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U ovom radu uspoređeni su ISO 20743:2013 (apsorpcijska i transfer metoda) i ISO 20645:2004 (metoda difuzije u agaru) standardi za određivanje antibakterijske učinkovitosti tekstilnih materijala. Testirana je antibakterijska učinkovitost nekoliko probnih materijala načinjenih na Tekstilno-tehnološkom fakultetu u sklopu projekta „Udobnost i antimikrobna svojstva tekstila i obuće“ prema bakterijama Staphylococcus aureus i Escherichia coli. Usporedbom triju metoda može se zaključiti kako za ispitane materijale nije uvijek moguće jednoznačno utvrditi pokazuju li antibakterijsku učinkovitost ili ne. Od ispitivanih materijala samo pozitivna kontrola, antibakterijski uložak za cipele, je pokazao antibakterijsku učinkovitost u sve tri metode i prema obje testirane bakterije. Ostali materijali su u nekim metodama pokazivali učinkovitost, a u nekima ne, a razlika je bilo i s obzirom na testiranu bakteriju. Kako bi se pouzdano odredila antibakterijska učinkovitost materijala, potrebno je paralelno izvoditi nekoliko prikladnih metoda i ispitati bar dvije bakterije, jer se na temelju samo jedne bakterije i jedne metode može pogrešno zaključiti o antibakterijskoj učinkovitosti nekog materijala.

Quercetin, a protective phenolic compound isolated from plants, exerts antibacterial activity against many strains of bacteria. It is discovered that some positive biological effects of free phenolics can be improved by their chemical derivatization with metals. Thus, we synthesized a complex of the phenolic compound quercetin (Q) with the rare earth metal europium (Eu3+) in a biological buffer, and compared its activity against Staphylococcus aureus cells with that of free Q. While free Q did not show antibacterial activity against S. aureus, its complex with Eu3+ did. Using confocal laser scanning microscope we screened for the ability of Q/Eu3+ complex, free Q and Eu3+ to penetrate into S. aureus cells. Images revealed all the three chemical forms penetrated into S. aureus cells. However, while free Q did not disrupt cell morphology, Q/Eu3+ complex and free Eu3+ caused degradation of cell structure – most of the cells were lysed, their structures were amorphous, and only few cells showed minor fluorescence signal corresponding to the Q/Eu3+ or free Eu3+. Thus, the antibacterial activity of Q/Eu3+ was attributed to degradation of complex and antibacterial effect of released free Eu3+. Confocal laser scanning microscopy was successfully employed for visualization of anti-S. aureus effect of Q/Eu3+ complex.

Natural zeolitized tuffs (NZ) rich in clinoptilolite are good adsorbents of heavy metals due to their high cation exchange capacity. The NZ containing 70 wt. % of clinoptilolite was obtained from the sedimentary deposit Zlatokop, Serbia. The particle size of the sample was in the range 0.063-0.1 mm. The NZ was firstly converted into the Na-form by treating the NZ with 2M of NaCl solution, in order to improve the clinoptilolite exchange capacity without affecting the crystallinity of NZ. The experiments on Zn2+ removal from water were performed at 30C during 24h using the batch method and ZnCl2 solution in concentration of 400 mg Zn/L with mass concentration of NZ of 1g/100mL. On this way NZ containing Zn2+ ions (named ClZn2+) was obtained. The ClZn2+ was than completely dehydrated at 550ºC under air. The dehydration led to a generation of ZnO nano-particles (about 5 nm) widespread over the surface of NZ. This material was assigned as ClZnO. The leaching test of ClZn2+ and ClZnO was performed using the 0.05M NaCl solution of pH 7.0 at 37C during 24h. The resulting leached concentration of Zn2+ in water was 0.67 mg/L for ClZn2+ whereas no leached Zn2+ was found for ClZnO. After the use of clinoptilolite for zinc removal from wastewater, we looked for future application of the used material, one of which can be removal of pathogenic bacteria in the tertiary stage of wastewater treatment. The antibacterial activity of ClZn2+ and ClZnO was tested against representative pathogenic Gram-negative (Escherichia coli, DSM 498) and Gram-positive (Staphylococcus aureus, clinical isolate at Institute of Public Health, Zagreb) bacteria. The antibacterial assay was carried out whit mass concentration of material of 1g/100mL in three different water media: Luria Bertani or LB medium, synthetic wastewater and real effluent water from the secondary stage of the biological wastewater treatment. The initial pH of used media was 7.0 and the chemical composition was as follows (in mg/L): LB medium (bacto-tryptone 10, 000 ; bacto-yeast extract 5, 000 ; NaCl 10, 000 ; COD 14, 000), synthetic