Enterobacter spp. resistant to beta-lactam antibiotics from urban and hospital wastewater

The increasing prevalence of antibiotic–resistant bacteria pose a huge risk for public health. Enterobacter spp. is a common in the environment but it is also documented as a frequent cause of human disease. Enterobacter spp. resistant to cephalosporins and carbapenems is increasingly detected globally both in and out of hospital setting. This thesis examines the occurrence of Enterobacter spp. strains resistant to –lactam antibiotics especially in wastewaters but also in treated water discharged into the environment. The aim of this study was to characterize a set of 205 strains of Enterobacter spp. selected on medium supplemented with cefotaxime and meropenem. The water samples were collected from three regional cities in the Czech Republic and originated from hospital wastewater, inflow and outflow of municipal wastewater treatment plant (WWTP), from a river above and below the WWTP. The isolates were screened for presence of eight genes encoding production of broad–spectrum –lactamases (ESBL; CTX-M, GES) and carbapenemases (GES, IMI/NMC-A, IMP, KPC, NDM, OXA-48, VIM) by the polymerase chain reaction (PCR) method. The phenotype of isolates carrying the gene of interest was tested for –lactamase production by the Mastdisc test and susceptibility to 24 different antibiotics by microdilution method. The phylogenetic relatedness was determined using restriction digestion in combination with pulsed-field gel electrophoresis (PFGE). From total of 205 strains, 36 isolates carried the gene of interest for –lactamase production. These isolates originated mostly from hospital wastewater (n=31) of all sampled sites (A, B and C). Also one isolate was detected in the WWTP influent in site B and four isolates came from the WWTP effluent in sites A and B. The following genes of interest were detected among the isolates: blaGES (n=16), blaCTX-M (n=24), blaOXA-48 (n=1) and blaNMC-A (n=1) a combination of two genes were detected in one isolate. All characterized isolates (n=36) showed a multidrug–resistant profile. High resistance to aminoglycosides was reported (76,9 %) with the exception of amikacin (19,4 %). In the case of carbapenems, resistance to ertapenem was observed (77,8 %), while a majority of strains were susceptible to meropenem, where resistance was detected only in 13,9 % of cases. Different resistance profile has been reported to cephalosporin antibiotics. All tested isolates showed resistance to antibiotics cefazolin and cefuroxime (100 %), lower rates of resistance were reported to antibiotics cefoperazone (80,6 %), cefotaxime (77,8 %) and cefepime (69,4 %). The lowest resistance in this group of antibiotics was found to cefoperazone/sulbactam (33,3 %). All selected isolates showed resistance to tetracycline antibiotics. Serine carbapenemase production (50 %) was the most frequently detected by phenotypic tests which is produced certain variants of –lactamase GES. Comparison of macrorestrictions profiles showed a high diversity among isolates represented by 20 different clusters, the related isolates originated from the same location. Wastewaters play a key role in the spread of antibiotics resistance. In this thesis, Enterobacter spp. strains with clinically significant resistance to cephalosporins and carbapenems were detected. These strains were detected mainly in hospital wastewaters but also in WWTP effluent. The current WWTP processes are unable to eliminate all bacteria that subsequently enter the environment