The Impact of Pseudomonas aeruginosa Infection in Adult Cystic Fibrosis Patients-A Single Polish Centre Study.

BACKGROUND: Pseudomonas aeruginosa (PA) is one of the most predominant pathogens of lung infections, often causing exacerbations in adult patients with cystic fibrosis (CF). MATERIALS AND METHODS: Microbiological characterization of 74 PA isolates and to evaluate the correlations between the bacterial features and 44 adult Polish CF cohort clinical parameters. RESULTS: The most common variant in the CF transmembrane conductance regulator (CFTR) gene was F508del (76.3%), followed by 3849+10kbC>T (26.3%). A total of 39.4% of the PA isolates showed multiple resistances. In patients with parameters pointing to a decline in lung function, there was a statistically significant moderate correlation with ß-lactam resistance and a weak correlation between hospital frequency and colistin resistance. The mucoidity did not correlate with the biofilm formation ability, which showed 41.9% of the isolates. Proteolytic activity, observed in 60.8% of the clinical isolates, was weakly associated with motility detected in 78.4% of the strains. The genetic profiles of the PA were highly heterogeneous, and a weak positive correlation was established between cluster group and biofilm formation. CONCLUSION: The findings suggest that there is a high variety in P. aeruginosa populations in adult CF patients. There is a need to monitor PA strains in groups of patients with cystic fibrosis, in particular, in terms of the occurrence of antibiotic resistance related to a decline in lung function.

Tannic Acid-Modified Silver Nanoparticles in Conjunction with Contact Lens Solutions Are Useful for Progress against the Adhesion of Acanthamoeba spp. to Contact Lenses.

Acanthamoeba spp. are amphizoic amoebae that are widely distributed in the environment and capable of entering the human body. They can cause pathogenic effects in different tissues and organs, including Acanthamoeba keratitis (AK), which may result in a loss of visual acuity and blindness. The diagnostics, treatment, and prevention of AK are still challenging. More than 90% of AK cases are related to the irresponsible wearing of contact lenses. However, even proper lens care does not sufficiently protect against this eye disease, as amoebae have been also found in contact lens solutions and contact lens storage containers. The adhesion of the amoebae to the contact lens surface is the first step in developing this eye infection. To limit the incidence of AK, it is important to enhance the anti-adhesive activity of the most popular contact lens solutions. Currently, silver nanoparticles (AgNPs) are used as modern antimicrobial agents. Their effectiveness against Acanthamoeba spp., especially with the addition of plant metabolites, such as tannic acid, has been confirmed. Here, we present the results of our further studies on the anti-adhesion potential of tannic acid-modified silver nanoparticles (AgTANPs) in combination with selected contact lens solutions against Acanthamoeba spp. on four groups of contact lenses. The obtained results showed an increased anti-adhesion activity of contact lens solutions in conjunction with AgTANPs with a limited cytotoxicity effect compared to contact lens solutions acting alone. This may provide a benefit in improving the prevention of amoebae eye infections. However, there is still a need for further studies on different pathogenic strains of Acanthamoeba in order to assess the adhesion of the cysts to the contact lens surface and to reveal a more comprehensive picture of the activity of AgTANPs and contact lens solutions.

Tannic acid-modified silver nanoparticles enhance the anti-Acanthamoeba activity of three multipurpose contact lens solutions without increasing their cytotoxicity.

