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Pandemija bolesti COVID-19 i danas nakon dvije godine ima mnoga otvorena pitanja, ali se očekuje da će bolest postati endemska. Pojavnost SARS-CoV- 2 u okolišu je do sada opisana puno puta, naročito pretraživanjem otpadnih voda gdje je moguće dokazati rastuću incidenciju čak 1-3 tjedna ranije u odnosu na kretanja u populaciji. Cilj predmetnog istraživanja je bio utvrditi cirkulaciju virusa SARS-CoV-2 u otpadnim i površinskim vodama u Republici Hrvatskoj. Istraživanje je obuhvatilo 13 uzoraka otpadnih i površinskih voda s početka epidemije i 17 uzoraka ulaznih i izlaznih otpadnih voda podrijetlom iz devet uređaja za pročišćavanje otpadnih voda (prvi do treći stupanj obrade) u sedam gradova (Zagreb, Vinkovci, Slavonski Brod, Koprivnica, Karlovac, Zadar i Split) tijekom drugog vala epidemije. Postupak koncentracije virusnih čestica proveden je prema normi EN ISO 15216-1 2017 uz primjenu mengovirusa kao procesne kontrole. SARS-CoV-2 je dokazivan primjenom metode kvantitativni RT-PCR (RT-qPCR) ili konvencionalni RT-PCR za gene koji kodiraju virusne proteine E, N (N1 i N2) i nsp14. Primijenjena je i metoda RT-PCR u stvarnom vremenu za dokazivanje dijela VP2 segmenta rotavirusa A (RVA) u svrhu dodatne provjere postupaka koncentracije virusnih čestica. Rezultati potvrđuju cirkulaciju SARS-CoV-2 u osam od devet uređaja za pročišćavanje otpadnih voda u svih sedam gradova obuhvaćenih ovim istraživanjem. Od 11 SARS-CoV-2 pozitivnih uzoraka, devet je ulaznih i dva izlazna uzorka nakon sekundarne obrade otpadnih voda. Svi uzorci otpadnih voda nakon tercijarne obrade su polučili negativan rezultat na SARS-CoV-2 kao i svi uzorci otpadnih i površinskih voda s početka epidemije. Osim jasne potvrde prisutnosti SARS-CoV-2 u otpadnim vodama sedam gradova RH, rezultati ukazuju i na doprinos sustava pročišćavanja otpadnih voda, naročito trećeg stupnja, u redukciji i uklanjanju ovog virusa. Rezultati dokazivanja RVA potvrđuju da se radi o izrazito rasprostranjenom enteričnom virusu s izraženom redukcijom u izlaznim vodama, ali ukazuju i na dinamiku te sezonalnost RVA infekcija u populaciji tijekom epidemije COVID-19. Zaključno je važno istaknuti da rezultati našeg istraživanja i dosadašnje spoznaje ukazuju na vrlo mali javnozdravstveni rizik otpadnih i površinskih voda kao mogućih izvora infekcije virusom SARS- CoV-2. Međutim potreban je oprez jer su istraživanja i dalje u tijeku, posebno po pitanju infektivnosti SARS-CoV-2 u predmetnim okolišnim uzorcima.

If not properly treated, hospital wastewaters become a route for dissemination of antibiotic-resistant bacteria to the environment. Even with disinfection, many hospital bacteria survive classical methods such as chlorination or UV irradiation. A novel, effective and economically feasible methods for removal of such bacteria from wastewaters would be desirable. One of the prominent hospital pathogens is bacterium Acinetobacter baumannii. Due to its extraordinary resistance to many classes of antibiotics, especially to carbapenems that were for long time the “last resort” drug, it has become common to detect multiple -, extensive- and even pan-drug resistant isolates of A. baumannii worldwide, both in hospital settings but also in natural environments. Over the last few years, it has been shown without a doubt that in Croatia, A. baumannii along with other Gram-negative pathogens readily disseminates to surface waters via hospital wastewaters, and that it readily passes wastewater treatment plant. Therefore, we tested natural mineral zeolite, enriched with silver by ion-exchange (AgNZ) as a substrate for disinfection of wastewater. The antibacterial efficacy of AgNZ was tested in flow system (biofilter) with pure bacterial cultures, but also with real effluent wastewater from Zagreb wastewater treatment plant. In experiments with real wastewater, we measured the numbers of all carbapenem-resistant bacteria, not just A. baumannii. The biofilter filled with AgNZ and constantly fed with bacterial suspension at 30 ml h-1 flow rate eliminated all of the A. baumannii cells (100 % removal) during 4 days of the experiment and remained effective by removing up to 3 Log values for 14 days. In experiments with real wastewater, the biofilter was effective in removing 90-100 % of carbapenem-resistant bacteria during 4 days. By using X-Ray microtomography it was also shown that particle size of biofilter filling is important from technological point of view, as small sized particles caused biofilter breakthrough by causing preferential flow-paths to emerge. However, after regeneration the biofilter became active again suggesting possile long-time use of AgNZ as biofilter filling. Here presented study is a contribution to “One Health” approach as it proposes a sustainable step forward in reduction of antibiotic resistance dissemination from hospitals to the environment and vice-versa.