wastewater (Na-propionate 1, 000 ; peptone 100 ; MgSO4 10 ; CaCl2 6 ; KCl 30 ; yeast extract 10 ; KH2PO4 20 ; COD 1, 206), effluent water (T-N 28.3 ; T-P 2.27 ; COD 31.4). All media and materials were autoclaved before the experiments were to commence. The experiments were carried out at 37C. The CFUs of bacteria were measured on LB agar at the start and after 24h of experiment. The antibacterial activity of material was expressed as percentage of log CFU reduction when compared to control where no material was added (Table 1). The percentage of inhibition of bacteria in the presence of ClZn2+ and ClZnO showed significantly negative correlation with the COD of water media used in assay (R = -0.987 and -0.949, respectively). The ClZn2+ showed better antibacterial activity than ClZnO, which is ascribed to the difference in leaching of Zn2+ ions from materials. The E. coli seems to be more resistant to Zn2+ ions than S. aureus. The difference in final pH among control and experimental reactors was not higher than 0.59 units. No significant antibacterial activity of materials was observed after 1h of contact with bacteria. Table 1. Antibacterial activity of ClZn2+ and ClZnO against E. coli and S. aureus after 24h of incubation in different water media when compared to control. c0 E. coli (106 CFU/mL) = 3.85±0.89 ; c0 S. aureus (107 CFU/mL) = 1.42±0.24 ; significantly different when compared to A-LB medium, B-synthetic wastewater, C-ClZn2+. The antibacterial activity of zinc containing clinoptilolite (mass concentration 1g/100mL, 24h of contact) was depended on the following factors in decreasing order: COD of the water medium, type of material and species of bacteria. The antibacterial activity of certain compound tested in the synthetic medium can be significantly different from those in real water medium.

Hidroksiapatit je obećavajući biomaterijal u regeneraciji kostiju zbog svojih bioaktivnih svojstva te strukturne i kemijske sličnosti s anorganskom fazom koštanog tkiva. Zbog nedostatka antibakterijskih svojstva, supstituira se ionom srebra za postizanje antibakterijske aktivnosti. Srebro posjeduje antibakterijska svojstva, no može biti citotoksično u većim koncentracijama. Za smanjenje citotoksičnosti srebrova iona, kosupstituira se stroncijevim ionom. Cilj ovog istraživanja bio je odrediti antibakterijsku aktivnost uzoraka hidroksiapatita obogaćenog ionima srebra i hidroksiapatita obogaćenog ionima srebra i stroncija metodom disk difuzije i određivanjem brojnosti bakterija u suspenziji. Testirane bakterije bile su Escherichia coli i Staphylococcus aureus kao predstavnici najčešćih patogena u infekcijama kostiju. Nesupstituirani hidroksiapatit nije pokazao antibakterijsku aktivnost. Svi uzorci hidroksiapatita supstituirani srebrovim ionom i uzorci hidroksiapatita supstituirani srebrovim i stroncijevim ionom pokazali su antibakterijsku aktivnost u obje metode što je vidljivo iz pojave zone inhibicije i analizom broja živih bakterija u bakterijskoj suspenziji nakon kontakta s uzorcima u određenom vremenu. Antibakterijska aktivnost uzoraka hidroksiapatita supstituiranih ionima srebra bila je ovisna o vremenu kontakta i količini supstituiranog srebrova iona. Hydroxyapatite is a promising biomaterial in bone regeneration due to its bioactive properties as well as its structural and chemical similarities to the inorganic phase of bone tissue. Due to lack of antibacterial properties, it is substituted with a silver ion to achieve antibacterial activity. Silver has antibacterial properties, but can be cytotoxic in higher concentrations. To reduce the cytotoxicity of silver ions, it is co-substituted with strontium ion. The aim of this study was to determine the antibacterial activity of samples of hydroxyapatite enriched with silver ions and hydroxyapatite enriched with silver and strontium ions using the disk diffusion method and determining the number of bacteria in suspension. The tested bacteria were Escherichia coli and Staphylococcus aureus as representatives of the most common pathogens in bone infections. Unsubstituted hydroxyapatite did not show antibacterial activity. All hydroxyapatite samples substituted with silver ion and hydroxyapatite samples substituted with silver and strontium ions showed antibacterial activity in both methods, which is evident from the appearance of the inhibition zone and the analysis of the number of living bacteria in the bacterial suspension after contact with the samples for a certain amount of time. The antibacterial activity of hydroxyapatite samples substituted with silver ions was dependent on the contact time and the amount of substituted silver ions.

