Wastewater (WW) is considered a source of antibiotic-resistant bacteria with clinical relevance and may, thus, be important for their dissemination into the environment, especially in countries with poor WW treatment. To obtain an overview of the occurrence and characteristics of carbapenem-resistant Gram-negative bacteria (CR-GNB) in WW of Belgrade, we investigated samples from the four main sewer outlets prior to effluent into international rivers, the Sava and the Danube. Thirty-four CR-GNB isolates were selected for antimicrobial susceptibility testing (AST) and whole-genome sequencing (WGS). AST revealed that all isolates were multidrug-resistant. WGS showed that they belonged to eight different species and 25 different sequence types (STs), seven of which were new. ST101 K. pneumoniae (bla_CTX-M-15/bla_OXA-48) with novel plasmid p101_srb was the most frequent isolate, detected at nearly all the sampling sites. The most frequent resistance genes to aminoglycosides, quinolones, trimethroprim-sulfamethoxazole, tetracycline and fosfomycin were aac(6′)-Ib-cr (55.9%), oqxA (32.3%), dfrA14 (47.1%), sul1 (52.9%), tet(A) (23.5%) and fosA (50%), respectively. Acquired resistance to colistin via chromosomal-mediated mechanisms was detected in K. pneumoniae (mutations in mgrB and basRS) and P. aeruginosa (mutation in basRS), while a plasmid-mediated mechanism was confirmed in the E. cloacae complex (mcr-9.1 gene). The highest number of virulence genes (>300) was recorded in P. aeruginosa isolates. Further research is needed to systematically track the occurrence and distribution of these bacteria so as to mitigate their threat.

Background: The screening for intestinal carriage of vancomycin-resistantEnterococcusspp. (VRE) among high risk patients in the Balkan region and molecular epidemiology of VRE is insufficiently investigated, yet it could be of key importance in infection control. The aim of this study was to provide baseline data on VRE intestinal carriage among high-risk patients in Serbian university hospitals, to determine the phenotypic/genotypic profiles of the isolated VRE, to obtain knowledge of local resistance patterns and bridge the gaps in current VRE surveillance.Methods: The VRE reservoir was investigated using stool samples from 268 inpatients. Characterization of isolated VRE stains consisted of BD Phoenix system, genotypic identification, glycopeptide and quinupristin–dalfopristin (Q–D) resistance probing, virulence gene (esp,hyl,efaA,asa1,gelE,cpd) detection and MLVA. Biofilm formation was evaluated by the microtiter plate method.Results: VRE carriage prevalence among at-risk patients was 28.7%. All VRE strains werevanA positive multidrug-resistantEnterococcus faecium(VRfm), harboringermB-1 (38.9%),esp(84%),efaA (71.2%),hyl(54.5%),asa1 (23.4%),gelE andcpd(11.6%) each. Ability of biofilm production was detected in 20.8%. Genetic relatedness of the isolates revealed 13 clusters, heterogeneous picture and 25 unique MTs profiles.Conclusion: The obtained prevalence of VRE intestinal carriage among high-risk inpatients in Serbia is higher than the European average, with high percentage of multidrug resistance. The emergence of resistance to Q–D is of particular concern. Close monitoring of pattern of resistance and strict adherence to specific guidelines are urgently needed in Serbia.

The predictors of intestinal carriage of vancomycin-resistant Enterococcus spp. (VRE) among high-risk patients in the counties of the Southeast Europe Region are insufficiently investigated, yet they could be of key importance in infection control. The aim of the study was to identify risk factors associated with fecal VRE colonization among high-risk inpatients in university hospitals in Serbia. The study comprised 268 inpatients from three university hospitals. Data on patient demographics and clinical characteristics, length of hospital stay, therapy, and procedures were obtained from medical records. Chi-squared tests and univariate and multivariate logistic regressions were performed. Compared to the hemodialysis departments, stay in the geriatric departments, ICUs, and haemato-oncology departments increased the risk for VRE colonization 7.6, 5.4, and 5.5 times, respectively. Compared to inpatients who were hospitalized 48 h before stool sampling for VRE isolation, inpatients hospitalized 3–7, 8–15, and longer than 16 days before sampling had 5.0-, 4.7-, and 6.6-fold higher risk for VRE colonization, respectively. The use of cephalosporins and fluoroquinolones increased the risk for VRE colonization by 2.2 and 1.9 times, respectively. The age ≥ 65 years increased the risk for VRE colonization 2.3 times. In comparison to the University Clinical Centre of Serbia, the hospital stays at Zemun and Zvezdara University Medical Centres were identified as a protector factors. The obtained results could be valuable in predicting the fecal VRE colonization status at patient admission and consequent implementation of infection control measures targeting at-risk inpatients where VRE screening is not routinely performed.

Background/Objectives: Long-term data on the molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) from the Western Balkans remain limited. This study investigated temporal changes in the molecular population structure and antimicrobial resistance of MRSA in Serbia over more than four decades. Methods: A total of 395 non-duplicate MRSA isolates from four temporally distinct collections (1975–1985, 1997–2003, 2009–2010, and 2018–2019) were analyzed. Isolates were characterized by antimicrobial susceptibility testing, spa typing, SCCmec typing, and detection of Panton–Valentine leukocidin (PVL) genes. Clonal complexes and putative MLST associations were inferred from spa repeat patterns and previously established molecular epidemiological associations rather than determined by direct MLST or whole-genome sequencing. Results: Marked differences in the molecular composition of MRSA were observed across the four collection periods. The 1975–1985 collection was dominated by isolates assigned to CC8-MRSA-I and ST239-related CC8-MRSA-III backgrounds. By 1997–2003, the early CC8-MRSA-I background was no longer prominent, while ST239-related CC8-MRSA-III and South German-related CC5-MRSA-I predominated. In 2009–2010, CC5-MRSA-I was the most frequently identified background, while ST239-related MRSA remained represented, and SCCmec IV/V-associated backgrounds were increasingly detected. By 2018–2019, greater molecular heterogeneity was observed, including a higher representation of CC45-MRSA-IV, changes in the distribution of CC5 backgrounds compared with the previously dominant CC5-t041-SCCmec I profile, continued detection of ST239-related CC8-MRSA-III, and an increased representation of ST152/CC152-associated isolates. PVL-positive isolates were absent from the first two collections, detected in 2009–2010 (4.0%), and more frequent in 2018–2019 (17.0%), predominantly within ST152/CC152- and CC1-associated backgrounds. Ciprofloxacin resistance increased from 0% in 1975–1985 to 84.0% in 2018–2019, while aminoglycoside resistance also increased markedly. Resistance to erythromycin and clindamycin remained consistently high, whereas no resistance to vancomycin, teicoplanin, or linezolid was detected among the investigated isolates. Conclusions: Over four decades, the investigated MRSA collections from Serbia showed substantial changes in molecular population structure, including the repeated detection of ST239-related MRSA, shifts in the predominant hospital-associated backgrounds, and greater molecular diversity in the later collections, with increased representation of SCCmec IV/V-associated and PVL-positive isolates. Historical population movements and healthcare connections within the former Yugoslavia may have contributed to the circulation of internationally recognized MRSA lineages in Serbia, although their role could not be directly established. Although the sampling design and typing resolution do not permit definitive conclusions regarding continuous clonal persistence or direct lineage replacement, these findings provide a valuable long-term perspective on MRSA molecular epidemiology in Southeastern Europe and highlight the importance of historical strain collections for investigating changes in antimicrobial-resistant bacterial populations.