Virus SARS-CoV-2 je uzrokovao pandemiju čija je dinamika, sada nakon gotovo dvije godine, i dalje neizvjesna. Iako primarno uzrokuje simptome respiratornog sustava, izlučuje se u produljenom trajanju putem probavnog sustava. To je ujedno bila i osnova za istraživanje otpadnih voda u kojima je dokazana prisutnost virusa SARS-CoV-2 diljem svijeta. Istraživanja moguće kontaminacije školjkaša virusom SARS-CoV-2 značajno zaostaju, sa svega dva do sada opisana istraživanja. Cilj predmetnog istraživanja je bio utvrditi cirkulaciju virusa SARS-CoV-2 u otpadnim i površinskim vodama u Republici Hrvatskoj te proširiti trenutne spoznaje o mogućnosti kontaminacije školjkaša u Jadranu tijekom drugog vala epidemije. Istraživanje je obuhvatilo 13 uzoraka otpadnih i površinskih voda s početka epidemije u Republici Hrvatskoj i 17 uzoraka ulaznih i izlaznih otpadnih voda podrijetlom iz devet uređaja za pročišćavanje otpadnih voda (prvi do treći stupanj obrade) u sedam gradova (Zagreb, Vinkovci, Slavonski Brod, Koprivnica, Karlovac, Zadar i Split) tijekom drugog vala epidemije. Tijekom tog razdoblja prikupljen je i veći dio od 77 uzoraka školjkaša, a zastupljene vrste su dagnje (Mytilus galloprovincialis, N= 52), kamenice (Ostra edulis, N= 23) i brbavice (Venus verrucosa) s komercijalnih uzgajališta. Postupak koncentracije virusnih čestica proveden je prema normi EN ISO 15216-1 2017 uz primjenu mengovirusa kao procesne kontrole. SARS-CoV-2 je dokazivan primjenom metode kvantitativni RT-PCR (RT-qPCR) ili konvencionalni RT-PCR za gene koji kodiraju virusne proteine E, N (N1 i N2) i nsp14. Također je primijenjena i metoda RT-PCR u stvarnom vremenu za dokazivanje dijela VP2 segmenta rotavirusa A (RVA) u svrhu dodatne potvrde postupaka koncentracije virusa. Dobiveni rezultati potvrđuju cirkulaciju virusa SARS-CoV-2 u osam od devet uređaja za pročišćavanje otpadnih voda u svih sedam gradova. Od 11 pozitivnih uzoraka na SARS-CoV-2, devet je ulaznih i dva izlazna uzorka nakon sekundarne obrade otpadnih voda. Svi uzorci otpadnih voda nakon tercijarne obrade polučili su negativan rezultat na SARS-CoV-2 kao i svi uzorci otpadnih i površinskih voda s početka epidemije. Nije utvrđena prisutnost kontaminacije školjkaša virusom SARS-CoV-2. Rotavirus A je potvrđen u svim uzorcima otpadnih voda (N= 21), u 22, 2% uzoraka površinskih voda i u 23% uzoraka školjkaša. Predmetnim istraživanjem dokazali smo cirkulaciju virusa SARS-CoV-2 u otpadnim vodama diljem Republike Hrvatske te utvrdili da sustav pročišćavanja otpadnih voda, naročito treći stupanj, učinkovito reducira ili uklanja virus. Nismo dokazali kontaminaciju školjkaša virusom SARS-CoV-2 iako su uzorci prikupljeni tijekom razdoblja visoke incidencije u okviru drugog vala epidemije. Rezultati dokazivanja RVA potvrđuju da se radi o izrazito rasprostranjenom enteričnom virusu s izraženom redukcijom u izlaznim vodama, ali ukazuju i na dinamiku te sezonalnost RVA u populaciji tijekom epidemije COVID-19. Rezultati našeg istraživanja i dosadašnje spoznaje ukazuju na vrlo mali javnozdravstveni rizik otpadnih voda i školjkaša kao mogućih izvora infekcije virusom SARS-CoV-2. Međutim potreban je oprez jer su istraživanja i dalje u tijeku, posebno po pitanju infektivnosti virusa SARS-CoV-2 u predmetnim okolišnim uzorcima.

