Rossella Grande

Potential antibacterial activity of carvacrol-loaded poly(DL-lactide-co-glycolide) (PLGA) nanoparticles against microbial biofilm.

The ability to form biofilms contributes significantly to the pathogenesis of many microbial infections, including a variety of ocular diseases often associated with the biofilm formation on foreign materials. Carvacrol (Car.) is an important component of essential oils and recently has attracted much attention pursuant to its ability to promote microbial biofilm disruption. In the present study Car. has been encapsulated in poly(dl-lactide-co-glycolide (PLGA) nanocapsules in order to obtain a suitable drug delivery system that could represent a starting point for developing new therapeutic strategies against biofilm-associated infections, such as improving the drug effect by associating an antimicrobial agent with a biofilm viscoelasticity modifier.

Bacterial response to the exposure of 50 Hz electromagnetic fields

To investigate the ability of prokaryotic microorganisms to activate strategies in adapting themselves to the environmental stress induced by exposure to extremely low frequency electromagnetic fields (ELF-EMF), cultures of Escherichia coli ATCC 700926 exposed at 50 Hz EMF (0.1, 0.5, 1.0 mT), and the respective sham-exposed controls were studied for: the total and culturable counts, the viability status, the antimicrobial susceptibility pattern, the morphological analysis, the genotypical and transcriptional profile. Exposed samples and controls displayed similar total and culturable counts, whereas an increased cell viability was observed in exposed samples re-incubated for 24 h outside of the solenoid compared to the corresponding controls. An exposure to 50 Hz EMF of 20-120 min produced a significant change of E. coli morphotype with a presence of coccoid cells also aggregated in clusters after re-incubation of 24 h outside of the solenoid. Atypical lengthened bacterial forms were also observed suggesting a probable alteration during cell division. No changes among DNA fingerprintings and some differences in RNA-AFLP analysis were observed for each 50 Hz EMF intensities evaluated. Our results indicate that an exposure to 50 Hz EMF acts as a stressing factor on bacteria which can represent a suitable model to investigate acute and chronic effects related to ELF-EMF exposure.

Extracellular DNA in Helicobacter pylori biofilm: a backstairs rumour

Aims:  This study detected and characterized the extracellular DNA (eDNA) in the biofilm extracellular polymeric substance (EPS) matrix of Helicobacter pylori and investigated the role of such component in the biofilm development.

Characterization of an Helicobacter pylori environmental strain

Aims:  To investigate the main genotypic virulence markers and the phenotypic features of an environmental Helicobacter pylori strain, named MDC1.

Analysis of genetic variability, antimicrobial susceptibility and virulence markers in Helicobacter pylori identified in Central Italy

Objective. To assess the relationship between the presence of mixed infection of Helicobacter pylori and both antimicrobial susceptibility and virulence markers. Material and methods. Thirty-six patients with H. pylori infection were included in the study. Three colonies were selected from each positive biopsy sample collected from each host for a total of 108 H. pylori strains. The genetic variability was evaluated through the amplified fragment length polymorphism (AFLP) analysis; the antibiotic susceptibility to amoxicillin, clarithromycin, moxifloxacin, rifabutin and tinidazole was determined using the minimum inhibitory concentrations (MICs) with the agar dilution method. Moreover, the vacA, cagA, iceA and babA2 statuse were detected by polymerase chain reaction (PCR). Results. There was a strong connection between mixed H. pylori infection and antimicrobial resistance. In particular, H. pylori strains with genetic variability, in the same host, expressed more resistance to clarithromycin, moxifloxacin and tinidazole than that expressed in strains with a unique genetic host pattern. VacA s1m1/s1m2 genotypes were found in 70% of strains isolated in mixed infection, whereas the same allelic combinations were found in 42% of strains, isolated in single infection. The cagA+ status prevailed both in patients with mixed (97%) and in those with single infection (85%) without significant differences. The iceA1 status was more commonly found in patients with mixed infection, whereas the babA2 status was significantly prevalent in single H. pylori infection. Conclusions. Mixed H. pylori infection harbouring in one patient is significantly related to strains that are more resistant to antibiotics and with a more virulent genotype (vacA s1m1/s1m2, cagA, iceA1) than strains responsible for single infection.

Viscoelastic properties of Staphylococcus aureus and Staphylococcus epidermidis mono-microbial biofilms

The viscoelastic properties of mono‐microbial biofilms produced by ocular and reference staphylococcal strains were investigated. The microorganisms were characterized for their haemolytic activity and agr typing and the biofilms, grown on stainless steel surface under static conditions, were analysed by Confocal Laser Scanning Microscopy. Static and dynamic rheometric tests were carried out to determine the steady‐flow viscosity and the elastic and viscous moduli. The analysed biofilms showed the typical time‐dependent behaviour of viscoelastic materials with considerable elasticity and mechanical stability except for Staphylococcus aureus ATCC 29213 biofilm which showed a very fragile structure. In particular, S. aureus 6ME biofilm was more compact than other staphylococcal biofilms studied with a yield stress ranging between 2 and 3 Pa. The data obtained in this work could represent a starting point for developing new therapeutic strategies against biofilm‐associated infections, such as improving the drug effect by associating an antimicrobial agent with a biofilm viscoelasticity modifier.

