Phage therapy combined with Gum Karaya injectable hydrogels for treatment of methicillin-resistant Staphylococcus aureus deep wound infection in a porcine model.

Skin and soft tissue infections (SSTIs) represent a significant healthcare challenge, particularly in the context of increasing antibiotic resistance. This study investigates the efficacy of a novel therapeutic approach combining bacteriophage (phage) therapy with a gum Karaya (GK)-based hydrogel delivery system in a porcine model of deep staphylococcal SSTIs. The study exploits the lytic activity and safety of the Staphylococcus phage 812K1/420 of the Kayvirus genus, which is active against methicillin-resistant Staphylococcus aureus (MRSA). The GK injectable hydrogels and hydrogel films, developed by our research group, serve as effective, non-toxic, and easy-to-apply delivery systems, supporting moist wound healing and re-epithelialization. In the porcine model, the combined treatment showed asynergistic effect, leading to a significant reduction in bacterial load (2.5 log CFU/gram of tissue) within one week. Local signs of inflammation were significantly reduced by day 8, with clear evidence of re-epithelialization and wound contraction. Importantly, no adverse effects of the GK-based delivery system were observed throughout the study. The results highlight the potential of this innovative therapeutic approach to effectively treat deep staphylococcal SSTIs, providing a promising avenue for further research and clinical application in the field of infections caused by antibiotic-resistant bacteria.

Enzyme-based treatment of skin and soft tissue infections.

Inflammatory diseases of the skin and soft tissues are an important group of human infections. The most common causes are the bacteria Staphylococcus aureus and Streptococcus pyogenes. Given the growing resistance of these pathogens to antimicrobials, the current research focuses on the search for novel therapeutic options that would be effective against infections refractory to conventional antimicrobials. A promising alternative is the use of enzyme-based antimicrobials (enzybiotics) that degrade the bacterial cell wall. They target the specific pathogen but do not affect the skin microbiome, thus helping the healing process. As enzymes can be poorly soluble, unstable, or subject to rapid elimination from the body, efforts are made to create biobetters, i.e., enzymes with improved characteristics. Emphasis is also put on the development of novel enzybiotic carriers or wound healing dressings with integrated enzymes.

Therapeutic potential of bacteriophages for staphylococcal infections and selected methods for in vitro susceptibility testing of staphylococci.

