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During November to December 2020, a high rate of COVID-19-associated pneumonia with bacterial superinfections due to multidrug-resistant (MDR) pathogens was recorded in a COVID-19 hospital in Zagreb. This study analyzed the causative agents of bacterial superinfections among patients with serious forms of COVID-19. In total, 118 patients were hospitalized in the intensive care unit (ICU) of the COVID-19 hospital. Forty-six out of 118 patients (39%) developed serious bacterial infection (VAP or BSI or both) during their stay in ICU. The total mortality rate was 83/118 (70%). The mortality rate due to bacterial infection or a combination of ARDS with bacterial superinfection was 33% (40/118). Six patients had MDR organisms and 34 had XDR (extensively drug-resistant). The dominant species was Acinetobacter baumannii with all isolates (34) being carbapenem-resistant (CRAB) and positive for carbapenem-hydrolyzing oxacillinases (CHDL). One Escherichia coli causing pneumonia harboured the blaCTX-M-15 gene. It appears that the dominant resistance determinants of causative agents depend on the local epidemiology in the particular COVID center. Acinetobacter baumannii seems to easily spread in overcrowded ICUs. Croatia belongs to the 15 countries in the world with the highest mortality rate among COVID-19 patients, which could be in part attributable to the high prevalence of bacterial infections in local ICUs. [ABSTRACT FROM AUTHOR]

Background: Recently, a dramatic increase of Klebsiella pneumoniae positive for OXA-48 β-lactamases was observed first in the hospital setting and later in the long-term care facilities (LTCFs) and community in the Zagreb County, particularly, in urinary isolates. The aim of the study was to analyse the epidemiology and the mechanisms of antibiotic resistance of OXA-48 carbapenemase producing K. pneumoniae strains isolated from urine of non-hospitalized elderly patients. Results: The isolates were classified into two groups: one originated from the LTCFs and the other from the community. Extended-spectrum β-lactamases (ESBLs) were detected by double disk-synergy (DDST) and combined disk tests in 55% of the isolates (51/92). The ESBL-positive isolates exhibited resistance to expanded-spectrum cephalosporins (ESC) and in majority of cases to gentamicin. LTCFs isolates showed a significantly lower rate of additional ESBLs and consequential resistance to ESC and a lower gentamicin resistance rate compared to the community isolates, similarly to hospital isolates in Zagreb, pointing out to the possible transmission from hospitals.ESBL production was associated with group 1 of CTX-M or SHV-12 β-lactamases. Ertapenem resistance was transferable from only 12 isolates. blaOXA-48 genes were carried by IncL plasmid in 42 isolates. In addition IncFII and IncFIB were identified in 18 and 2 isolates, respectively. Two new sequence types were reported: ST4870 and ST4781. Conclusions: This study showed eruptive and extensive diffusion of OXA-48 carbapenemase to LTCFs and community population in Zagreb County, particularly affecting patients with UTIs and urinary catheters. On the basis of susceptibility testing, β-lactamase production, conjugation experiments, MLST and plasmid characterization it can be concluded that there was horizontal gene transfer between unrelated isolates, responsible for epidemic spread of OXA-48 carbapenemase in the LTCFs and the community The rapid spread of OXA-48 producing K. pneumoniae points out to the shortcomings in the infection control measures. [ABSTRACT FROM AUTHOR]

[Display omitted] • 148 carbapenem-resistant Enterobacterales were isolated from treated wastewater. • bla NDM-1 , bla OXA-48 and/or bla KPC-2 were detected in most isolates. • Most identified sequence types (n = 14) were associated with human-associated clones. • Possible patogenicity was detected in all sequenced Klebsiella isolates. • mcr-4.3 was identified for the first time in K. pneumoniae and on plasmid IncHI1B. Among the most problematic bacteria with clinical relevance are the carbapenem-resistant Enterobacterales (CRE), as there are very limited options for their treatment. Treated wastewater can be a route for the release of these bacteria into the environment and the population. The aim of this study was to isolate CRE from treated wastewater from the Zagreb wastewater treatment plant and to determine their phenotypic and genomic characteristics. A total of 200 suspected CRE were isolated, 148 of which were confirmed as Enterobacterales by MALDI-TOF MS. The predominant species was Klebsiella spp. (n = 47), followed by Citrobacter spp. (n = 40) and Enterobacter cloacae complex (cplx.) (n = 35). All 148 isolates were carbapenemase producers with a multidrug-resistant phenotype. Using multi-locus sequence typing and whole-genome sequencing (WGS), 18 different sequence types were identified among these isolates, 14 of which were associated with human-associated clones. The virulence gene analysis of the sequenced Klebsiella isolates (n = 7) revealed their potential pathogenicity. PCR and WGS showed that the most frequent carbapenemase genes in K. pneumoniae were bla OXA-48 and bla NDM-1 , which frequently occurred together, while bla KPC-2 together with bla NDM-1 was mainly detected in K. oxytoca , E. cloacae cplx. and Citrobacter spp. Colistin resistance was observed in 40% of Klebsiella and 57% of Enterobacter isolates. Underlying mechanisms identified by WGS include known and potentially novel intrinsic mechanisms (point mutations in the pmrA/B , phoP/Q , mgrB and crrB genes) and acquired mechanisms (mcr-4.3 gene). The mcr-4.3 gene was identified for the first time in K. pneumoniae and is probably located on the conjugative IncHI1B plasmid. In addition, WGS analysis of 13 isolates revealed various virulence genes and resistance genes to other clinically relevant antibiotics as well as different plasmids possibly associated with carbapenemase genes. Our study demonstrates the important role that treated municipal wastewater plays in harboring and spreading enterobacterial pathogens that are resistant to last-resort antibiotics. [ABSTRACT FROM AUTHOR]