Kvaliteta života i antropogene aktivnosti utječu na složenost sastava komunalnih otpadnih voda. Pročišćavanje komunalnih otpadnih voda predstavlja jedan od osnovnih uvjeta održivog upravljanja vodenim resursima te unaprjeđenje zaštite okoliša. Proces s aktivnim muljem je najčešći izbor za uklanjanje organskog opterećenja iz otpadnih voda. Mikroorganizmi i održavanje aktivne populacije su ključni dio procesa. Na aktivnost mikroorganizama utječu mnogi procesni parametri čime se izravno utječe na učinkovitost postupka pročišćavanja, a u konačnici rezultira određenom kvalitetom pročišćene vode. U ovom radu praćena je promjena organskog opterećenja u komunalnim otpadnim vodama iz Centralnog uređaja za pročišćavanje otpadnih voda grada Zagreba. Tijekom procesa su praćene vrijednosti kemijske potrošnje kisika i petodnevne biokemijske potrošnje kisika, taložive krute tvari, ukupne suspendirane tvari te koncentracije ukupnog dušika i ukupnog fosfora. Mikroskopskom analizom je dobiven uvid u učinkovitost pročišćavanja otpadnih voda. Učinkovitosti uklanjanja taložive krute tvari i ukupne suspendirane tvari te smanjenja KPK i BPK5 iznosile su više od 90 % u prosjeku, a koncentracija ukupnog dušika i ukupnog fosfora smanjena je za 44 %, odnosno 45 %. Mikroskopskom analizom omogućen je brzi uvid u proces s aktivnim muljem kako bi se na vrijeme mogli osigurati optimalni uvjeti za biološku obradu komunalne otpadne vode.

Od pojave prvog slučaja bolesti krajem 2019. godine u Kini, virus SARS-CoV-2 je uzrokovao pandemiju bez presedana. Unatoč velikom napretku u razvoju i primjeni cjepiva, očekuje se da će bolest postati endemska s puno otvorenih pitanja glede dinamike infekcija virusom SARS-CoV-2. Pojavnost ovog virusa u okolišu je opisana puno puta naročito pretraživanjem otpadnih voda te se smatra da je na taj način moguće dokazati rastuću incidenciju bolesti COVID-19 čak 1-3 tjedna prije pojave u populaciji ljudi. Pretraživanje školjkaša na moguću kontaminaciju virusom SARS-CoV-2 značajno zaostaje, sa svega dva do sada opisana istraživanja. Cilj našeg istraživanja je bio utvrditi cirkulaciju virusa SARS-CoV-2 u otpadnim i površinskim vodama u Republici Hrvatskoj te proširiti trenutne spoznaje o mogućnosti kontaminacije školjkaša u Jadranu tijekom drugog vala epidemije. Ovo istraživanje obuhvatilo je 13 uzoraka otpadnih i površinskih voda s početka epidemije u Republici Hrvatskoj i 17 uzoraka ulaznih i izlaznih otpadnih voda podrijetlom iz devet pročišćivača u sedam gradova (Zagreb, Vinkovci, Slavonski Brod, Koprivnica, Karlovac, Zadar i Split) tijekom drugog vala epidemije. Pročišćivači otpadnih voda su bili različitog stupnja obrade, od prvog do trećeg stupnja. Tijekom tog razdoblja potječe i većina od 77 uzoraka školjkaša, a zastupljene vrste su dagnje (Mytilus galloprovincialis, N= 52), kamenice (Ostra edulis, N= 23) i brbavice (Venus verrucosa). Postupak koncentracije virusnih čestica, u gore navedenim uzorcima, proveden je djelomično modificiranim postupkom opisanim u normi EN ISO 15216-1 2017 uz primjenu mengovirusa kao procesne kontrole. Dokazivanje virusa SARS-CoV-2 provedeno je primjenom metode RT-PCR u stvarnom vremenu ili konvencionalno za gene koji koji kodiraju virusne proteine E, N i nsp14. Također je primijenjena i metoda RT-PCR u stvarnom vremenu za dokazivanje dijela genomskog segmenta VP2 rotavirusa A (RVA) u svrhu dodatne potvrde postupaka koncentracije virusa. Dobiveni rezultati potvrđuju cirkulaciju virusa SARS-CoV-2 u osam od devet pročišćivača otpadnih voda lociranih u svih sedam gradova. Od 11 SARS-CoV-2 pozitivnih uzoraka, devet je ulaznih i dva izlazna uzorka nakon sekundarne obrade otpadnih voda. Svi uzorci otpadnih voda nakon tercijarne obrade su polučili negativan rezultat na SARS-CoV-2 kao i svi uzorci otpadnih i površinskih voda s početka epidemije. Nije utvrđena prisutnost kontaminacije školjkaša virusom SARS-CoV-2. Rotavirus A je potvrđen u svim uzorcima otpadnih voda (N= 21), u 22, 2% uzoraka površinskih voda i u 23% uzoraka školjkaša. SARS-CoV-2 dokazano cirkulira u otpadnim vodama diljem Republike Hrvatske, a sustav pročišćavanja, naročito treći stupanj učinkovito reducira ili uklanja virus. Naši rezultati su u suglasju s mnogim istraživanjima provedenim u svijetu. Uzorci otpadnih voda tijekom početka epidemije su negativni ponajviše zbog strategije uzorkovanja usmjerene na izlazne vode gdje zbog niske incidencije u populaciji nije bilo moguće dokazati prisutnost virusa. Važno je istaknuti da dosadašnje spoznaje ne ukazuju na infektivnost otpadnih voda pozitivnih na RNA virusa SARS-CoV-2, ali pozivaju na oprez, poglavito djelatnika u postrojenjima pročišćivača. Negativan rezultat na prisutnost virusa SARS-CoV-2 u školjkašima u Jadranu je sličan rezultatima provedenim u Francuskoj. Važno je istaknuti da je naše istraživanje po prvi puta obuhvatilo razdoblje uzorkovanja tijekom jeseni i zime kada su enterične infekcije najčešće, a ujedno je to bilo i razdoblje drugog i najizraženijeg vala epidemije virusa SARS-CoV-2. Međutim, pokusno je dokazana mogućnost kontaminacije kamenica inaktiviranim virusom SARS-CoV-2, a u Španjolskoj je dokazana prisutnost neaktivnih virusnih čestica SARS-CoV-2 u školjkama u uzgajalištu, ali u području ušća rijeke pod utjecajem izljeva otpadnih voda. Rezultati dokazivanja RVA potvrđuju da se radi o široko rasprostranjenom enteričnom virusu s izraženom redukcijom u izlaznim vodama. Povrh toga ovi rezultati ukazuju i na dinamiku te sezonalnost RVA u populaciji tijekom epidemije koronavirusa. Zaključno možemo istaknuti da naši rezultati u kombinaciji s dosadašnjim spoznajama ukazuju na vrlo mali javnozdravstveni rizik otpadnih voda i školjkaša kao mogućih izvora infekcije virusom SARS-CoV-2. Međutim kako su istraživanja i dalje u tijeku, posebno po pitanju infektivnosti virusa SARS-CoV-2 u navedenim matriksima, potreban je oprez.