AIM: Staphylococcus aureus strains are the cause of frightening hospital and community infections, especially when they are resistant to antimicrobials, have important pathogenicity factors, or have biofilm production ability. Looking for novel therapeutic options which would be effective against such strains is one of the highest priorities of medicine and medical research. The study aim was to describe the occurrence of S. aureus strains and proportion of methicillin resistant strains (MRSA) detected in laboratories of the Microbiological Institute, Faculty of Medicine, Masaryk University (FM MU) and St. Anne's University Hospital, Brno in 2011-2018. Selected strains of S. aureus were tested for biofilm production ability and susceptibility to antimicrobials and Stafal®, a phage therapeutic agent. A prerequisite was to develop a simple routine method suitable for phage susceptibility testing of bacteria. MATERIAL AND METHODS: Altogether 867 clinical isolates of S. aureus and 132 strains of other species of the genus Staphylococcus (isolated in 2011-2017) were tested for susceptibility to the phage therapy preparation Stafal® using the double-layer agar method. All strains of S. aureus were tested for biofilm production ability by the modified Christensen method with the use of titration microplates and for susceptibility to antistaphylococcal antibiotics by the disk diffusion test. For 95 S. aureus strains, the outcome of the double-layer agar method (DAM) was compared with that of our newly designed method (ODM) based on optical density decrease of the bacterial suspension. RESULTS: During the study period, the laboratories of the Faculty of Medicine, Masaryk University (FM MU) and St. Anne's University Hospital, Brno detected 2900 strains of S. aureus per year on average. The proportion of MRSA among S. aureus isolates from blood culture and venous catheters ranged between 8.8-15.2 %. S. aureus strains recovered from venous catheters and blood culture were confirmed as stronger biofilm producers than those from other clinical specimens. MRSA strains showed higher biofilm production than methicillin susceptible strains (MSSA). As many as 90.4 % of S. aureus strains tested susceptible to the Stafal® preparation. Even a higher proportion, i.e. 99.0 %, of MRSA strains were Stafal® susceptible. No relationship was found between Stafal® susceptibility and biofilm production ability. Although Stafal® targets primarily S. aureus, some susceptibility (26.5 %) was also found for other staphylococcal species. A novel simple method designed for routine testing of susceptibility to phage therapy preparations based on optical density decrease was comparably sensitive and reliable as the commonly used double-layer agar method (DAM) and, in addition to being easy and rapid to perform, after prolonged suspension culture and at higher measurement frequency, it has an extra advantage of providing the possibility for monitoring also phage action dynamics. CONCLUSIONS: The proportion of MRSA strains detected in this study is comparable to that reported for the whole Czech Republic, and the biofilm production data are consistent with scientific evidence. The host range of the Stafal® preparation is relatively wide and covers most strains of S. aureus and some coagulase negative staphylococci. The highest efficiency of Stafal® (99.4 %) was observed against MRSA strains with multiple types of antibiotic resistance. In vitro testing of 867 strains of S. aureus and 132 other staphylococcal species has shown the phage therapy preparation Stafal® to be a suitable candidate therapeutic option for the treatment of staphylococcal infections, especially in case of failure of conventional antibiotic therapy. Moreover, a simple method for routine phage susceptibility testing of clinical bacterial isolates has been designed, which is an essential tool to be used in phage therapy.

Antimicrobial effect of novel hydrogel matrix based on natural polysaccharide Sterculia urens.

INTRODUCTION: Materials for modern wound-management are a very broad and heterogeneous group. One of the most important representatives is natural materials, or more precisely polysaccharides isolated from various plants and animals. With the increasing resistance of pathogens to established antimicrobial agents, there is also an attempt to discover new mechanisms of the effects of these materials. Gum karaya (GK) is a very promising representative of the natural polysaccharides group and, since it is obtained from Sterculia urens as resin, it is also possible to assume its certain antimicrobial activity. MATERIAL AND METHODOLOGY: The antimicrobial potential of GK and chitosan (Ch) has been tested on several preselected strains to match the real epidemiological situation of the agents of infectious complications in the field of burned wounds. Tested strains included representatives of gram-positive and gram-negative bacteria as well as selected yeasts. Methicillin susceptible Staphylococcus aureus CCM 4223 (ATCC 29213), methicillin resistant Staphylococcus aureus CCM 4750 (ATCC 43300), Klebsiella pneumoniae CCM 4985 (ATCC 700603), Candida albicans CCM 8261 (ATCC 90028), Pseudomonas aeruginosa CCM 3955 (ATCC 27853) were obtained from the Czech Collection of Microorganisms. Pseudomonas aeruginosa FF 1, Pseudomonas aeruginosa FF 2 and Pseudomonas aeruginosa FF 3 (all multi-resistant clinical strains), Staphylococcus epidermidis A 013, Staphylococcus epidermidis A 117, and Candida parapsilosis BC 11 were obtained from the Collection of Microorganisms at the St. Annes University Hospital, Brno. Antimicrobial tests were performed using the disk diffusion test methodology. Another set of antimicrobial tests was obtained by measuring the growth curves. RESULTS: Bacteriostatic activity testing showed 1% GK concentration and both 1% and 0.5% chitosan concentration effective against all pathogens tested. The combination of GK50/Ch50 in concentrations of 1% and 0.5% had similar or better effect. Lower concentrations of the combined material are poorly effective against tested strains. Bactericidal activity testing has not produced positive results, except for Candida spp., where only a partial effect of GK50/Ch50 was observed at 1% concentration. In the growth curve test, the efficiency of both GK alone and chitosan was found to be significantly higher in gram-positive bacteria compared to gram-negative ones. In the case of this experiment, only a one-tenth concentration was used compared to the disk diffusion test concentration. This results correspond with the data from the bacteriostatic activity testing. CONCLUSION: This is the first publication that attempts to comprehensively define the potential for GK antimicrobial activity and also the possible potentiation of this activity with the use of chitosan. Further experiments are needed to extend the antimicrobial efficiency to gram-negative bacteria.

