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Escherichia coli i Klebsiella pneumoniae su među najznačajnijim uzročnicima infekcija kod ljudi. Problem predstavljaju multirezistentni sojevi koji se javljaju ne samo u bolničkom nego i u vanbolničkom okruženju. Karbapenemi, beta-laktami sa najširim spektrom delovanja, spadaju u lekove poslednje linije odbrane. Rezistencija na karbapeneme među enterobakterijama je u porastu širom sveta. Može nastati usled prisustva karbapenemaza, enzima koji degradiraju karbapeneme, ili usled hiperprodukcije AmpC cefalosporinaza ili beta-laktamaza proširenog spektra uz gubitak porina. Geni koji kodiraju karbapenemaze se nalaze na mobilnim genetičkim elementima koji im omogućavaju brz prenos. Najčešće karbapenemaze su KPC, NDM, VIM, IMP i OXA-48 enzimi. Detekcija sojeva koji produkuju karbapenemaze nije moguća samo na osnovu profila rezistencije izolata, s obzirom da minimalne inhibitorne koncentracije karbapenema mogu biti u referentnom opsegu. Svaki izolat sa smanjenom osetljivošću na karbapeneme bi trebalo ispitati kako bi se sprečilo njihovo širenje. Detekcija karbapenemaza može da se zasniva na fenotipskim i genotipskim metodama. Ciljevi istraživanja su bili da se utvrdi postojanje rezistencije na karbapeneme kod multirezistentnih izolata Escherichia coli i Klebsiella pneumoniae iz kliničkih uzoraka, da se dokaže produkcija karbapenemaza korišćenjem fenotipskih i genotipskih testova, kao i da se analizira osetljivost izolata Escherichia coli i Klebsiella pneumoniae sa molekularno dokazanim karbapenemazama. Istraživanje je sprovedeno kao prospektivna studija u periodu 01.11.2013. do 01.11.2014. godine u Centru za mikrobiologiju Instituta za javno zdravlje Vojvodine u Novom Sadu. U istraživanje je bilo uključeno 300 multirezistentnih izolata Escherichia coli i Klebsiella pneumoniae konsekutivno izolovanih iz kliničkih uzoraka (krv, punktat, sekret iz donjeg respiratornog trakta, urin i sekret rana) hospitalizovanih pacijenata. Identifikacija do nivoa vrste je vršena klasičnim bakteriološkim metodama. Za ispitivanje osetljivosti korišćeni su disk difuziona metoda i gradijent testovi. Vrednosti minimalnih inhibitornih koncentracija su ispitane automatizovanim Vitek 2 sistemom (BioMérieux, Francuska), a interpretacija izvršena u skladu sa preporukama CLSI (Clinical Laboratory Standards Institute). Za fenotipsko testiranje prisustva betalaktamaza proširenog spektra korišćen je kombinovani disk test. Za fenotipsko testiranje prisustva karbapenemaza kod sojeva rezistentnih na karbapeneme korišćen je kombinovani disk test i test sinergizma sa dva diska. Detekcija gena za beta-laktamaze blaCTXM, gena za karbapenemaze blaKPC, blaVIM, blaNDM, blaIMP i blaOXA-48-like izvršena je metodom lančane reakcije polimeraze. Genotipizacija odabranih izolata Klebsiella pneumoniae izvršena pomoću repetitivne lančane reakcije polimeraze korišćenjem DiversiLab sistema (BioMérieux, Francuska). Od 300 multirezistetntnih izolata, bilo je 242 (80,7%) Klebsiella pneumoniae i 58 (19,3%) Escherichia coli izolovanih iz kliničkih uzoraka. Smanjenu osetljivost na bar jedan karbapenem (imipenem, meropenem, ertapenem) pokazalo je 179 (59,7%) izolata. Fenotipski test za dokazivanje produkcije betalaktamaza proširenog spektra bio je pozitivan kod 87/171 (50,9%) izolata. Gen blaCTX-M je dokazan kod 111/121 (91,7%) izolata. Fenotipski test za dokazivanje karbapenemaza bio je pozitivan kod 65/179 (36,3%) izolata, kod 63 (96,9%) je ukazivao na prisustvo metalo-beta laktamaza, a kod 2 (3,1%) na prisustvo karbapenemaza iz grupe A. Senzitivnost fenotipskog testa za dokazivanje karbapenemaza klase A i B iznosila je 100,0%, specifičnost 96,6%, a ukupna tačnost 97,6%. Karbapenemaze su nađene kod 79/179 (44,1%) izolata rezistentnih na karbapeneme. Gen blaNDM nađen je kod 58 (32,4%) izolata, blaOXA- 48-like kod 11 (6,1%), a blaKPC kod 2 (1,1%) izolata. Geni blaVIM i blaIMP nisu detektovani. Kod 8 (4,5%) izolata nađena su 2 gena koja kodiraju karbapenemaze, blaNDM i blaOXA-48-like. Određivanjem osetljivosti disk difuzionom metodom i automatizovanim Vitek 2 sistemom, izolati koji produkuju karbapenemaze pokazivali su smanjenu osetljivost na sve testirane beta-laktame i gentamicin, odnosno tobramicin. Visok procenat