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Enterobacterales are known for harboring resistance to various antibiotics, especially to carbapenems. Moreover, treated wastewater are hotspots for their dissemination into the environment. Therefore, this work aims to reveal the occurrence of carbapenemase-producing Enterobacterales from treated wastewater of a wastewater treatment plant (WWTP), in Zagreb, Croatia (Winter 2020). 200 presumptive Enterobacterales species were isolated from treated wastewater (secondary effluent) on CHROMagarmSuperCARBA plates and identified by MALDI-TOF MS. Antimicrobial susceptibility profiles and carbapenemase production was assessed. Carbapenemases and mcr genes were detected by PCR. Genetic relatedness was explored by puls-field gel electrophoresis (PFGE). Chosen isolates underwent whole genome sequencing (WGS) for the determination of resistance genes and multilocus-sequence typing (MLST). 148/200 Enterobacterales from treated wastewater, were confirmed to be carbapenemase-producing Enterobacterales. The predominant taxa included Klebsiella spp. (32 %), Citrobacter spp. (27 %), and Enterobacter spp. (24 %). All isolates were multi-drug resistant, with 29 % resistance to colistin. In a subset of 83 isolates, selected according to their resistance profiles, blaKPC-2, blaNDM-1, and blaOXA-48 were predominant genes, while blaVIM-1 and blaIMP-13 were detected sporadically. Colistin resistance gene mcr-4.3 was detected in three Klebsiella pneumoniae. PFGE revealed five pulsotypes in K. pneumoniae belonging to ST1803, ST3590, and ST1697, while two went undetermined. Four pulsotypes of Klebsiella oxytoca belonged to ST43, ST1697, ST17, and ST68. In Enterobacter spp, eight pulsotypes belonged to ST277, ST339, and ST32. All Escherichia coli belonged to ST8889. Eleven isolates (1 E. coli, 5 K. pneumoniae, 2 K. oxytoca, and 3 Enterobacter spp.) were chosen for WGS, according to their pulsotypes and detected genes. Ten isolates harbored at least one carbapenemase gene in co- occurrence with other β-lactamase genes. All isolates carried resistance genes to aminoglycosides, fluoroquinolones, trimethoprim, sulfonamides, macrolides, and/or biocides. The pan-drug K.pneumoniae harbored mcr-4.3 with co-occurrence of blaOXA-48, blaCTX-M-15, and blaSHV-81 genes. This is the first report of K. pneumoniae harboring the plasmid-mediated mcr-4.3 gene with the co-occurrence of carbapenemase genes. The improvement of WWTP technologies is needed to minimize the release of highly resistant pathogens into the receiving water bodies which can contribute spread of the resistance.

The emergence of extended-spectrum β-lactamases (ESBLs)- and especially carbapenemases in Enterobacterales has led tolimited therapeutic options ; therefore, it is critical to fully understand all potential pathways of their transmission, especiallyin high-risk sources such as hospital wastewater. The aim of this study was to isolate and characterize ESBL- andcarbapenemase-producing Enterobacterales (CRE) from untreated wastewater of two major hospitals in Zagreb, Croatia. Phenotypic testing was performed to identify ESBL or carbapenemase producers. Antibiotic susceptibility was tested by agardisk diffusion and broth microdilution methods. Genotypic antibiotic resistance profiles of isolates were analyzed by PCRand whole genome sequencing (WGS), while molecular epidemiology was studied by PFGE and MLST. Sixty-nine ESBL and 90 CRE isolates were isolated from hospital wastewater ; all were multidrug resistant and were mostlyidentified as Escherichia coli, Klebsiella pneumoniae, Citrobacter, and Enterobacter cloacae complex. Among ESBL isolates, blaCTX-M-15 was the most prevalent ESBL gene, while among CRE isolates, bla KPC-2 and bla NDM-1 were the mostfrequently detected carbapenemases, especially in Citrobacter spp. followed by bla OXA-48, especially in K. pneumoniae . Themost frequently detected sequence types included clinically relevant variants E. coli ST131 ( bla CTX-M-15/ bla TEM-116) andST541 ( bla KPC-2), K. pneumoniae ST101 ( bla OXA-48/ bla NDM-1) and E. cloacae ST277 ( bla KPC-2/ bla NDM-1). WGS alsoshowed that these isolates contained resistance genes to multiple antibiotic classes and a diverse plasmidome previouslyassociated with clinically relevant ESBL or carbapenemases. Finally, analysis of the genomic context of bla CTX-M, bla OXA-48, and bla KPC-2 showed that these genes were associated with mobile genetic elements, particularly transposons andinsertion sequences, indicating the potential for mobilisation. Conclusions Our results suggest that hospital wastewater is a reservoir for critical priority pathogens that exhibit resistance mechanismsto antibiotics critical to human health and provide a pathway for their dissemination in water systems. Effective treatment ofthis wastewater with advanced treatment methods such as UV and ozone treatment must therefore be ensured to reduce orstop the spread of resistant bacteria and ARGs in the natural environment.