U disertaciji je istražena mogućnost priprave visokokvalitetnog pjenostaklenog materijala iz pepela dobivenog spaljivanjem mulja nastalog pročišćavanjem komunalnih otpadnih voda, koji zbog rastućih količina i nepovoljnog kemijskog sastava predstavlja opasnost za okoliš. Uz pepeo za pripravu pjenostakla korišteni su otpadno ambalažno staklo i otpadni vapnenac kao agens za pjenjenje. Utvrđeno je da se pjenostaklo ujednačene poroznosti i volumne stabilnosti te poboljšane tlačne čvrstoće (≈ 30 MPa) u odnosu na komercijalne pjenostaklene proizvode može pripraviti brzinom zagrijavanja od 5 °C/min do temperature od 850 °C uz zadršku od 15 min na vršnoj temperaturi. U pjenostaklu može se zbrinuti visoki maseni udio pepela (do 28 %) s niskim masenim udjelom vapnenca (1–2 %). Izluživanjem teških metala iz uzoraka pjenostakla utvrđeno je da su koncentracije teških metala u eluatima unutar zakonskih dopuštenih granica za neopasne otpade te da pjenostakla pokazuju antibakterijsku aktivnost prema klinički relevantnoj bakteriji Acinetobacter baumannii. Poboljšana mehanička svojstva dobivenog pjenostakla posljedica su stvaranja novih mineralnih faza tijekom toplinske obrade. Dodatkom pepela dolazi do pojačane kristalizacije natrijevog-kalcijevog silikata, α-kvarca, diopsida i volastonita. Kristalizacija ovih faza osim što doprinosi poboljšanju tlačne čvrstoće materijala istovremeno smanjuje izluživanje teških metala iz pjenostaklene matrice. Antibakterijska aktivnost posljedica je visoke pH-vrijednosti eluata uzrokovane otapanjem nevezanog kalcijevog oksida čije su čestice utvrđene elektronskom mikroskopijom na stijenkama pora.