BACKGROUND: Free-living amoebae of the genus Acanthamoeba are cosmopolitan, widely distributed protozoans that cause a severe, vision-threatening corneal infection known as Acanthamoeba keratitis (AK). The majority of the increasing number of AK cases are associated with contact lens use. Appropriate eye hygiene and effective contact lens disinfection are crucial in the prevention of AK because of the lack of effective therapies against it. Currently available multipurpose contact lens disinfection systems are not fully effective against Acanthamoeba trophozoites and cysts. There is an urgent need to increase the disinfecting activity of these systems to prevent AK infections. Synthesized nanoparticles (NPs) have been recently studied and proposed as a new generation of anti-microbial agents. It is also known that some plant metabolites, including tannins, have anti-parasitic activity. The aim of this study was to evaluate the anti-amoebic activity and cytotoxicity of tannic acid-modified silver NPs (AgTANPs) conjugated with selected multipurpose contact lens solutions. METHODS: The anti-amoebic activities of pure contact lens care solutions, and NPs conjugated with contact lens care solutions, were examined in vitro by a colorimetric assay based on the oxido-reduction of alamarBlue. The cytotoxicity assays were performed using a fibroblast HS-5 (ATCC CRL-11882) cell line. The results were statistically analysed by ANOVA and Student-Newman-Keuls test using P < 0.05 as the level of statistical significance. RESULTS: We show that the NPs enhance the anti-Acanthamoeba activities of the tested contact lens solutions without increasing their cytotoxicity profiles. The activities are enhanced within the minimal disinfection time recommended by the manufacturers. CONCLUSIONS: The conjugation of the selected contact lens solutions with AgTANPs might be a novel and promising approach for the prevention of AK infections among contact lens users.

Evaluation of in vitro effects of selected physical and chemical agents on detected in Poland Acanthamoeba strains ­ factors of increasing threats for public health

Acanthamoeba keratitis, the vision-threatening corneal disease reported with increasing frequency in Poland is difficult to treat due to extremely high resistance of the amoeba cysts to chemicals. The agents of possible anti-amoebic activity are still tested. Pathogenic Acanthamoeba samples/isolates acquired from severe cases of keratitis examined by molecular techniques to determine genotypes, compared to one another as well as to the environmental Acanthamoeba castellanii Neff strain were included in the studies. These strains were in vitro examined in terms of their sensitivity/resistance to selected chemicals and tolerance to temperature changes. Samples of the strains cultivated in vitro under bacteria-free conditions were monitored during different growth phases. Higher amoebic population dynamics was observed in both pathogenic Acanthamoeba strains during transfer to 37°C. Agents tested influenced population dynamics in different degree; they showed amoebostatic or amoebicidal effects, however a tendency toward induction of encystment also appeared. Because activation of the dormant cysts can lead to repeated development of amoebae, very important is cysticidal efficacy of chemicals. Further in vitro investigations on various Acanthamoeba strains with different chemicals are still necessary.

Evaluation of in vitro effect of selected contact lens solutions conjugated with nanoparticles in terms of preventive approach to public health risk generated by Acanthamoeba strains.

INTRODUCTION: Various Acanthamoeba species are free-living organisms widely distributed in the human environment. Amphizoic amoebae as facultative parasites may cause vision-threatening eye disease - Acanthamoeba keratitis, mostly among contact lens wearers. As the number of cases is increasing, and applied therapy often unsuccessful, proper hygienic measures and effective contact lenses disinfection are crucial for the prevention of this disease. Available contact lens solutions are not fully effective against amphizoic amoebae; there is a need to enhance their disinfecting activity to prevent amoebic infections. The use of developing nanotechnology methods already applied with success in the prevention, diagnostic and therapy of other infectious diseases might be helpful regarding amoebic keratitis. This study assesses the in vitro effect of selected contact lens solutions conjugated with nanoparticles against Acanthamoeba trophozoites. MATERIAL AND METHODS: Three selected contact lens solutions conjugated with silver and gold nanoparticles in concentration of 0.25-2.5 ppm were used in vitro against the axenically cultured ATCC 30010 type Acanthamoeba castellanii strain. The anti-amoebic efficacy was examined based on the oxido-reduction of AlamarBlue. The cytotoxicity tests based on the measurement of lactate dehydrogenase (LDH) activity were performed using a fibroblast HS-5 cell line. RESULTS: Enhancement of the anti-amoebic activity of contact lens solutions conjugated with selected nanoparticles expressed in the dose dependent amoebic growth inhibition with a low cytotoxicity profile was observed. CONCLUSIONS: Results of the study showed that conjugation of selected contact lens solutions with silver nanoparticles might be a promising approach to prevent Acanthamoeba keratitis among contact lens users.