Bacterial carbapenem resistance, especially among Enterobacteriaceae, is a major public health problem and wastewater treatment plants (WWTPs) can promote its spread in the environment. In this work we describe the occurrence of carbapenem-resistant coliforms and carbapenemase genes and the characteristics of carbapenemase- producing enterobacteria from municipal wastewater in Croatia. Conventional culturing was performed to monitor coliform bacteria and quantitative PCR to monitor 5 carbapenemase (CP) genes in the influent and effluent of 7 Croatian WWTPs in two seasons. Carbapenem-resistant enterobacteria were isolated on CHROMagar mSuperCARBA plates from the effluent of the largest WWTP (Zagreb) and identified by MALDI-TOFF. Antimicrobial susceptibility profiles and carbapenemase production were assessed phenotypically. Detection of CP genes was performed by PCR and sequencing of selected amplicons or by whole genome sequencing (WGS). Molecular epidemiology was assessed by PFGE and WGS. In general, levels of total and carbapenem- resistant coliforms were significantly reduced but not eliminated by conventional treatment in most WWTPs. CP genes, blaKPC-3, blaNDM and blaOXA-48- like, were sporadically detected, while blaIMP and blaVIM were frequently enriched during treatment and were influenced by specific features of the WWTPs. A total of 200 carbapenem-resistant enterobacteria were isolated from Zagreb WWTP effluent. Most isolates (144/200) produced carbapenemases and were confirmed as Enterobacteriaceae, with the predominant taxa identified as Klebsiella (33%), Enterobacter (24%), and Citrobacter (25%). All Klebsiella isolates were multidrug-resistant, of which 6% were pandrug-resistant. Most Klebsiella isolates were grouped into 8 pulsotypes ; among these, two were predominant and belonged to a novel Klebsiella lineages, ST1697 and ST3590, which may be a hybrid of the recombination of the Klebsiella quasipneumoniae and Klebsiella pneumoniae genomes. The predominant CP genes among these isolates were blaOXA-48, blaNDM-1 and blaKPC-2, whereas blaVIM-1 and blaIMP-13 were rarely detected. 40% of Klebsiella isolates were resistant to colistin, and a novel plasmid-encoded mcr-4.3 gene was detected for the first time in pandrug-resistant Klebsiella pneumoniae. Our results suggest that improvements in WWTP technologies are needed to minimize the risk of environmental contamination with key opportunistic enteric pathogens and CP genes and the resulting public health impact.

U stolici hospitaliziranog pacijenta s kliničkom slikom bacilarne dizenterije izolirana je Shigella sonnnei. Fenotipskim testom potvrđena je produkcija bet-laktamaze proširenog spektra. Nađeno je da izolat pripada sekvencijskom tipu 152 koji je dominantan u Europi i Sjevernoj americi i često se nalazi u MSM populaciji. Prema nešem saznanju to je prvo izviješće o ESBL u S. sonnei u Hrvatskoj.

