Grigore Mihaescu

Modulation of virulence and antibiotic susceptibility of enteropathogenic Escherichia coli strains by Enterococcus faecium probiotic strain culture fractions

The increasing rate of antimicrobial resistance drastically reduced the efficiency of conventional antibiotics and led to the reconsideration of the interspecies interactions in influencing bacterial virulence and response to therapy. The aim of the study was the investigation of the influence of the soluble and cellular fractions of Enterococcus (E.) faecium CMGB16 probiotic culture on the virulence and antibiotic resistance markers expression in clinical enteropathogenic Escherichia (E.) coli strains. The 7 clinical enteropathogenic E. coli strains, one standard E. coli ATCC 25,922 and one Bacillus (B.) cereus strains were cultivated in nutrient broth, aerobically at 37 °C, for 24 h. The E. faecium CMGB16 probiotic strain was cultivated in anaerobic conditions, at 37 °C in MRS (Man Rogosa Sharpe) broth, and co-cultivated with two pathogenic strains (B. cereus and E. coli O28) culture fractions (supernatant, washed sediment and heat-inactivated culture) for 6 h, at 37 °C. After co-cultivation, the soluble and cellular fractions of the probiotic strain cultivated in the presence of two pathogenic strains were separated by centrifugation (6000 rpm, 10 min), heat-inactivated (15 min, 100 °C) and co-cultivated with the clinical enteropathogenic E. coli strains in McConkey broth, for 24 h, at 37 °C, in order to investigate the influence of the probiotic fractions on the adherence capacity and antibiotic susceptibility. All tested probiotic combinations influenced the adherence pattern of E. coli tested strains. The enteropathogenic E. coli strains susceptibility to aminoglycosides, beta-lactams and quinolones was increased by all probiotic combinations and decreased for amoxicillin-clavulanic acid. This study demonstrates that the plurifactorial anti-infective action of probiotics is also due to the modulation of virulence factors and antibiotic susceptibility expression in E. coli pathogenic strains.

Immunomodulatory effect of non-viable components of probiotic culture stimulated with heat-inactivated Escherichia coli and Bacillus cereus on holoxenic mice

Background Competition of probiotic bacteria with other species from the intestinal microbiota involves different mechanisms that occur regardless of probiotics’ viability. The objective of this paper was to assess the cytokine serum levels in holoxenic mice after oral administration of non-viable components (NVC) of Enterococcus faecium probiotic culture stimulated with heat-inactivated Escherichia coli and Bacillus cereus in comparison to NVC of unstimulated E. faecium probiotic culture. Methods Probiotic E. faecium CMGb 16 culture, grown in the presence of heat-inactivated cultures of E. coli and B. cereus CMGB 102, was subsequently separated into supernatant (SN) and heat-inactivated cellular sediment (CS) fractions by centrifugation. Each NVC was orally administered to holoxenic mice (balb C mouse strain), in three doses, given at 24 hours. Blood samples were collected from the retinal artery, at 7, 14, and 21 days after the first administration of the NVC. The serum concentrations of IL-12 and tumor necrosis factor-alpha (TNF-α) interleukins were assessed by ELISA method. Results After the oral administration of SN component obtained from the probiotic culture stimulated with heat-inactivated cultures of B. cereus CMGB 102 and E. coli O28, the serum concentrations of IL-12 were maintained higher in the samples collected at 7 and 14 days post-administration. No specific TNF-α profile could be established, depending on stimulated or non-stimulated probiotic culture, NVC fraction, or harvesting time. Conclusion The obtained results demonstrate that non-viable fractions of probiotic bacteria, stimulated by other bacterial species, could induce immunostimulatory effects mediated by cytokines and act, therefore, as immunological adjuvants.

Influence of hybrid inorganic/organic mesoporous and nanostructured materials on the cephalosporins' efficacy on different bacterial strains

The aim of this study was to investigate the effect of different hybrid inorganic-organic micro- and nanomaterials (Fe(3)O(4)/PEG(600), Fe(3)O(4)/C(12), ZSM-5) on the antibacterial activity of different cephalosporins against Gram-positive and Gram-negative bacterial strains. The synergic effect of the studied materials was demonstrated by the increase in the growth inhibition zones diameter. All tested hybrid micro- and nanomaterials increased the activity of cefotaxime against Staphylococcus aureus. ZSM-5 increased the activity of cefotaxime and ceftriaxone and Fe(3)O(4)/C(12) that of ceftriaxone against Pseudomonas aeruginosa and S. aureus. The anti-Pseudomonas, anti-Klebsiella pneumoniae and anti-Bacillus subtilis activity of cefoperazone was increased by Fe(3)O(4)/C(12) nanoparticles, while the ZSM-5 improved its anti-Escherichia coli, K. pneumoniae, S. aureus and B. subtilis activity, whereas Fe(3)O(4)/PEG(600) against K. pneumoniae. The anti-K. pneumoniae activity of cefepime was increased by all tested nanoparticles, whereas its anti-B. subtilis and anti-E. coli activity was improved by Fe(3)O(4)/C(12) and Fe(3)O(4)/PEG(600) nanoparticles. In conclusion, both magnetic Fe(3)O(4) nanoparticles, charged outside as extra-shell with the antibiotic as well as ZSM-5 microparticles carrying the antibiotic inside the pores, significantly and specifically improved cephalosporin efficacy. A probable explanation for the increase in the antibiotic efficiency is the better penetration through the cellular wall of the antibiotic charged nanoparticles.