Effect of povidone iodine, chlorhexidine digluconate and toyocamycin on amphizoic amoebic strains, infectious agents of Acanthamoeba keratitis - a growing threat to human health worldwide.

INTRODUCTION: Free-living amoebae, ubiquitous in outer environments, in predisposing circumstances may exist as parasites, infectious agents of Acanthamoeba keratitis. In recent decades, the vision-threatening corneal infection is a growing human health threat worldwide, including Poland. The applied therapy is often ineffective due to diagnostic mistakes, various pathogenicity of Acanthamoeba strains and high resistance of cysts to drugs; many agents with possible anti-amoebic activity are still being tested. In the presented study, selected chemicals are investigated in terms of their in vitro effect on corneal and environmental Acanthamoeba strains. MATERIAL AND METHODS: Samples of a corneal isolate from a patient with severe Acanthamoeba keratitis,of assessed on the basis of genotype associations of 18S rRNA and the type strain, Acanthamoeba castellanii Neff cultivated in bacteria-free condition, were exposed to povidone iodine, chlorhexidine digluconate or toyocamycin. In vitro population dynamics of the strains were monitored and compared to those of control cultures. RESULTS: All chemicals showed anti-amoebic effects with different degrees of effectiveness. Significant differences were observed in the in vitro population dynamics, and the morpho-physiological status of A. castellanii Neff T4 and corneal strains determined as A. polyphaga T4 genotype, exposed to povidone iodine or toyocamycin, in comparison with chlorhexidine taken as reference. CONCLUSIONS: Time-dependent amoebstatic in vitro effects were demonstrated for all agents, in particular, the results of assays with povidone iodine are promising. No significant stimulation of encystation appeared; however, as cysticidal efficacy of chemicals is expected, complementary research is needed on different Acanthamoeba strains with modified agent concentrations and method application.

Comparison of chlorhexidine disinfectant in vitro effect on environmental and ocular Acanthamoeba strains, the amoebic agents of human keratitis − an emerging sight-threatening corneal disease in Poland

Small amoebae belonging to the Acanthamoeba genus complete their life cycles in different environmental niches as free-living protists however some of them are facultative parasites that can cause severe disease in humans. The sight-threatening Acanthamoeba keratitis develops in immune-competent persons, mainly in contact lens wearers; it is detected with increasing frequency along with the spread of contact lens use. The high abundance of the amoebae in the environment is important for dispersion and transmission of the infections among humans. Emerging threats for the public health generated by these amoebae is the serious medical problem worldwide. Nonspecific symptoms, similar to those occurring in the other eye diseases, diagnostic mistakes, the delay of an appropriate treatment, an exceptional high resistance of the amoebae to chemicals and drugs result in a prolonged course of the disease and often unsuccessful therapeutic management. Thus, different chemicals are still examined for their potential activity in vitro against various species, strains/isolates of Acanthamoeba. As the prolonged therapy often induces encystation subsequently leading to excystment and recurrences of amoebic keratitis, apart from anti-amoebic activity, cysticidal effect of examined agents is desirable. In the present study, results of our comparative investigations showed that cationic antiseptic chlorhexidine digluconate indicated in vitro anti-amoebic effect on environmental Acanthamoeba castellanii Neff strain and pathogenic corneal Acanthamoeba polyphaga T4 genotype. Amoebostatic effect of the disinfectant was expressed in reduced number of surviving amoebae in comparison to the respective control cultures; simultaneously, despite prolonged incubation with the agent no stimulation of encystation was noted. The corneal strain was more resistant to the tested compound than the Neff strain. The cysticidal efficacy of chemicals is very expected, thus further in vitro studies on pathogenic Acanthamoeba strains with different application chemicals pattern are needed.