Determination of ciprofloxacin and levofloxacin in human sputum collected from cystic fibrosis patients using microextraction by packed sorbent-high performance liquid chromatography photodiode array detector

This paper reports a new, easy, cheap, and fast MEPS-HPLC-PDA method for the simultaneous analysis of ciprofloxacin and levofloxacin, two fluoroquinolones (FLQs) commonly used for the treatment of pulmonary infections in cystic fibrosis (CF) patients. The FLQs were resolved on a Discovery C8 column (250mm×4.6mm; 5μm particle size) using an isocratic elution with a run time of 15min, without further purification. The method was validated over concentrations ranging from 0.05 to 2μg/mL for both analytes in human sputum, and enrofloxacin was used as internal standard. This method was successfully tested to detect FLQs in sputum collected from CF patients. The MEPS-HPLC-PDA method was validated using biological samples collected from CF patients orally or intravenously injected with FLQs. The resultant data showed that the method is selective, sensitive and robust over range of concentrations for both FLQs. The limit of quantification of the method was 0.05μg/mL for both analytes (comparable to more complex and expensive instrument configurations), weighted-matrix-matched standard curves showed a good linearity up to 2μg/mL, and parallelism tests were also successfully assessed. The intra- and inter-day precision (RSD%) values were ≤10.4% and ≤11.1%, respectively, for all range of analysis. The intra- and inter-day trueness (Bias%) values are ranged from -11.8% to 7.25% for both antibiotic drugs. At the best of our knowledge, this is the first MEPS-HPLC-PDA based method that uses MEPS procedure for simultaneous determination of ciprofloxacin and levofloxacin in human sputum. The method was tested successfully on real sputum samples by following a conventional drug administration. Furthermore, the MEPS-HPLC-PDA based method provides more advantages to detect and analyze quickly the antibiotic drugs in biological matrices than other analytical procedures reported in literature.

Dynamic colonization of Helicobacter pylori in human gastric mucosa.

Objective. To investigate the dynamic behaviour of Helicobacter pylori in the colonization of the human gastric mucosa in patients previously treated for H. pylori infection. Material and methods. Twenty-one dyspeptic patients were included in the study. Biopsies from each individual were taken and analysed for H. pylori detection using cultural, molecular and ultrastructural methods. Results. Through culture, H. pylori was isolated in 7 out of 21 patients and the detection of the minimum inhibitory concentration (MIC) against drugs commonly used in H. pylori therapy revealed a susceptibility panel in which only one strain was multidrug resistant. By studying the expression of the H. pyloriglmM constitutive gene, viable H. pylori cells were detected in 19 out of 21 analysed biopsies. In these positive cases, the expression of the Quorum-Sensing related gene, luxS, was always detected. The analysis of glmM and luxS sequences confirmed the H. pylori identity. Scanning electron microscopy (SEM) analysis of biopsies from patients harbouring culturable bacteria showed a prevalent “S-shape” H. pylori morphotype co-existent with coccoid aggregated bacteria embedded in an abundant matrix; while samples from patients shown as H. pylori-positive only through the molecular method showed clustered coccoid bacteria arranged in a microbial biofilm. Conclusions. In the present work we describe a new scenario in H. pylori mucosa colonization suggesting, in infection recalcitrance, the planning of more efficacious protocols in order also to identify camouflaged and protected clustered bacteria, taking into account this serious microbial problem in medicine in the recommendation of therapeutic regimens.

Laser irradiation effect on Staphylococcus aureus and Pseudomonas aeruginosa biofilms isolated from venous leg ulcer

Chronic wounds, including diabetic foot ulcers, pressure ulcers and venous leg ulcers, represent a significant cause of morbidity in developed countries, predominantly in older patients. The aetiology of these wounds is probably multifactorial, but the role of bacteria in their pathogenesis is still unclear. Moreover, the presence of bacterial biofilms has been considered an important factor responsible for wounds chronicity. We aimed to investigate the laser action as a possible biofilm eradicating strategy, in order to attempt an additional treatment to antibiotic therapy to improve wound healing. In this work, the effect of near‐infrared (NIR) laser was evaluated on mono and polymicrobial biofilms produced by two pathogenic bacterial strains, Staphylococcus aureus PECHA10 and Pseudomonas aeruginosa PECHA9, both isolated from a chronic venous leg ulcer. Laser effect was assessed by biomass measurement, colony forming unit count and cell viability assay. It was shown that the laser treatment has not affected the biofilms biomass neither the cell viability, although a small disruptive action was observed in the structure of all biofilms tested. A reduction on cell growth was observed in S. aureus and in polymicrobial biofilms. This work represents an initial in vitro approach to study the influence of NIR laser treatment on bacterial biofilms in order to explain its potentially advantageous effects in the healing process of chronic infected wounds.

Biofilm formation and modulation of lux S and rpo D expression by Helicobacter pylori

Bacteria often choose a sessile biofilm lifestyle as a strategy to overcome environmental stress. In this study, we describe biofilm formation by Helicobacter pylori on a polystyrene surface, evaluating the viability and the morphological dynamics of bacterial cells during multicellular aggregate development. Moreover, we investigate expression of the lux S and rpo D genes, which are involved in biofilm formation. Two clinically susceptible and resistant strains of H. pylori were analyzed, as well as H. pylori ATCC 43629 for reference. The dominant form of expression, clustered bacterial cells arranged in an abundant matrix, was recorded after 2 days of incubation without shaking. Coccoid (spherical) cells with a “wrinkled” aspect presented the prevalent morphology (59.26%) among cells in the biofilm structure as observed by scanning electron microscopy. In aged H. pylori cultures, death occurred in single cells or cells grouped into microcolonies in which degenerated bacteria were localized inside the aggregates. The expression of lux S and rpo D genes among the sessile bacterial population reached a maximum after 2 days, with a significant reduction at subsequent time-points. No differences in gene expression and biofilm formation were recorded in the three evaluated strains. The morphological fickleness expressed in the life cycle by H. pylori strains emphasizes the bacteriums ability to overcome environmental stress, balancing its spread both outside and inside the host.