Is the level of pollutants a risk factor for exercise-induced asthma prevalence?

Increased prevalence of asthma is commonly though to be associated with increased air pollution and with lifestyle changes. This study evaluates a possible relationship of levels of exposure to outdoor pollutants and exercise-induced asthma prevalence. Two groups of students (age 14-17) in distant urban areas were randomly selected (n = 649): Group I (n = 430), Vancouver, B.C., Canada; Group II (n = 219), Prague, Czech Republic. The free running asthma screening test was performed at both locations; self-report questionnaires were distributed to all participants. Peak expiratory flow rates were used to evaluate baseline and postexercise spirometry. A 15% decrease was considered a positive response to exercise challenge. The information on ambient air quality (SO2, NO2, O3, PM10) was obtained from appropriate institutions. Of 649 subjects tested [Group I: n = 430, F = 251 (58.4%,) M = 179 (41.6%) and Group II: n = 219, F = 112 (51.6%), M = 107 (48.4%)], 81 of the participants (13.2%) responded positively to the exercise challenge [Group I: n = 55 (12.8%) and Group II: n = 26 (11.8%)]. Of the population tested, 150 (23.6%) participants self-reported a history of allergic disease [Group I: 94 (21.9%) and Group II: 56 (25.6%)]. The levels of air pollutants obtained for Group II significantly exceeded those obtained for Group I. This study reports a prevalence of 12.3% of exercised-induced asthma among high school students, when free running asthma screening test and peak expiratory flow rate are used. Significantly different levels of common pollutants do not correlate with asthma prevalence. Although it is recognized that air pollution is a well-known asthma trigger, it cannot be a simple explanation for the continued problem of rising asthma prevalence.

Incidence of exercise-induced asthma in high school population in British Columbia.

The Free Running Asthma Screening Test (FRAST) was performed on 830 randomly selected volunteers (age 14-17) chosen from British Columbia school students. An educational workshop on asthma was presented before testing. A self-reported questionnaire was distributed to evaluate predisposing factors. Air quality parameters were obtained on days of testing. FRAST was used to challenge exercise induced asthma (EIA). Peak expiratory flow rate (PEFR) was used to obtain baseline and post exercise spirometry. A 15% decrease in PEFR was considered a positive challenge result. Of 830 subjects tested, 110 (13.2%) responded positively to exercise challenge. Of the population tested, 21.8% self-reported a history of allergic disease, 76.4% of whom were from the positive responders' group. From the positive group only 72 (65.4%) were previously diagnosed as asthmatic. History of allergic disease was the strongest predisposing factor. The results indicate that inexpensive screening may improve early diagnosis and thus better preventive management.

[Protective effect of carvedilol on renal circulation and tissue injury in reperfusion of ischemic kidneys in rats]. / Ochranný vliv carvedilolu na prutok krve a poskození tkáne pri reperfuzi ischemické ledviny krysy.