Bakterijski biofilm je kompleks građen od jedne ili češće više vrsta bakterija koje su ugrađene u matriks izvanstanične polimerne tvari (eng. extracellular polymeric substance EPS) kojeg same proizvode. Takvi bakterijski sustavi rade velike štete kako na medicinskoj opremi, tako i u ljudskim organizmima. Razumijevanje mehanizma formacije viševrsnog biofilma može znatno olakšati razvoj metoda za borbu protiv bakterijskih biofilmova u bolnicama, okolišu ili industrijama. Za istraživanje su korištene bakterije Staphylococcus aureus i Pseudomonas aeruginosa. Cilj istraživanja bio je napraviti model biofilma kojeg tvore te vrste bakterija, vizualizirati ga, usporediti rast bakterija na granici voda – zrak kod uzoraka u kojima se bakterijama mijenja medij s uzorcima u kojima se medij ne mijenja, te usporediti količine proizvedenog EPS-a. Obzirom da se pratila biomasa bakterija na pojedinim područjima na predmetnom stakalcu (vodeni medij, interfaza i zrak), te da se pratila količina nastalog EPS-a, bakterije su se bojile metodama po Gramu i Alcian blue radi bolje vidljivosti EPS-a i usporedbe uzoraka u navedena dva slučaja. Zaključeno je da se većina biofilma nalazi na interfazi, te da mijenjanje medija više pogoduje bakteriji P. aeruginosa, a ne mijenjanje medija više pogoduje bakteriji S. aureus. Bakterija S. aureus u oba slučaja nakon nekog vremena nadvlada bakteriju P. aeruginosa. Bacterial biofilm is a complex of one or more species of bacteria that are embedded within an extracellular matrix composed of extracellular polymeric substances (EPS) produced by the cells. Such bacterial systems are doing significant damage both on medical equipment and in human organisms. Understanding the mechanism of multiple-species biofilm formation can ease the development of methods for bacterial biofilm prevention in hospitals, environment or industry. In this research, bacteria Staphylococcus aureus and Pseudomonas aeruginosa were used. The goal was to determine a model of biofilm created by these bacteria, visualize it, compare the growth of bacteria on air–liquid interface in systems in which the medium was continuously being replenished and when it is not, and compare the amount of produced EPS. Given that we monitored bacterial biomass in different areas (liquid medium, interface and air), and the amount of produced EPS, bacteria were Gram-stained, but also stained with Alcian blue to achieve better visibility of EPS and to compare samples in two aforementioned cases. It was concluded that biofilm is mostly located on the interface and that changing the medium was more beneficial to the growth of P. aeruginosa, while not changing the medium was more beneficial to the growth of S. aureus. Eventually, S. aureus outgrew P. aeruginosa in both cases.