BACKGROUND AND AIM The patient was a 30 year old male, who inhaled sewage during work due to the rupture of the sewage pipeline. He developed bacillary dysenthery with frequent stools mixed with blood and mucus and was hospitalized in Sestre Milosrdnice Hospital in Zagreb, Croatia. He was released after 1 day without antibiotic therapy. Shigella sonnei was isolated from the stool specimen. MATERIAL AND METHODS The antibiotic susceptibility testing to a wide range of antibiotics was performed by disk- diffusion and broth microdilution method. Double disk synergy test and combined disk test with clavulanic acid were done to determine the production of an extended-spectrum beta-lactamase (ESBL). The transferability of cefotaxime and ciprofloxacin resistance was done by broth mating method at 35°C employing E. coli J65 recipient strain resistant to sodium-azide. The transconjugants were selected on MacConkey agar containing either ampicillin (0.5 mg/L) or ciprofloxacin (0.5 mg/L) and sodium azide (100 mg PCR was applied to detect genes encoding ESBLs(blaSHV, blaTEM, blaCTX-M, blaOXA-9, blaOXA-1 and blaPER-1) and fluoroquinolone resistance (qnrA, qnrB, qnrS). The CTX-M β-lactamase cluster was detected with multiplex PCR including five primer pairs: CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25. The genetic context of blaCTX-M genes was determined by PCR mapping with forward primer for insertion sequences ISEcp1 and IS26 combined with primer MA-3 (universal reverse primer for blaCTX-M genes) Amplicons were visualised after electrophoresis in 1 % agarose by staining it with ethidium bromide. WGS was done using the IonTorrent PGM platform (Life Technologies, Carlsbad, USA) according to the manufacturer's instructions. Plasmids were extracted from ampicillin resistant donor strains and their respective transconjugants with Qiagen plasmid mini kit according to the manufacturer’s instructions (Hilden, Germany) PCR-based replicon typing (PBRT) was applied to determine the plasmid content of the tested strains RESULTS The strain exhibited resistance to amoxicillin, cephalexin, cefuroxime, azythromycin, tetracycline, cefotaxime, ceftriaxone, and ciprofloxacin but remained susceptible to ceftazidime, cefepime, amoxicillin/clavulanic acid, piperacillin/tazobactam, imipenem, meropenem and gentamicin. Cotrimoxazole and cefoxitin tested resistant and susceptibile in disk-difusion test with the inhibition zones of 6 and 25 mm, respectively. Phenotypic tests confirmed production of an ESBL. PCR identified blaCTX-M gene belonging to the cluster 1. WGS demonstrated a variety of aminoglycoside (aadA, aph(6), aph(3), β-lactam (blaCTX-M-27), macrolide (Erm(B, ) Mph(A)), sulphonamide (sul 1), trimethoprime (dfrA17, dfrA) and tetracycline (tet A). Four different plasmids were found: IncFIB, IncFII, IncI1 and IncCol. The isolate was found to belong to the widespread ST152 which is dominant in Europe and North America and usually associate with MSM population. CONCLUSIONS To our best knowledge, this is the first report of an ESBL in Shigella spp from Croatia. This allelic variant was recently described in S. sonnei in UK and Australia. Plasmid-mediated resistance determinants to fluoroquinolones were not found and thus it is very likely that ciprofloxacin resistance was associated with mutations of gyrA and parC genes. This report emphasizes the ability of S. sonnei belonging to the widespread ST 152 to accumulate various resistance determinants.

