain that was thoroughly analysed through various methods. The bacterial strain was incubated for 48 h in brain-heart infusion (BHI) broth before being concentrated and injected intracisternally to bypass the blood-brain barrier in CD-1 mice. This prolonged incubation time was a key factor in increasing the virulence of the bacterial strain. A total of three more differently prepared inoculums were tested to further solidify the importance of the protocol (a 24-h incubated inoculum, a diluted inoculum, and an inactivated inoculum). Antibiotic treatment groups were also established. The clinical parameters and number of deaths were recorded over a period of 5 days, and comatose mice with no chance of recovery were euthanised. RESULTS: The bacterial strain was confirmed to have no capsule but was found to harbour a total of 56 genes coding virulence factors, and its antibiotic susceptibility was established. Meningitis was confirmed through positive tissue culture and histological evaluation, where specific lesions were observed, such as perivascular sheaths with inflammatory infiltrate. In the treatment groups, survival rates were significantly higher (up to 81.25% in one of the treatment groups compared to 18.75% in the control group). CONCLUSION: We managed to successfully develop a cost-efficient murine (using simple CD-1 mice instead of expensive transgenic mice) meningococcal meningitis model using an unencapsulated strain with a novel method of preparation.

Medical and surgical management of a rare and complicated case of multivisceral hydatidosis; 18 years of evolution.

Hydatidosis (echinococcosis) is a parasitic disease caused by the development in the human host of the larval form of the Echinococcus spp. tapeworm. Among the parasitic diseases transmitted from animal to human, hydatidosis represents the main Romanian helminthic zoonosis in humans, due to the severity of the clinical presentation and the complications of this illness. Before 1995-2000, surgical care was considered the only treatment available for the disease in Romania. Recently, the association of surgical procedures with pre and postoperative benzimidazolic drugs has been imposed. We describe the case of a patient diagnosed in 1995, when he had already presented a form of multivisceral hydatidosis, and we also emphasize the development of this disease in this transition period, which concerns the change in the hydatidosis approach.

Comparative analysis of Staphylococcus aureus and Escherichia coli microcalorimetric growth.

BACKGROUND: Microcalorimetric bacterial growth studies have illustrated that thermograms differ significantly with both culture media and strain. The present contribution examines the possibility of discriminating between certain bacterial strains by microcalorimetry and the qualitative and quantitative contribution of the sample volume to the observed thermograms. Growth patterns of samples of Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) were analyzed. Certain features of the thermograms that may serve to distinguish between these bacterial strains were identified. RESULTS: The thermograms of the two bacterial strains with sample volumes ranging from 0.3 to 0.7 ml and same initial bacterial concentration were analyzed. Both strains exhibit a roughly 2-peak shape that differs by peak amplitude and position along the time scale. Seven parameters corresponding to the thermogram key points related to time and heat flow values were proposed and statistically analyzed. The most relevant parameters appear to be the time to reach a heat flow of 0.05 mW (1.67 ± 0.46 h in E. coli vs. 2.99 ± 0.53 h in S. aureus, p < 0.0001), the time to reach the first peak (3.84 ± 0.5 h vs. 5.17 ± 0.49 h, p < 0.0001) and the first peak value (0.19 ± 0.02 mW vs. 0.086 ± 0.012 mW, p < 0.0001). The statistical analysis on 4 parameters of volume-normalized heat flow thermograms showed that the time to reach a volume-normalized heat flow of 0.1 mW/ml (1.75 ± 0.37 h in E. coli vs. 2.87 ± 0.65 h in S. aureus, p < 0.005), the time to reach the first volume-normalized peak (3.78 ± 0.47 h vs. 5.12 ± 0.52 h, p < 0.0001) and the first volume-normalized peak value (0.35 ± 0.05 mW/ml vs. 0.181 ± 0.040 mW/ml, p < 0.0001) seem to be the most relevant. Peakfit® decomposition and analysis of the observed thermograms complements the statistical analysis via quantitative arguments, indicating that: (1) the first peak pertains to a faster, "dissolved oxygen" bacterial growth (where the dissolved oxygen in the initial suspension acts as a limiting factor); (2) the second peak indicates a slower "diffused oxygen" growth that involves transport of oxygen contained in the unfilled part of the microcalorimetric cell; (3) a strictly fermentative growth component may slightly contribute to the observed complex thermal signal. CONCLUSION: The investigated strains of Staphylococcus aureus and Escherichia coli display, under similar experimental conditions, distinct thermal growth patterns. The two strains can be easily differentiated using a selection of the proposed parameters. The presented Peakfit analysis of the complex thermal signal provides the necessary means for establishing the optimal growth conditions of various bacterial strains. These conditions are needed for the standardization of the isothermal microcalorimetry method in view of its further use in qualitative and quantitative estimation of bacterial growth.