In one-kidney rats the resting blood flow in the renal artery was recorded for 10 minutes then the artery was clipped for one hour and the following reperfusion blood flow was registered again for 10 minutes. The animals were divided into three groups: Series A, untreated control animals. Series B, two weeks before renal ischemia the rats were fed a diet containing 0.1% of BM-14.190, a derivate of carvedilol. Series C the rats received a diet with 0.1% of carvedilol. The resting renal blood flow was 4.58 +/- 0.13 ml/min (x +/- S.E.M.) in series A whereas 6.36 +/- 0.41 ml/min. in series B and 5.87 +/- 0.59 ml/min. in series C (p < 0.01), also the reperfusion blood flow in series B and C was significantly higher (p < 0.01) i.e. 3.65 +/- 0.25 ml/min. and 3.08 +/- 0.33 ml/min. respectively than in series A, 1.08 +/- 0.13 ml/min. The ischemia and the ensuing reperfusion elicited in series A serious injury, especially in renal proximal tubules and their reticulin skeleton whereas the renal injury in series B and C was of a markedly lesser degree. Carvedilol and its derivate BM-14.190 possess beside vasodilatory potency and alpha- and beta-adrenoceptor blocking activity a potent scavenger property. This may be the cause of the protective effect preventing the ischemia and reperfusion injury in the renal tissue.

[Endothelial cells in tumors of the rectum and large intestine]. / Výskyt endoteliálních bunek pri nádorech konecníku a tlustého streva.

In 40 controls and 102 patients with tumours of the rectum and large intestine the number of circulating anuclear cell residues of endothelial cells was investigated. The number of these endothelia in the blood of patients was significantly higher (p < 0.001) than in healthy subjects. After successful surgery of the tumour and subsequent chemotherapy the number of endothelia in venous blood reached normal levels with in 1-2 months. Conversely in patients where despite surgery secondaries were detected in other organs, the number of endothelia remained elevated.

[The occurrence of endothelial cells in the blood in deep venous inflammation]. / Výskyt endoteliálních bunëk v krvi pri hlubokém zánetu zil.

The number of anuclear residues of endothelial cells in the blood stream was investigated in 22 patients suffering from deep phlebitis and no other disease. The level of endothelaemia in these patients was in all instances higher than in 24 healthy subjects. Assessment of the endothelaemia in patients with deep phlebitis makes more accurate assessment of the diagnosis and the follow-up of regression of the disease possible.

The effect of prostaglandins E2 and F2 alpha on carotid blood flow in rats with renovascular hypertension.

The effect of i.v. bolus administration of PGE2 and PGF2 alpha on carotid blood flow (Q) and mean arterial blood pressure (MAP) was recorded in 21 anaesthetized normotensive control (N) and 12 rats with 1K1C renovascular hypertension (RH). From the measured parameters the regional vascular impedance (PVI) and the change in blood volume were calculated. In normotensive animals both PGs elicited a dose-dependent initial fast increase of Q (threshold dose 0.4 ng/kg) and a decrease of MAP and PVI (threshold dose 0.4 micrograms/kg). Subsequently, Q decreased below the initial level. MAP and PVI remained depressed after E2 but increased after F2 alpha. The time course of the Q and MAP responses was analyzed in more detail at a standard dose 4 micrograms/kg. The average time to peak of the first phase was 12 s and of the second approximately 80 s. The initial levels of Q and MAP were reestablished within 3 to 4 minutes. The total volume of carotid blood flow obtained by planimetric integration was unaltered after F2 alpha but depressed after E2. In hypertensive animals both phases of the response to E2 were significantly retarded and the Q response was nearly abolished. On the other hand, the time course of the reaction to F2 alpha was unchanged but the magnitude of the second pressoric phase was reduced. Thus, the capacity of the carotid vascular bed to dilate remains the same in RH while the ability to constrict is limited. It is concluded that the response of MAP and Q to both PGs are relatively independent.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional differences of cardiovascular effects of diltiazem in the rat.

