UNLABELLED: IncF plasmids are mobile genetic elements found in bacteria from the Enterobacteriaceae family and often carry critical antibiotic and virulence gene cargo. The classification of IncF plasmids using the plasmid Multi-Locus Sequence Typing (pMLST) tool from the Center for Genomic Epidemiology (CGE; https://www.genomicepidemiology.org/) compares the sequences of IncF alleles against a database to create a plasmid sequence type (ST). Accurate identification of plasmid STs is useful as it enables an assessment of IncF plasmid lineages associated with pandemic enterobacterial STs. Our initial observations showed discrepancies in IncF allele variants reported by pMLST in a collection of 898 Escherichia coli ST131 genomes. To evaluate the limitations of the pMLST tool, we interrogated an in-house and public repository of 70,324 E. coli genomes of various STs and other Enterobacteriaceae genomes (n = 1247). All short-read assemblies and representatives selected for long-read sequencing were used to assess pMLST allele variants and to compare the output of pMLST tool versions. When multiple allele variants occurred in a single bacterial genome, the Python and web versions of the tool randomly selected one allele to report, leading to limited and inaccurate ST identification. Discrepancies were detected in 5,804 of 72,469 genomes (8.01%). Long-read sequencing of 27 genomes confirmed multiple IncF allele variants on one plasmid or two separate IncF plasmids in a single bacterial cell. The pMLST tool was unable to accurately distinguish allele variants and their location on replicons using short-read genome assemblies, or long-read genome assemblies if the same allele variant was present more than once. IMPORTANCE: Plasmid sequence type is crucial for describing IncF plasmids due to their capacity to carry important antibiotic and virulence gene cargo and consequently due to their association with disease-causing enterobacterial lineages exhibiting resistance to clinically relevant antibiotics in humans and food-producing animals. As a result, precise reporting of IncF allele variants in IncF plasmids is necessary. Comparison of the FAB formulae generated by the pMLST tool with annotated long-read genome assemblies identified inconsistencies, including examples where multiple IncF allele variants were present on the same plasmid but missing in the FAB formula, or in cases where two IncF plasmids were detected in one bacterial cell, and the pMLST output provided information only about one plasmid. Such inconsistencies may cloud interpretation of IncF plasmid replicon type in specific bacterial lineages or inaccurate assumptions of host strain clonality.

• Klíčová slova
• Enterobacteriaceae, IncF, antibiotic resistance, pMLST, plasmids,
• MeSH
• alely MeSH
• Enterobacteriaceae genetika   MeSH
• Escherichia coli genetika   MeSH
• genetická variace MeSH
• genom bakteriální * MeSH
• lidé MeSH
• multilokusová sekvenční typizace * metody   MeSH
• plazmidy * genetika   MeSH
• počítačová simulace MeSH
• replikon * genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Multidrug-resistant (MDR) bacteria pose a significant challenge to the treatment of infectious diseases. Of particular concern are members of the Klebsiella pneumoniae species complex (KpSC), which are frequently associated with hospital-acquired infections and have the potential to spread outside hospitals via wastewaters. In this study, we aimed to investigate the occurrence and phylogenetic relatedness of MDR KpSC from patients with urinary tract infections (UTIs), hospital sewage, municipal wastewater treatment plants (mWWTPs) and surface waters and to evaluate the clinical relevance of the KpSC subspecies. METHODS: A total of 372 KpSC isolates resistant to third-generation cephalosporins and/or meropenem were collected from patients (n = 130), hospital sewage (n = 95), inflow (n = 54) and outflow from the mWWTPs (n = 63), river upstream (n = 13) and downstream mWWTPs (n = 17) from three cities in the Czech Republic. The isolates were characterized by antimicrobial susceptibility testing and whole-genome sequencing (Illumina). The presence of antibiotic resistance genes, plasmid replicons and virulence-associated factors was determined. A phylogenetic tree and single nucleotide polymorphism matrix were created to reveal the relatedness between isolates. RESULTS: The presence of MDR KpSC isolates (95%) was identified in all water sources and locations. Most isolates (99.7%) produced extended-spectrum beta-lactamases encoded by blaCTX-M-15. Resistance to carbapenems (5%) was observed mostly in wastewaters, but carbapenemase genes, such as blaGES-51 (n = 10), blaOXA-48 (n = 4), blaNDM-1 (n = 4) and blaKPC-3 (n = 1), were found in isolates from all tested locations and different sources except rivers. Among the 73 different sequence types (STs), phylogenetically related isolates were observed only among the ST307 lineage. Phylogenetic analysis revealed the transmission of this lineage from patients to the mWWTP and from the mWWTP to the adjacent river and the presence of the ST307 clone in the mWWTP over eight months. We confirmed the frequent abundance of K. pneumoniae (K. pneumoniae sensu stricto and K. pneumoniae subsp. ozaenae) in patients suffering from UTIs. K. variicola isolates formed only a minor proportion of UTIs, and K. quasipneumoniae was not found among UTIs isolates; however, these subspecies were frequently observed in hospital sewage communities during the first sampling period. CONCLUSION: This study provides evidence of the transmission and persistence of the ST307 lineage from UTIs isolates via mWWTPs to surface waters. Isolates from UTIs consisted mostly of K. pneumoniae. Other isolates of KpSC were observed in hospital wastewaters, which implies the impact of sources other than UTIs. This study highlights the influence of urban wastewaters on the spread of MDR KpSC to receiving environments.

• Klíčová slova
• Klebsiella spp. subspecies, bla CTX−M−15, Urinary tract infections, Wastewater treatment plants,
• MeSH
• antibakteriální látky * farmakologie   MeSH
• bakteriální proteiny genetika   MeSH
• beta-laktamasy * genetika   MeSH
• fylogeneze * MeSH
• infekce bakteriemi rodu Klebsiella * mikrobiologie   epidemiologie   MeSH
• infekce močového ústrojí mikrobiologie   epidemiologie   MeSH
• infekce spojené se zdravotní péčí mikrobiologie   epidemiologie   MeSH
• Klebsiella pneumoniae * účinky léků   genetika   izolace a purifikace   klasifikace   MeSH
• lidé MeSH
• mikrobiální testy citlivosti * MeSH
• mnohočetná bakteriální léková rezistence * MeSH
• nemocnice * MeSH
• odpadní voda * mikrobiologie   MeSH
• odpadní vody mikrobiologie   MeSH
• sekvenování celého genomu MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika epidemiologie   MeSH
• Názvy látek
• antibakteriální látky * MeSH
• bakteriální proteiny MeSH
• beta-laktamasy * MeSH
• odpadní voda * MeSH
• odpadní vody MeSH

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.

• Klíčová slova
• mcr, Antimicrobial resistance, Carbapenemase, Environment,
• MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální léková rezistence genetika   MeSH
• bakteriální proteiny genetika   MeSH
• beta-laktamasy * genetika   MeSH
• Enterobacter * genetika   účinky léků   izolace a purifikace   MeSH
• fylogeneze MeSH
• kolistin * farmakologie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• odpadní voda * mikrobiologie   MeSH
• plazmidy genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antibakteriální látky MeSH
• bakteriální proteiny MeSH
• beta-laktamasy * MeSH
• carbapenemase MeSH Prohlížeč
• kolistin * MeSH
• odpadní voda * MeSH
• VIM-1 metallo-beta-lactamase MeSH Prohlížeč