Multi-drug resistant (MDR) Acinetobacter baumannii were found in untreated as well as in biologically or chemically treated hospital and municipal wastewaters. However, there is no evidence about the fate of A. baumannii in the wastewater treatment process. The aim of this study was to screen the sewage sludge after its stabilization by anaerobic mesophilic digestion for the presence of A. baumannii. On 9 sampling occasions at the municipal wastewater treatment plant of the City of Zagreb, 17 isolates of A. baumannii were recovered form digested sludge with MALDI-TOF MS score values ranging from 2.026-2.288. 3/17 isolates were sensitive to all antibiotics, while 14 MDR isolates shared the resistance to carbapenems and fluoroquinolones but sensitivity to colistin. Technology of anaerobic sludge digestion was performed at 36°C, neutral pH and digestion time of 21-36 days. In laboratory controlled anaerobic conditions isolates were able to survive on Nutrient agar in anaerocultA system during 30 days, after which multiplied normally in aerobic conditions. However, isolates were not able to multiply directly in anaerocultA. The finding confirms the need of proper management and disposal of sewage sludge generated at wastewater treatment plants in order to prevent the spread of MDR A. baumannii in nature.

Acinetobacter baumannii is an emerging human pathogen. Biofilm formation is an important virulence factor which contributes to the pathogenesis of A. baumannii, but data regarding the isolates recovered from the natural environment are missing. The biofilm formation at the solid-liquid/air-liquid interfaces of environmental isolates of A. baumannii, as well as the influence of natural zeolitizied tuff (NZ) on its formation was determined. In total, 24 environmental isolates of A. baumannii (14 multi-drug resistant, 10 sensitive to antibiotics) were recovered from different stages of the secondary type of municipal wastewater treatment plant. Isolates sensitive to antibiotics were statistically significantly more hydrophobic and formed stronger biofilm than multi-drug resistant isolates. Biofilm formation was significantly positive correlated with hydrophobicity of cells. Biofilm formation at solid-liquid interface was significantly inhibited by the addition of 1% of NZ into the growth medium due to the immobilization of bacterial cells onto the NZ particles. Biofilm formation at air- liquid interface was inhibited only by the addition of 10% of NZ. NZ could find application in control of the biofilm formation of this pathogen on abiotic surfaces.

Acinetobacter baumannii is an emerging opportunistic pathogen associated mainly with hospital infections. Since there is insufficient data regarding the successfulness of A. baumannii in the natural environment, the aim of this investigation was to examine the survival rates of A. baumannii in different types of natural water media. Multi-drug resistant (MDR) environmental isolates were cultivated on CHROMagar Acinetobacter at 42°C/24h. Bacterial suspension was inoculated into bottles containing the 100 mL of sterile spring water, seawater and effluent water from the secondary type of municipal wastewater treatment plant, and incubated at 20°C with stirring (150 rpm) during 28 days. Number of bacteria was followed by cultivation on Nutrient agar plates at 42°C/24h, from which the survival rates were calculated. In effluent water the successful multiplication of A. baumannii was evident (124% survival rate after 28 days), while in natural spring water and seawater no multiplication occurred. The survival rate in spring water was constant (100%) with a slight decrease (94%) after 21 days and 89% after 28 days, while in seawater the survival rate was constant for the first 2 days (100%), and decreased to 64% after 28 days. The chemical oxygen demand of effluent was considerably higher (24 mgO2/L) than the spring (3 mgO2/L) and seawater (4 mgO2/L). These findings indicate that MDR isolates of A. baumannii successfully persist in the natural water media and that the survival rates are dependent on the availability of nutrients.

Management of waste materials for sustainable development imply their recycling and reuse. In this work the foam glass (FG) was prepared from powder mixture of waste materials: sewage sludge ash (SSA), recycled bottle glass culet, and carbonate foaming agent through one-step thermal process at temperatures up to 850°C. Prepared FG, with wollastonite as a major mineral phase, had open and connected pores from several mm to closed small pores ˂1mm and can be classified as macro- or meso-porous materials. FG fulfil requirements for masonry mortars. Low leaching of heavy metals confirmed its safely incorporation inside glass matrix. FG exhibited the antibacterial activity against emerging pathogenic bacterium Acinetobacter baumannii due to the alkaline reaction of FG in water medium. SSA and bottle glass could be successfully transformed by simple production method to FG as environmental friendly material.

The carbapenem resistance in Gram-negative bacteria, which is usually associated with resistance to several other classes of antibiotics, has become a global problem of this century in hospital settings. Reports regarding the occurrence of viable carbapenem- resistant bacterial population (CRBP) outside medical institutions are globally scarce. The aim of this study was to quantify the numbers of viable CRBP through the secondary type of municipal wastewater treatment process (WWTP). The study was performed at the WWTP of the Croatian capital city of Zagreb, on 20 sampling occasions from September 2015 to June 2016. The CRBP was enumerated on CHROMagar Acinetobacter supplemented with CR102, which allows the growth of carbapenem-resistant isolates after incubation at 37C/72 h. The CRBP were found in influent, effluent, fresh activated sludge, anaerobically digested sludge, but not in lime- treated stabilized sludge of pH 12. Similar abundance (p>0.05) of CRBP was detected in influent and fresh activated sludge (median value 4.0 log CFU/mL) and significant decrease (p