rezistenicije izolati su pokazali u odnosu na ciprofloksacin, levofloksacin i trimetoprim/sulfametoksazol. Najefikasniji antibiotski lekovi su bili amikacin, tigeciklin, fosfomicin i kolistin. Poređenjem minimalnih inhibitornih koncentracija izolata koji produkuju i izolata koji ne produkuju karbapenemaze utvrđena je statistički značajna razlika za meropenem, imipenem, ertapenem, amikacin, gentamicin. Genotipizacijom odabranih izolata Klebsiella pneumoniae korišćenjem DiversiLab sistema klonalno širenje je dokazano među izolatima koji produkuju NDM i OXA-48-like karbapenemaze u okviru iste zdravstvene institucije, ali i među različitim zdravstvenim ustanovama. Među izolatima rezistentnim na karbapeneme Klebsiella pneumoniae se češće izoluje od Escherichia coli. Kod izolata koji su pokazali smanjenu osetljivost prema bar jednom karbapenemu, karbapenemaze su detektovane u manje od polovine izolata. Kod ostalih izolata dokazane su beta-laktamaze proširenog spektra koje uz gubitak porina mogu uzrokovati rezistenciju na karbapeneme. Kod izolata Klebsiella pneumoniae sa dokazanim genima koji kodiraju karbapenemaze detektovani su pojedinačni blaKPC, blaNDM i blaOXA-48-like geni, kao i kombinacija gena blaNDM i blaOXA-48-like. Kod izolata Escherichia coli nađeni su samo blaNDM geni. Najefikasniji antibiotski lekovi za izolate koji produkuju karbapenemaze su amikacin, tigeciklin, fosfomicin i kolistin. Izolati sa dokazanim karbapenemazama pokazuju rezistenciju na veći broj antibiotika u odnosu na izolate koji ne produkuju karbapenemaze. Dokazano je klonalno širenje izolata Klebsiella pneumoniae koji produkuju karbapenemaze. Testove za fenotipsku detekciju karbapenemaza bi trebalo koristiti i u rutinskim mikrobiološkim laboratorijama u skladu sa EUCAST (European Committee on Antimicrobial Susceptibility Testing) preporukama, a konačnu potvrdu treba izvršiti molekularnim metodama u referentnoj laboratoriji., Escherichia coli and Klebsiella pneumoniae are among the most common human pathogens. Multiresistant strains are emerging not only in hospital settings, but also in the community representing a major concern. Carbapenems, beta-lactams with the broadest spectrum of activity are considered to be antibiotics of last resort. Resistance to carbapenems among enterobacteria is spreading worldwide. It is mainly caused by carbapenemases, enzymes capable of degrading carbapenems or by hyperproduction/overexpression of AmpC betalactamases or extended spectrum betalactamases with porin loss. Carbapenemaseencoding genes are usually located on mobile genetic elements providing their fast transfer. The most common carbapenemases are KPC, NDM, VIM, IMP and OXA-48. The detection of carbapenemase-producer cannot rely only on the resistance profile as their minimal inhibitory concentration values may sometimes lay within the susceptibility range. Therefore, every multidrug-resistant isolates with lower susceptibility to carbapenems should be tested for the presence of carbapenemases in order to prevent further spreading. The detection of carbapenemases is based on phenotypic and genotypic methods. The aims of the study were to determine the occurrence of carbapenem resistance in multidrug-resistant Escherichia coli and Klebsiella pneumoniae isolated from clinical samples, to detect carbapenemase production using both phenotypic and genotypic methods and to analyze the susceptibility of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae. The study was conducted from 1st November 2013 to 1st November 2014 at the Center for Microbiology in the Institute for Public Health of Vojvodina, Novi Sad, Serbia. The study included 300 nonrepetitive multidrug-resistant strains of Escherichia coli and Klebsiella pneumoniae isolated from clinical specimen (blood, aspirates, lower respiratory tract secretions, urine and wound secretion) of hospitalized patients. Identification of isolated strains was done using conventional bacteriological methods. Antimicrobial susceptibility was tested using the disk diffusion method and MIC test strips. Minimal inhibitory concentrations were determined using Vitek 2 Compact automated system (BioMérieux, France), interpreted according to the CLSI (Clinical and Laboratory Standards Institute) recommendations. Phenotypic