The dose-response relations of the central and peripheral effects of diltiazem were studied in 26 anaesthetized rats. Measured were the heart rate (HR), atrioventricular conduction time (PR), mean arterial blood pressure (BP), carotid and renal blood flow (Fc, Fr) and the corresponding relative regional resistance (RRc, RRr). The effects were evaluated by their maxima regularly reached 15-20 s after the i.v. bolus administration. The minimum dose which produced a significant HR decrease and PR prolongation were 0.4 and 2.0 mg/kg, respectively. In the mg/kg dose range a transient second degree AV block was regularly recorded. The lethal dose (cardiac arrest) was 20 mg/kg. BP significantly already decreased after 4 micrograms/kg. The dose-dependent decrease of Fr matched the hypotensive effect in the whole range due to unchanged RRr. In contrast Fc invariably increased at lower doses reflecting the RRc decay. Only in the mg/kg dose range Fc decreased in accord with BP since RRc dropped to a constant value (50% of control) with each administration. The peripheral reactions were significantly augmented in rats with renovascular hypertension. It is concluded that, in this model, the peripheral effects of diltiazem evidently surpass the central ones. The regional difference between the inert renal and responsive carotid vasculature might be due to a different mode of regulation of the respective vascular tone, hypothetically reflecting different density of membrane, potential-dependent Ca2+ channels.

[Aging changes in conjunctival blood vessels]. / Alternsveränderungen der konjunktivalen Blutgefässe.

The relation of the changes in the conjunctival blood vessels to the age were studied in a group of 217 persons aged 20 to 79 years. All persons suffering from arterial hypertension, diabetes, systemic diseases and manifested forms of arteriosclerosis were excluded. It has been proved that examination of the conjunctival blood vessels by means of the slot-lamp facilitates the study of the changes in the microscopic blood vessels in relation to the age. Most conspicuous was the increase of the meander course of the blood vessels, also the increasing number of blood vessels is more frequent in the group of older persons. Clusters of vessels and a narrowing of the diameter of the arterioles was seen only in persons over fifty years of age. The "upright" course of the arterioles was found only in individual cases and without any special dependence to age. Sacculations on the small veins and microaneurysms on the arterioles were not observed in any of the examined group of healthy persons.

Distribution of microspheres in the brain of hypertensive rats.

The blood perfusion of different parts of the brain tissue was examined by means of microspheres 15 and 50 micron in diameter, in normotensive control rats and in animals with experimental renovascular hypertension. The microspheres were labelled with fluorescein isothiocyanate and their numbers in the tissue were determined in consecutive histological sections by UV microscopy. In the control rats, the incidence of wedged microspheres per 1 mm3 tissue was high in the cerebellum, cerebral cortex, subcortical tissue and pons Varolii, but low in the thalamic and hypothalamic regions, indicating that these parts were relatively poorly perfused with blood. The significantly greater accumulation of microspheres in the cortex and subcortical tissue of hypertensive rats seems to have been due to hypertensive narrowing of the arterioles. Conversely, the diminished incidence of microspheres in the thalamus and hypothalamus may have been due partly to microsphere trapping in the narrowed upstream blood vessels and partly to thinning of the capillary network. Total microsphere recovery in the brains of the control and the hypertensive rats was almost identical, implying that only the distribution of brain blood perfusion is altered in experimental hypertension.

Early stages of experimental atheromatosis.

In female Wistar rats, fed an atherogenic diet, the early changes of serum lipids and the deviation of the vasomotor response to adrenaline and acetylcholine were compared with morphological manifestations. It has been found that the first sign may be detected as the shift in serum lipoproteins and lipids just in a few days. Merging and decrease of alpha-lipoproteins and an increase of the beta-lipoprotein fraction proved as the most sensitive indicator. In the course of several weeks, an alteration of hemodynamic response to adrenaline and acetylcholine occured. The animals revealed a diminished depressoric response to acetylcholie, whereas their blood pressure response to adrenaline in the pressoric phase was decreased but the depressoric phase was markedly expressed. The morphological impairment of the vascular system followed with a delay of several months, although a lipoid infiltration of the liver appeared after 6 weeks of the atherogenic dietary regimen.