Molecular determination of suspected alveolar echinococcosis requiring surgical treatment in human cases from Poland

In this study five cases of suspected alveolar echinococcosis from Poland in which surgical treatment was needed, previously diagnosed by means of imaging and serological techniques, were analyzed in terms to identify the causative agent. Samples of the parasite tissues taken perioperatively from the liver lesions were used for the histopathological and molecular examinations. The sequences of all isolates were identical to Echinococcus multilocularis; all nad1 sequences have been deposited in GenBank The histopathological examination revealed Passpositive fragments of laminated layers typical for E. multilocularis metacestode. Obtained results confirm that the use of imaging techniques only may be insufficient to diagnose alveococcosis thus the recognition of the zoonosis should base on several procedures; especially valuable are highly sensitive and specific molecular methods.

Amoebicidal or amoebostatic influence of disinfectants used in health facilities and laboratories on corneal strains of Acanthamoeba

Different Acanthamoeba species are amphizoic organisms distributed in wide range of habitats in natural and man-made environments; they are also detected on surfaces of equipment and accessories in health facilities. Some strains of the amoebae are causative agents of the vision-threatening human disease Acanthamoeba keratitis, mainly reported in contact lens wearers. An exceptional high resistance of Acanthamoeba trophozoites and particularly cysts to chemicals, disinfectants and drugs is believed as influencing difficulty resulting in unsuccessful therapeutic management. As Acanthamoeba keratitis is the serious medical problem worldwide, different chemicals with possible activity against environmental and clinical Acanthamoeba strains are tested. In our study, selected disinfectants used in health care settings and laboratories were tested and their efficacy against the corneal strains Acanthamoeba castellanii and A. polyphaga , and environmental A. castellanii Neff strain was assessed. Comparative assessment of results of the assays show that, apart from amoebistatic effects, the disinfectants indicated expected cysticidal efficacy.

In vivo confocal microscopy and in vitro culture techniques as tools for evaluation of severe Acanthamoeba keratitis incidents

Amphizoic amoebae belonging to the genus Acanthamoeba are known as etiological agents of sight-threatening Acanthamoeba keratitis. The leading risk factor for the development of this serious human disease is contact lens wearing which popularity increases worldwide, also in Poland. The disease with active epithelial inflammations, corneal ulcers, including loss of the visual acuity is a serious medical problem as an emerging threat for the public health related to improper contact lens hygiene. The treatment of the amoebic keratitis is difficult, often unsuccessful due to delayed proper diagnosis. The clinical picture of the disease, often with severe course is nonspecific, similar to that occurring in viral, fungal or bacterial keratitis, thus clinical symptoms alone are not sufficient to identify the causative agent of the amoebic infection. Early diagnosis is decisive for the suitable therapeutic management and the treatment efficacy. In our studies, several complicated, difficult to treat Acanthamoeba keratitis incidences pertaining Polish patients using contact lenses have been retrospectively analyzed in terms of the usefulness of non-invasive methods of in vivo confocal microscopy and in vitro culture techniques applied for diagnosis. Hyper-reflective double-walled spherical Acanthamoeba cysts, with a more reflective outer wall were detected in the epithelium and anterior layers of the corneal stroma. In vivo confocal microscopy, if available, may be a valuable, sensitive tool for diagnosis in late identified severe infections mainly with strong viability strains, however confoscan may offer limited value at lowintensity amoebic infections. The microscopic visualization of amoebae in slides prepared directly from corneal scraping and laboratory examinations of specimens from in vitro cultivated corneal isolates allow to confirm or verify results of in vivo examinations, furthermore to identify directly the pathogens and to clarify previous misdiagnoses.