Background: Acinetobacter calcoaceticus- baumannii complex (ABC) includes six genetically closely related but phenotypically undistinguishable species: A. calcoaceticus, A. baumannii, A. pittii, A. nosocomialis, genomic species “close to 13TU”, and genomic species “between 1 and 3”. A. baumannii is most often studied as an emerging hospital pathogen. Reports on the occurrence of ABC in wastewater treatment plants are scarce. The objective of this study was to screen the municipal wastewater of Zagreb, the capital of Croatia, for the presence of viable ABC and for its possible discharge into a natural recipient after the wastewater passage through the treatment process. Material/methods: The sampling was done at the secondary type of wastewater treatment plant of the City of Zagreb (capacity 1, 200.000 population equivalents) where the municipal wastewater treated consists of domestic, industrial, hospital and storm wastewaters. The composite 24h samples of the influent and effluent wastewater were collected during 6 months in 2014. The isolation of ABC from wastewater was performed at 42C/48h on CHROMagar Acinetobacter with or without the addition of commercial supplement CR102 (CHROMagar) allowing the growth of carbapenemresistant isolates. Presumptive ABC colonies were characterized phenotypically by routine bacteriological techniques and by using Vitek 2 system. Further identification was carried out by MALDI-TOF MS and RNA polymerase β-subunit (rpoB) gene analyses. Antibiotic resistance profiles were determined for carbapenems meropenem and imipenem by Vitek 2 system and interpreted according to EUCAST criteria. Results: On 6 sampling occasions 34 isolates belonging to ABC, as determined by Vitek 2 system, were recovered: 28 from influent and 6 from effluent wastewater. The comparison of ribosomal proteins by MALDI-TOF MS with strains of bacteria in MALDI Biotyper database gave the score values form 2.013 - 2.409 identifying the ABC isolates as A. baumannii or A. pittii. Phylogenetic analysis of the rpoB gene fragment confirmed the identity of isolates as A. baumannii or A. pittii and showed their close relatedness to the clinical isolates with 100% sequence ID. Molecular identification of ABC confirmed that 22 and 6 isolates of A. baumannii were recovered from influent and effluent wastewater, respectively, while 6 A. pittii were recovered only from influent wastewater. The 28/34 isolates (27 A. baumannii and 1 A. pittii) were resistant to carbapenems. Conclusions: Municipal wastewaters of Zagreb are continuously polluted with ABC probably due to the input of untreated hospital wastewaters. Among the ABC, only A. baumannii and A. pittii species are present with A. baumannii as a predominant species. More frequent isolation of A. baumannii from influent than from effluent suggests its moderate elimination, but also its persistence in the secondary type wastewater treatment system. The absence of A. pittii isolation from effluent suggests its complete elimination in the secondary type of wastewater treatment plant.

Acinetobacter baumannii is an emerging hospital pathogen causing outbreaks in Croatia since 2002 and is still present in Croatian hospitals. Clinical isolates of A. baumannii in Croatian hospitals are usually multi-drug resistant(MDR), with resistance to carbapenems dramatically increasing from 10% in 2008 to 82% in 2014. Although A. baumannii has been isolated from patients and hospital environment during outbreaks, crucial questions regarding its epidemiology remains incompletely understood. The aim of this study was to screen the hospital and municipal wastewater of the City of Zagreb for the presence of viable A. baumannii and carbapenems. Sampling of hospital wastewater was performed on 2 occasions in 2015 at the central manhole of one Zagreb’s hospital from which the clinical isolates of A. baumannii were recovered. Sampling of municipal wastewater was performed on 6 occasions in 2014/15 at the influent and effluent of the central Zagreb’s wastewater treatment plant. Concentration of carbapenems in wastewater was measured by UHPLC Q-TOF MS. The isolation of A. baumannii from wastewater was performed at 42C/48h on CHROMagar Acinetobacter without or with the addition of commercial supplement CR102 which allows the growth of carbapenem- resistant isolates. Presumptive A. baumannii colonies were characterized phenotypically, by using Vitek2 system, and MALDI-TOF MS. Antibiotic resistance profiles were determined by Vitek2 system and interpreted according to EUCAST criteria. Hospital and municipal wastewaters contained high concentrations of imipenem and meropenem which averaged: 894 and 129ng/L in hospital wastewater, 3060 and 380ng/L in raw and 497 and 311ng/L in treated municipal wastewater, respectively. From hospital wastewater, raw and treated municipal wastewater 8, 30 and 7 isolates of A. baumannii were recovered, respectively. All isolates from hospital wastewater and majority (33/37) of isolates from municipal wastewater were resistant to carbapenems and majority of tested antibiotics except colistin. The 7 MDR clinical isolates recovered in the same period showed comparable levels of antibiotic resistance to MDR isolates from hospital and municipal wastewater. These suggest that A. baumannii is able to survive in environment outside hospitals. However, 4 isolates from raw municipal wastewater were susceptible to carbapenems and other antibiotics. This finding opens the possibility that A. baumannii could have natural habitat in sewage system.