Epidemiological and molecular investigation of a rubella outbreak, Romania, 2011 to 2012

We describe a rubella outbreak that occurred in Romania between September 2011 and December 2012. During this period 24,627 rubella cases, 41.1% (n=10,134) of which female, were notified based on clinical criteria, and a total of 6,182 individuals were found serologically positive for IgM-specific rubella antibody. The median age of notified cases was 18 years (range: <1–65) and the most affected age group 15 to 19 years (n=16,245 cases). Of all notified cases, 24,067 cases (97.7%) reported no history of vaccination. Phylogenetic analysis of 19 sequences (739 nucleotides each), from 10 districts of the country revealed that the outbreak was caused by two distinct rubella virus strains of genotype 2B, which co-circulated with both temporal and geographical overlap. In addition to the 6,182 IgM-positive rubella cases, 28 cases of congenital rubella syndrome (CRS) were identified, including 11 neonatal deaths and one stillbirth. The outbreak underscores the need to encourage higher vaccination uptake in the population, particularly in women of reproductive age, and to strengthen epidemiological and laboratory investigations of suspected rubella cases. Genetic characterisation of wild-type rubella virus is an essential component to enhance surveillance and here we report rubella virus sequences from Romania.

Antibiotic Drug Delivery Systems for the Intracellular Targeting of Bacterial Pathogens

Intracellular bacterial pathogens are hard to treat because of the inability of conventional antimicrobial agents belonging to widely used classes, like aminoglycosides and β-lac‐ tams, fluoroquinolones, or macrolides to penetrate, accumulate, or be retained in the mammalian cells. The increasing problem of antibiotic resistance complicates more the treatment of the diseases caused by these agents. In many cases, the increase in therapeu‐ tic doses and treatment duration is accompanied by the occurrence of severe side effects. Taking into account the huge financial investment associated with bringing a new antibi‐ otic to the market and the limited lifetime of antibiotics, the design of drug delivery sys‐ tems to enable the targeting of antibiotics inside the cells, to improve their activity in different intracellular niches at different pH and oxygen concentrations, and to achieve a reduced dosage and frequency of administration could represent a prudent choice. An ideal drug delivery system should possess several properties, such as antimicrobial activ‐ ity, biodegradability, and biocompatibility, making it suitable for use in biomedical and pharmaceutical formulations. This approach will allow reviving old antibiotics rendered useless by resistance or toxicity, rescuing the last line therapy antibiotics by increasing the therapeutic index, widening the antimicrobial spectrum of antibiotics scaffolds that failed due to membrane permeability problems, and thus reducing the gap between in‐ creasingly drug-resistant pathogens and the development of new antibiotics. Different improved drug carriers have been developed for treating intracellular pathogens, includ‐ ing antibiotics loaded into liposomes, microspheres, polymeric carriers, and nanoplexes. The purpose of this chapter is to present the limitations of each class of antibiotics in tar‐ geting intracellular pathogens and the main research directions for the development of drug delivery systems for the intracellular release of antibiotics.

Comparative study of primary and secondary tumors from patients with laryngeal and oropharyngeal cancer, using transmission electron microscopy

In modern laboratories, the study of cancer is performed using a series of cellular and molecular methods based on optical instruments measurements. Optical and electron microscopy are valuable tools for revealing morphological features of cancer cells. Our study was focused on laryngeal and oropharyngeal cancers, which have nowadays an increased incidence, especially for women, due to unhealthy habits like tobacco and alcohol consumption. We used transmission electron microscopy (TEM) for highlighting the ultrastructural features of cancer cells, both in primary and secondary tumors. The primary tumor is considered that which appears for the first time, at a certain organ; the secondary tumor is that which reappears at the same region or neighbouring regions, at a certain interval of time after the primary one has been surgically removed. The differences between the inner architecture of the cells from primary and secondary tumors where correlated with the expression of some genes (oncogenes and tumor suppressor factors), in order to establish the aggressiveness of the tumor, in different disease stages. The main stress in the study is placed upon electron microscopy, in order to achieve a more precise characterization of both these type of cancer cells. These ultrastructural data complete the image of laryngeal and pharyngeal cancer cells, along with molecular data obtained by Real-Time PCR.