BACKGROUND: Wastewaters are considered as important players in the spread of antimicrobial resistance, thus affecting the health of humans and animals. Here, we focused on wastewaters as a possible source of carbapenemase-producing Enterobacterales for the environment. METHODS: A total of 180 presumptive coliforms from hospital and municipal wastewaters, and a river in the Czech Republic were obtained by selective cultivation on meropenem-supplemented media and tested for presence of carbapenemase-encoding genes by PCR. Strains carrying genes of interest were characterized by testing antimicrobial susceptibility, carbapenemase production and combination of short- and long- read whole-genome sequencing. The phylogenetic tree including publicly available genomes of Enterobacter asburiae was conducted using Prokka, Roary and RAxML. RESULTS: Three VIM-producing Enterobacter asburiae isolates, members of the Enterobacter cloacae complex, were detected from hospital and municipal wastewaters, and the river. The blaVIM-1 gene was located within a class 1 integron that was carried by different F-type plasmids and one non-typeable plasmid. Furthermore, one of the isolates carried plasmid-borne colistin-resistance gene mcr-10, while in another isolate chromosomally located mcr-9 without colistin resistance phenotype was detected. In addition, the analysis of 685 publicly available E. asburiae genomes showed they frequently carry carbapenemase genes, highlighting the importance of this species in the emergence of resistance to last-line antibiotics. CONCLUSION: Our findings pointed out the important contribution of hospital and community wastewaters in transmission of multi-drug resistant pathogens.

• Keywords
• mcr, Antimicrobial resistance, Carbapenemase, Environment,
• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Drug Resistance, Bacterial genetics   MeSH
• Bacterial Proteins genetics   MeSH
• beta-Lactamases * genetics   MeSH
• Enterobacter * genetics   drug effects   isolation & purification   MeSH
• Phylogeny MeSH
• Colistin * pharmacology   MeSH
• Humans MeSH
• Microbial Sensitivity Tests MeSH
• Wastewater * microbiology   MeSH
• Plasmids genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Bacterial Proteins MeSH
• beta-Lactamases * MeSH
• carbapenemase MeSH Browser
• Colistin * MeSH
• Wastewater * MeSH
• VIM-1 metallo-beta-lactamase MeSH Browser

The resistance to carbapenems is usually mediated by enzymes hydrolyzing β-lactam ring. Recently, an alternative way of the modification of the antibiotic, a β-lactone formation by OXA-48-like enzymes, in some carbapenems was identified. We focused our study on a deep analysis of OXA-48-like-producing Enterobacterales, especially strains showing poor hydrolytic activity. In this study, well characterized 74 isolates of Enterobacterales resistant to carbapenems were used. Carbapenemase activity was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), liquid chromatography/mass spectrometry (LC-MS), Carba-NP test and modified Carbapenem Inactivation Method (mCIM). As meropenem-derived β-lactone possesses the same molecular weight as native meropenem (MW 383.46 g/mol), β-lactonization cannot be directly detected by MALDI-TOF MS. In the spectra, however, the peaks of m/z = 340.5 and 362.5 representing decarboxylated β-lactone and its sodium adduct were detected in 25 out of 35 OXA-48-like producers. In the rest 10 isolates, decarboxylated hydrolytic product (m/z = 358.5) and its sodium adduct (m/z = 380.5) have been detected. The peak of m/z = 362.5 was detected in 3 strains co-producing OXA-48-like and NDM-1 carbapenemases. The respective signal was identified in no strain producing class A or class B carbapenemase alone showing its specificity for OXA-48-like carbapenemases. Using LC-MS, we were able to identify meropenem-derived β-lactone directly according to the different retention time. All strains with a predominant β-lactone production showed negative results of Carba NP test. In this study, we have demonstrated that the strains producing OXA-48-like carbapenemases showing false-negative results using Carba NP test and MALDI-TOF MS preferentially produced meropenem-derived β-lactone. We also identified β-lactone-specific peak in MALDI-TOF MS spectra and demonstrated the ability of LC-MS to detect meropenem-derived β-lactone.

• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Bacterial Proteins * analysis   MeSH
• beta-Lactamases analysis   MeSH
• Enterobacteriaceae * MeSH
• Carbapenems pharmacology   MeSH
• Meropenem pharmacology   MeSH
• Microbial Sensitivity Tests MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Bacterial Proteins * MeSH
• beta-Lactamases MeSH
• carbapenemase MeSH Browser
• Carbapenems MeSH
• Meropenem MeSH