Over the last decade hospital-acquired infections due to Acinetobacter baumannii are increasing worldwide. Clinical isolates of A. baumannii are usually multi-drug resistant (MDR), with resistance to carbapenems increasing drastically in Croatia. The most important mechanism of carbapenem resistance in A. baumannii is the enzymatic hydrolysis mediated by oxacillinases encoded by blaOXA genes. Although A. baumannii has been isolated from patients and hospital environment during outbreaks, its epidemiology remains incompletely understood. Here we report the finding of MDR carbapenem resistant A. baumannii in treated municipal wastewater related to clinical isolates. The isolate of A. baumannii named EF2 was recovered from the secondary treated municipal wastewater of the City of Zagreb. The isolation was performed at 42°C/48h on CHROMagar Acinetobacter supplemented with CR102 and cefsulodin sodium salt hydrate. By phenotypical analyses and Vitek 2 system the isolate was determined as A. calcoaceticus-baumannii complex. MALDI-TOF MS analysis gave the reliable score value of 2.352 identifying it as A. baumannii. Phylogenetic analysis of the RNA polymerase β-subunit (rpoB) gene fragment confirmed the identity as A. baumannii species and showed 100% sequence ID to the clinical isolates. According to MIC values determined by Vitek 2 system or E-test and interpreted according to EUCAST criteria, isolate was susceptible to amikacin, trimethoprim- sulfamethoxazole and colistin, but resistant to carbapenems (imipenem, meropenem), fluoroquinolones (ciprofloxacin, levofloxacin), aminoglycosides (gentamicin, tobramycin) and therefore could be classified as MDR. Multiplex PCR and sequencing confirmed the presence of both intrinsic blaOXA-51 and acquired blaOXA-23 genes. Phylogenetic analyses of blaOXA genes from environmental isolate showed association with those previously described from clinical isolates. Isolate multiplied in the batch system containing autoclaved water from which it was recovered up to 50 days of monitoring at 16.7°C, when its number was 9% higher than initial number. These results suggest that MDR A. baumannii recovered from treated municipal wastewater is most probably of clinical origin and is able to survive in environment outside hospital.

Acinetobacter baumannii is a human pathogen, emergence of which in hospital acquired infections increased dramatically over the last decade, both in Croatia and worldwide. Resistance of A. baumannii to antibiotics, disinfectants, and adverse conditions lead to its long persistence in hospitals. Twitching and swarming surface motility together with biofilm formation are virulence factors that contribute to its pathogenesis. Inhibition of twitching motility as a prerequisite for biofilm formation is promising tool to suppress the virulence of A. baumannii. Influence of micronized natural zeolitized tuff (NZ) on twitching and swarming surface motility of 17 isolates of A. baumannii was tested. The NZ at concentration 1-3% reduced or completely blocked the twitching motility of A. baumannii on polystyrene due to the immobilization of bacterial cells onto NZ particles. The swarming motility on the surface of semisolid medium was not reduced. Micronized NZ could find application in control of the adherence and subsequent biofilm formation of pathogenic bacteria on abiotic surfaces.

Acinetobacter pittii is genetically closely related to A. baumannii, A. calcoaceticus, A. nosocomialis, genomic species “close to 13TU”, and genomic species “between 1 and 3”. Therefore, these six species of Acinetobacter spp. are grouped into A. calcoaceticus- baumannii complex. Human diseases have been attributed mainly to A. baumannii, and to a lesser extent to A. pittii. The latter is sensitive to antibiotics unlike A. baumannii which is usually multi-drug resistant. However, recent molecular studies revealed that A. pittii is often misidentified by routine laboratory methods. The prevalence of A. pittii in nature and its epidemiology are undefined to date. The objective of this study was to screen the municipal wastewater of Zagreb, the capital city of Croatia, for the presence of A. pittii. The sampling was done at the influent of the largest Croatian wastewater treatment plant of the City of Zagreb, where the municipal wastewater consisting of domestic, industrial, hospital and storm wastewaters are treated. In the composite 24h samples the physico-chemical and microbiological parameters were determined. The isolation of A. pittii was done on CHROMagar Acinetobacter supplemented with 15mg/L of cefsulodin after incubation at 42C/48h. The presumptive A. pittii colonies were firstly characterized to possess the following characteristics: Gram negative coccobacilli, negative oxidase, positive catalase reaction, no reaction on the Kligler Iron Agar. Further identification was carried out by using Vitek 2 system, and confirmed by MALDI-TOF MS and RNA polymerase β-subunit (rpoB) gene sequencing. Four isolates of A. pittii were obtained from municipal wastewater with high MALDI-TOF MS score values (2.156- 2.291). Isolates were sensitive to all 9 antibiotics tested by Vitek 2 system and E- test. One isolate showed MIC value of meropenem close to intermediate values (1.5mg/L) and twice as high MICs values of levofloxacin and colistin (0.5mg/L) than other three isolates. MALDI-TOF MS and PFGE dendrograms grouped three isolates with lower MICs in cluster different form one isolate with higher MICs values. Phylogenetic analysis has shown the close relatedness of the isolate with higher MICs values to the A. pittii strain CIP 70.15, with 100% sequence ID of the rpoB gene fragment. The isolate of A. pittii with higher MICs values was probably of clinical origin and entered the municipal wastewater trough hospital sewage. The finding of isolates with low MICs values suggests that A. pittii could also be an autochthonous species in water.