Comparative assessment of selected intraoral microorganisms ­ potential factors for peri-surgical management complications

In this research, a comparative analysis of results of investigations involving different human populations, in terms of a relation between the oral cavity health and the species composition of mouth microbiota is reported. The purpose of this analysis was to identify and assess microorganisms that could cause health complications in patients with neoplasm requiring dental problem-related surgical management. The patients with the oral cancer surgically treated and those without neoplasm were assessed for their oral health: status of teeth, gingiva, periodontium, and occurrence of inflammatory processes. From each patient, microorganisms isolated of periodontium, dental plaque, and dental pocket swabs were identified in wet and stained microscopic preparations; standard microbiology in vitro techniques were also applied to determine the fungal and bacterial strains. The comparative analysis of results of direct microscopic examinations and in vitro cultures assessment indicated significant differences in prevalences of fungi, parasitic oral protozoans and bacteriae in particular patient's groups. Yeast-like fungi belonging to Candida genus, mostly of C. albicans group, were identified in 93.75% patients with the oral cancer, while in 25% of individuals assessed without neoplasm. E. gingivalis amoebae were only found in 12.5% patients with the serious disease; no trichomonads were detected in all patients analyzed. Among bacteria species, potentially pathogenic Enterobacteriaceae were found in the patients with oral cancer. The pronounced shift in the microbiota species composition in the patients who needed prolonged treatment due to oral cavity cancer, compared to other generally healthy persons has been showed in this analysis.

Assessment of in vitro dynamics of pathogenic Acanthamoeba strains originating from contact lens wearers with infectious keratitis

Recently, incidents of Acanthamoeba keratitis, the vision-threatening eye disease, are reported with increasing frequency worldwide, particularly in contact lens wearers. In our study, the retrospective assessment of in vitro dynamics of subsequent pathogenic Acanthamoeba isolates cultured at 24°C, detected in Polish contact lens wearers with keratitis is presented and results compared with those of environmental A. castellanii Neff strain. There were delayed the proper diagnosis that influenced prolonged and severe course of this eye disease and treatment difficulties. The corneal material was examined directly to visualize developmental amoeba stages for diagnose verification, microbiologically tested for the specific identification of bacteriae and fungi, and in vitro grown in culture medium in temperature 24°C. Among twenty-six keratitis incidents analyzed, eleven were cases of Acanthamoeba keratitis; in the six of them, Acanthamoeba strains and concomitant bacterial and/or fungal infectious agents were detected. In vitro assays showed variability in population density of several clinical strains in the exponential growth phase expressed in various range of overall amoeba number and different proportion between trophozoites and cysts. The clear influence of temperature on the in vitro cultivation of the amoebae was observed: statistically significant lower population dynamics was revealed by most of pathogenic clinical isolates in comparison with those showed by environmental strain. The in vitro monitoring of dynamics of Acanthamoeba strains isolated from infected eyes may be helpful for diagnostics verification, especially in mixed infectious keratitis.

Acanthamoeba castellanii: in vitro effects of selected biological, physical and chemical factors.

Trophozoites and cysts of free-living Acanthamoeba castellanii present a serious risk to human health as causative agents of human diseases such as fatal granulomatous amoebic encephalitis and Acanthamoeba keratitis that is reported from various part of the world, also in Poland, with increasing frequency, particularly in the contact lens wearers. The amphizoic amoebae are generally extremely resistant to different chemical agents, however, several strains/isolates within A. castellanii may differ in virulence. Among the features considered as associated with the amoeba pathogenicity, temperature tolerance and resistance to different environmental conditions are reported. In the present study, A. castellanii strain cultured in 26 degrees C after several year passages were tested for sensibility/tolerance to instant temperature changes as well as exposition to deuterium oxide, D2O. Significant decrease of number of viable amoebae during in vitro exposition to D2O occurred, but no changes in trophozoites/cysts ratio. The ability of the strain examined to develop in higher temperature may indicate a wide adaptation reserve and its pathogenic potential.