testing of extended-spectrum beta-lactamases production was done using combined disk test. Phenotypic testing of carbapenemase production was done by combined disk test and double-disk synergy test. Detection of blaCTX-M, gene encoding extended-spectrum beta-lactamases and blaKPC, blaVIM, blaNDM, blaIMP i blaOXA-48-like, genes encoding carbapenemases was done using PCR. Genotyping of selected Klebsiella pneumoniae isolates was done by repPCR using DiversiLab system (BioMérieux, France). From the total of 300 multiresistant isolates, 242 (80.7%) were Klebsiella pneumoniae and 58 (19.3%) were Escherichia coli obtained from clinical samples. Reduced susceptibility to at least one carbapenem (imipenem, meropenem, ertapenem) was found in 179 (59.7%) isolates. Phenotypic test for extended-spectrum betalactamases production was positive in 87/171 (50.9%) isolates. A total of 111/121 (91.7%) isolates harbored blaCTX-M. Phenotypic test for carbapenemase production was positive in 65/179 (36.3%) isolates, 63 (96.9%) indicating the presence of metallo-beta-lactamases and 2 (3.1%) indicating the presence of class A carbapenemases. Sensitivity of the phenotypic test for carbapenemase production of class A and B was 100.0%, specificity 96.6% and overall accuracy 97.6%. Carbapenemases were detected in 79/179 (44.1%) carbapenemresistant isolates. Gene blaNDM was found in 58 (32.4%) isolates, blaOXA-48-like in 11 (6.1%) and blaKPC in 2 (1.1%) isolates. Genes blaVIM and blaIMP were not detected. In 8 (4.5%) isolates 2 genes encoding carbapenemases were found, blaNDM and blaOXA-48-like. Using both disk diffusion method and Vitek 2 automated system for antimicrobial susceptibility testing carbapenemase-producing isolates were resistant to all beta-lactams and also to gentamicin and tobramicin respectively. Resistance rates were high for ciprofloxacin, levofloxacin and cotrimoxazole. Good activity maintained for amikacin, tigecycline, fosfomycin and colistin. Comparing minimal inhibitory concentrations of carbapenemaseproducing isolates and non-carbapenemase producers, significant difference was found for meropenem, imipenem, ertapenem, amikacin and gentamicin. Genotyping of selected Klebsiella pneumoniae isolates using DiversiLab system, revealed the clonal spread of NDM- and OXA-48-like-producers not only within one healthcare-setting, but also between different healthcare centers. Among carbapenem-resistant isolates, Klebsiella pneumoniae was found more often than Escherichia coli. Carbapenemases were detected in less than 50% of isolates resistant to at least one carbapenem. In other carbapenem resistant isolates extended-spectrum betalactamases were confirmed most likely causing carbapenem-resistance with porin deficiency or porin loss. Among carbapenemase-producing Klebsiella pneumoniae blaKPC, blaNDM and blaOXA-48-like genes were detected, as well as combination of 2 genes blaNDM and blaOXA-48-like. In carbapenemase-producing Escherichia coli only blaNDM was found. The most efficient antimicrobial drugs among tested carbapenemase-producing isolates were amikacin, tigecycline, fosfomycin and colistin. Carbapenemase-producing isolates were resistant to more antimicrobial agents compared to non-carbapenemase producers. Clonal dissemination of carbapenemase-producing Klebsiella pneumoniae was confirmed. Phenotypic detection of carbapenemase production should be done in routine microbiology laboratories according to EUCAST (European Committee on Antimicrobial Susceptibility Testing) recommendations. Final confirmation should be done by molecular methods in the reference laboratory.