A study of trace metals fluxes in the wastewater treatment plant (WWTP) of the city of Zagreb was performed over a period of 8 months in 2009. Total and dissolved concentration of 26 elements ( Ag, Al, As, Ba, Bi, Cd, Co, Cr, Cs, Cu, Fe, Li, Mn, Mo, Ni, Pb, Rb, Sb, Se, Sn, Sr, Ti, Tl, U, V, Zn) were determined by high-resolution inductively coupled plasma mass spectrometry (HRICPMS) in the composite samples of raw wastewater and secondary effluent. For the determination of total metals in wastewater samples a novel ultrasonic acid digestion procedure was developed. The observed total and dissolved metal concentrations in raw wastewater were in typical ranges reported for municipal effluents with no indication of a significant industrial input [1]. Due to the high suspended solid load and organic matter content in the raw wastewater, only few of the determined metals were present predominately in the dissolved form (Li, Rb, Sr, U, Se). The majority of metals were partly (Mo, Sb, Cs, As, Ba, Ni, V, Co, Sn, Zn) or nearly completely (Zn, Cr, Tl, Cd, Cu, Pb, Ag, Ti, Fe, Bi, Al) bound to particles. High affinity of these metals for particulate matter is the main mechanism controlling the efficiency of their removal in wastewater treatment. Removal of the most toxic metals (Cr, Cd, Cu, Pb, Ag or Bi) in the investigated WWTP is higher than 85%, but for some other toxic elements, such as As, Sb, Ni, V and Zn, the removal is significantly lower (50- 70%). Due to the much lower particle load, the relative percentage of dissolved metals in secondary effluents (40-100 % for all measured elements) is much higher than in the raw wastewater. For most of the elements (with exception of Zn, Pb and Ag) the concentrations of the dissolved species in secondary effluents were found to be of the same order of magnitude as their concentrations in the Sava River water, indicating that the contribution of the effluents from WWTP of the city of Zagreb to the total metals fluxes in the Sava River was probably not very high.

U radu je istraživana mogućnost priprave staklokeramike uporabom otpadnog materijala nastalog pri proizvodnji stakla. Taj otpad sadrži znatne količine kationa i aniona, koji su stalna prijetnja zagađenju okoliša, a njihovo zbrinjavanje na prikladno uređenim odlagalištima predstavlja veliko financijsko opterećenje. S obzirom da stakleni otpad sadrži veliki udio kalcijevih i silicijevih iona, taj se otpad može iskoristiti kao sirovina za proizvodnju staklokeramike na bazi beta- volastonita (beta-CaSiO_3). U ovom su radu modificiranjem sastava staklenog otpada pripravljena stakla koja su potom termički obrađivana. Sastav stakla bio je w(CaO) + w(SiO_2) = 87 %, w(Al_2O_3) = 5 %, w(ZnO) = 5 %, w(Na_2O) = 3 %, pri čemu je množinski omjer n(CaO) : n(SiO_2) varirao između 0, 35 i 1, 10. Kao primarni nukleator korišten je cinkov oksid. Istraživan je utjecaj sastava stakala na njihovo ponašanje tijekom termičke obrade, proces nukleacije i kristalizacije te vrstu i relativni udio kristalnih faza. Kod svih je stakala utvrđeno nastajanje beta-volastonita, a ovisno o sastavu stakla, uočena je i pojava hardistonita (Ca_2ZnSi_2O_7), anortita (CaAl_2Si_3O_8) i kristobalita (alfa-SiO_2). Karaktrizacija uzoraka te ispitivanje ponašanja stakala tijekom termičke obrade provedeno je rendgenskom difrakcijskom analizom i diferencijalnom termičkom analizom.