Svrha ovog završnog rada je dati osnovne informacije o električnim motorima koji su važan segment u funkcioniranju raznih postrojenja, industrija, strojeva, uređaja, ali i prikazati primjenu električnih motora na realnom primjeru, odnosno na primjeru automobila. Kako se u današnje vrijeme vodi briga o okolišu sve više raste potreba za vozilima koji u što manjoj mjeri zagađuju okoliš prilikom eksploatacije istih. U tu svrhu razvijaju se električni automobili čije je srce zapravo sam električni motor. U ovom radu poseban osvrt je dan na glavnu podjelu električnih motora, odnosno podjelu na motore istosmjerne struje i motore izmjenične struje te na njihovo upravljanje koje se odnosi na pokretanje elektromotora i podešavanje brzine vrtnje. Također, opisane su osnovne zakonitosti kojih je se važno pridržavati i koje je važno poznavati prilikom rada sa elektromotorima. Pri tome je važno i poznavanje osnovnih fizikalnih procesa, poznavanje elektrotehnike i strojarstva. Na kraju rada predočen je realan primjer primjene elektromotora u automobilima, točnije u novom automobilu „Nevera“. Taj automobil koristi elektromotore sa permanentnim magnetima čiji se magneti koriste za generiranje konstantnog magnetskog toka. Također, prikazane su i opisane određene performanse koje karakteriziraju taj automobil., The purpose of this final work is to provide basic informations about electric motors that are an important segment in the functioning of various plants, industries, machines, devices, but also to show the application of electric motors on a real example, or on the example of cars. As the environment is being taken care of nowadays, the need for vehicles that pollute the environment as little as possible during their operation is growing. To this end, electric cars are being developed whose heart is actually the electric motor itself. In this work, special attention is given to the main division of the electric motors, respectively the division into DC motors and AC motors and their control that is related to starting electric motors and adjusting the speed. Also, the basic laws that are important to follow and that are important to know when working with electric motors are described. Knowledge of basic physical processes, knowledge of electrical engineering and mechanical engineering are also important. At the end of the work, a real example of the application of electric motors in cars is presented, more precisely in the new car "Nevera". That car uses electric motors with permanent magnets whose magnets are used to generate a constant magnetic flux. Also, certain performances that characterize that car are shown and described.

Krajem 12/2019 u kineskoj provinciji Haubei, u nekoliko zdravstvenih ustanova u gradu Wuhanu dijagnosticirana je u više skupina bolesnika bilateralna pneumonija , svi oboljeli su bili epidemiološki povezani s tržnicom živih životinja u Wuhanu. U 01/2020 izoliran je uzročnik SARS-CoV-2, RNA virus iz porodice Coronavirinae, a bolest karakterizirana bilateralnom atipičnom pneumonijom koju uzrokuje je u MKB nomenklaturi registrirana kao COVID-19. Paralelno s kasnijim razvojem epidemije odnosno pandemije COVID-19 u svijetu raslo je i znanje o ovom oboljenju, tako da je bolest koja je u početku shvaćena prvenstveno kao respiratorna s vremenom postala multisistemska i imunološka. U skladu s navedenim i samim mehanizmom „atakiranja“ virusa na stanice domaćina putem ACE2 receptora razvidno je da je kardiovaskularni sustav uvelike zahvaćen virusnim odnosno imunološki posredovanim mnogobrojnim oštećenjima. Pokazalo se da su brojni bolesnici, naročito oni u visoko rizičnim skupinama (arterijska hipertenzija, šećerna bolest tip 2, pretilost, aterosklerotska bolest, kardiomiopatije) za razvoja teških oblika COVID-19 bolesti imali mnogobrojne kardiovaskularne komplikacije poput venskih odnosno arterijskih tromboembolija s posljedičnim infarktima u više organskih sustava Pored cirkulacijskih komplikacija zabilježene su i mnogobrojne aritmijske: od bradiaritmija kakav je totalni AV blok kao najgora do tahiaritmijskih poput ventrikularne tahikardije odnosno fibrilacije ventrikule kao glavnih uzroka iznenadne smrti u ovih bolesnika. Potrebno je naglasiti i razvoj srčane insuficijencije odnosno pogoršanje iste kod bolesnika s od ranije poznatim kardiomiopatijama uvjetovane SARS-CoV-2 virusnom infekcijom. Zaključno, možemo reći da su oštećenja unutar kardiovaskularnog sustava posredovana samim SARS-CoV-2 virusom i imunološkim odgovorom na njega mnogobrojna i može se slobodno reći ključna za razvoj ishoda ove bolesti jer i samo akutno pogoršanje respiratorne funkcije uslijed ARDSa ima podlogu u poremećaju plućne mikrocirkulacije zbog infekcije i imunološkog odgovora na istu.