Procjena antimikrobne aktivnosti tekstilnih materijala se provodi standardiziranim metodama koje donose različite organizacije za standardizaciju jedna od kojih je i ISO – Internacionalna organizacija za standardizaciju. Ciljevi ovog istraživanja su bili uvođenje i optimizacija ISO 20743:2013 i ISO 20645:2004 standarda za određivanje antibakterijske aktivnosti tekstilnih materijala u Laboratorij za bakteriologiju na Zavodu za mikrobiologiju Prirodoslovno-matematičkog fakulteta te određivanje antibakterijske aktivnosti materijala načinjenih na Tekstilno-tehnološkom fakultetu u sklopu projekta „Udobnost i antimikrobna svojstva tekstila i odjeće“ Hrvatske zaklade za znanost. Istraženi materijali su pamuk, sirova i obojena viskoza, tencel, mikromodal, modal, materijal X, materijal sa srebrom Aquacel Ag i antibakterijski uložak za cipele. Aquacel Ag, materijal X i uložak su pokazali antibakterijska svojstva na vrstama Staphylococcus aureus i Escherichia coli u sve tri korištene metode, a modal i mikromodal u dvije metode. Obojena i sirova viskoza su imale antibakterijska svojstva u jednoj od metoda za svaku bakteriju, a pamuk samo na vrsti E. coli u jednoj metodi. Tencel nije pokazao značajnija antibakterijska svojstva. Da bi se dobio točan rezultat, potrebno je paralelno koristiti nekoliko metoda i ispitati bar dvije vrste bakterija, jer se na temelju samo jedne vrste i jedne metode može pogrešno zaključiti o antibakterijskim svojstvima materijala. An assessment of the antimicrobial activity of textile materials is carried out by standardized methods that are provided by different organizations, such as ISO - International Organization for Standardization. The aim of this study was to introduce and optimize ISO 20743: 2013 and ISO 20645:2004 standards for the determination of antibacterial activity of textile materials in the Bacteriological Laboratory at the Department of Microbiology of the Faculty of Science and to determine the antibacterial activity of materials made at the Faculty of Textile Technology as part of the project "Textiles and footwear comfort and antimicrobial properties". The investigated materials were cotton, crude and dyed viscose, tencel, micromodal, modal, material X, silver-containing material Aquacel Ag and antibacterial insoles for shoes. Aquacel Ag, material X and antibacterial insoles showed antibacterial properties against Staphylococcus aureus and Escherichia coli in all three methods, and modal and micromodal in the two applied methods. Dyed and crude viscose had antibacterial properties in one of the methods for each bacterium, and cotton only against E. coli in one method. Tencel showed no significant antibacterial properties. In order to obtain precise and relevant result, it is necessary to use several methods and at least two bacteria species, because only one species and one method can misconstrue the antibacterial properties of a material.

Teški metali zbog svoje toksičnosti, stabilnosti i sklonosti nakupljanja u živim organizmima, predstavljaju ozbiljni ekološki problem. Stoga ih je potrebno ukloniti iz otpadnih voda prije ispuštanja u prirodni prijemnik. Prirodni zeolitni tuf pokazao se kao pogodan materijal s velikim kapacitetom vezanja iona Cu2+ iz otpadnih voda. U radu je ispitana antibakterijska aktivnost zeolitnog tufa koji sadrži 2,60 % masenog udjela (eng. percentage by weight, wt.%) Cu2+ u odnosu na bakterije Escherichia coli i Staphylococcus aureus, nakon 1 h i 24 h kontakta s zeolitom u tri vodena medija: hranjivom mediju Luria Bertani (LB), sintetskoj otpadnoj vodi i realnoj otpadnoj vodi. Utvrđeno je da zeolitni tuf obogaćen bakrom pokazuje antibakterijsku aktivnost u odnosu na Gram - pozitivnu bakteriju Staphylococcus aureus i Gram - negativnu bakteriju Escherichia coli. Nakon 24 h kontakta dolazi do redukcije broja bakterija u sintetskoj (87,35 – 95,67%) i realnoj otpadnoj vodi (86,79 – 93,54%), dok u Luria Bertani (LB) mediju (5,54 – 10,69%) redukcija nije bila učinkovita. Ne dolazi do izlučivanja toksičnih Cu2+ iona u vodenu otopinu što zeolit s Cu2+ čini obećavajućim antibakterijskim materijalom za dezinfekciju otpadnih voda. Heavy metals pollution is one of the most important environmental problems, due of their toxicity, stability, and tendency to accumulate in living organisms. Therefore, heavy metals should be removed from the wastewater before it can be discharged into a natural recipient. Natural zeolitized tuff was proved to be a suitable material with high capacity binding of Cu 2+ ions from wastewater. The antibacterial activity of natural zeolitized tuff containing 2.60 percentage by weight (wt.%) Cu2+ were tested against bacteria Escherichia coli and Staphylococcus aureus after 1 h and 24 h of contact with zeolit in three different media, namely: Luria Bertani (LB) medium, synthetic wastewater and real wastewater. The copper – loaded zeolitized tuff showed antibacterial activity against Gram - positive bacteria Staphylococcus aureus and Gram - negative bacteria Escherichia coli, after 24 h of contact. The reduction of bacterial numbers was efficient in synthetic water (87,35 – 95,67%) and real effluent water (86,79 – 93,54%), while in the Luria Bertani (LB) medium the reduction was not efficient (5,54 – 10,69%). Since there was not leaching of toxic Cu2 + ions in the aqueous solution, Cu - loaded zeolite can be regarded as promising antibacterial material for disinfection of wastewater.