[Microcalorimetry -- a new method for bacterial characterisation]. / Microcalorimetria -- metoda noua de caracterizare bacteriana.

The microcalorimetry is a method used for recording of the heat produced by a thermodinamic system in a scale of micronanojouls. One of the domains in which this method is used is the one called bacterial microcalorimetry, which studies the heat generated by the bacterial populations. The process of bacterial growth can be monitored in real time by the recording a graph of the generated power over time. The modern isothermal microcalorimeters allow the detection of a signal variation of only one microwatt. The estimated generated power of a bacteria is approximately 1-4pW thus only a small number of bacteria is necessary for the experiments. Recent studies in the field of bacterial microcalorimetry have demonstrated that, in standard conditions, this method can be reproductible and can be used to detect and characterize bacterial growth (through the study of the microcalorimetric growth curve particular to a bacterial species which is called a microcalorimetric fingerprint) and offers the new information in regards to bacterial metabolism. Also, microcalorimetry can offer information about bacterial interaction with different factors in the medium (for example, antibioticsubstances, in which case an antibiogram is obtained in 4-5 hours). In conclusion, we can say that microcalorimetry is a reproducible method, which offers an interesting perspective on bacterial characterization, with great scientific potential, and there are sufficient arguments to continue studies in this field.

Serodiagnosis of environmental mycobacterial infections.

To demonstrate the usefulness of enzyme-linked immunosorbent assay for serodiagnosis of mycobacterioses due to environmental mycobacteria we utilized a panel of glycolipid antigens selective for Mycobacterium avium-intracellulare, Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium scrofulaceum and Mycobacterium gordonae. The levels of circulating antibodies were determined against the environmental mycobacteria, and Mycobacterium tuberculosis in human immunodeficiency virus-negative and -positive patient sera. The method used immunomagnetic separation of the antigens, with covalent immobilization of antibodies to superparamagnetic amine and carboxyl terminated particles in solutions of the specific antigens. Enzyme-linked immunosorbent assay was performed on 195 patient sera: 34 with infections due to environmental mycobacteria, 114 with tuberculosis, 47 with other respiratory diseases. There were 46 human immunodeficiency virus-1 infected individuals. Among the 34 infections due to environmental mycobacteria, 9 patients were singularly infected with an environmental mycobacterium, and 25 co-infected with both M. tuberculosis and an environmental mycobacterium. Sensitivity, specificity and false positivity ranges were determined for each of the volunteer groups: tuberculosis positive, human immunodeficiency virus negative; tuberculosis positive, human immunodeficiency virus positive; those with infections due to individual environmental mycobacteria (such as M. scrofulaceum and M. kansasii); and those with other respiratory diseases. We demonstrate that such multiple assays, can be useful for the early diagnosis of diverse environmental mycobacterial infections to allow the start of treatment earlier than henceforth.

MicroDSC study of Staphylococcus epidermidis growth.

BACKGROUND: A microcalorimetric study was carried out using a Staphylococcus epidermidis population to determine the reproducibility of bacterial growth and the variability of the results within certain experimental parameters (temperature, bacterial concentration, sample thermal history). Reproducibility tests were performed as series of experiments within the same conditions using either freshly prepared populations or samples kept in cold storage. In both cases, the samples were obtained by serial dilution from a concentrated TSB bacterial inoculum incubated overnight. RESULTS: The results show that experiments are fairly reproducible and that specimens can be preserved at low temperatures (1 - 2°C) at least 4 days. The thermal signal variations at different temperatures and initial bacterial concentrations obey a set of rules that we identified. CONCLUSION: Our study adds to the accumulating data and confirms available results of isothermal microcalorimetry applications in microbiology and can be used to standardize this method for either research or clinical setting.