Achieving and maintaining good health and prosperity requires a clear and harmonized environment. Studying the environment to protect human health aims to determine the type of pollutants and to monitor their impact on humans. Pollutants include the chemical, physical and microbiological pollutants. It is believed that the best way of studying the interaction between the population and the environment is the method that allows selection of representative, easily measurable and routinely controlled environmental indicators and health status. The World Health Organization recommends "DPSEEA" (Driving Force, Pressure, State, Exposure, Effect and Action) system. "DPSEEA" describes a closed circuit system of interdependent factors of the environment and human health which can be defined as a cause _ effect model. Application of "DPSEEA" system to assess the influence factors of the environment on human health is defined by studying the air quality of the environment, water and sanitation, noise and health, housing, traffic accidents, chemical accidents, and radiation. The use of complex indicators by "DPSEEA" system in our country so far has been used to assess the environmental situation, but not for systematic assessment of the impact factors of the environment on human health. AIM: Aims of the research are based on determining the type of microbiological, physical and chemical hazards in the environment of Novi Sad, exposure of the population and assess the impact of identified hazards to human health, as well as determining the significance of the application of environmental factors as indicators of impacts on human health. METHODOLOGY: Identifying hazards present in the environment of the City of Novi Sad (air quality, drinking water quality, noise) in 2006 were carried out based on the statutory methodology for sampling, microbiological, physical and chemical analysis and preparation of expert opinions on the hazards present in the environment. There were sampled, analyzed and professionally studied 9936 air samples, 6474 samples of drinking water and 648 individual measurements of daily levels of noise. Determination of population-level exposure to environmental noise survey was conducted by applied poll concerning subjective population experience of noise and subjective assessment of noise impact on human health, based on 1079 valid completed questionnaires. Assessment of exposure to microbial, physical and chemical hazards from the environment and determine the impact of identified hazards from the environment on human health is determined in accordance with the "DPSEEA" methodology by using the 15 indicators. To assess the impact of air, water and noise on human health following indicators have been considered: Air_Exl, Air_El, Air_E2, Air_E3, WatSan_S2,WatSan_S3, WatSan_Exl, WatSan_Ex2, WatSan_Ex3, WatSan_Ex4, WatSan_El,WatSan_E2, WatSan_E3, Noise_El and Noise_E2. The questionnaire of the population of the subjective experience of noise and subjective assessment of noise impact on human health was conducted with the help of specially composed questionnaire, based on national surveys conducted. RESULTS: Out of the 15 indicators of "DPSEEA" system in our research 87 percents is considered accessible, 67 percents quality and 73 percents usable. Monitoring of air quality in the City of Novi Sad in 2006 indicates that average annual concentrations of sulfur dioxide (0.05). Reducing the average population exposure to suspended particles in 10 µg/m3 in the City of NoviSad, reducing the expected total number of deaths from 131 to 115, the expected number of deaths from cardiopulmonary disease (MKB 10:JOO-99 and MKB 1 0:120-25) in people aged above 30 with 42 to 37 and expected number of deaths from respiratory diseases (MKB 1 0:JOO-99) of children under five years from 0.036 to 0.03, statistically significant Cp>0.05). Increase the total suspended particles (TSP) In air of the City of Novi Sad for 10 µg/m causes the Increase rate of total mortality in 0.047 without statistical significance (p=0.29299) and increased rates of mortality from cardiopulmonary disease (MKB 10:JOO-99 and MKB 10:120-25) of people aged above 30 to 0.016, with no statistical significance . The decrease of total suspended particles (TSP) in air of the City of Novi Sad for 10 µg/m3 induced reduction of total mortality rate of 0.051 with no statistical significance (p=0.26355) and the reduction of mortality from cardiopulmonary disease (MKB 10:JOO-99 and MKB 1 0:120-25) of people aged above 30 years to 0.016 with no statistical significance (p=OA04055). By monitoring the presence of microbiological and physical-chemical hazards in treated drinking water from the water plant and water system of central water supply infrastructure and sewerage in the City of Novi Sad in 2006 showed the absence of microbiological and physical-chemical hazards in 94.50 percents (p