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

Wastewaters belong among the most important sources of environmental pollution, including antibiotic-resistant bacteria. The aim of the study was to evaluate treated wastewaters as a possible transmission pathway for bacterial colonisation of gulls occupying the receiving river. A collection of antibiotic-resistant Escherichia coli originating both from treated municipal wastewaters discharged to the river Svratka (Czech Republic) and nestlings of Black-headed Gull (Chroicocephalus ridibundus) living 35 km downstream of the outlet was obtained using selective cultivation. Isolates were further characterised by various phenotyping and genotyping methods. From a total of 670 E. coli isolates (450 from effluents, 220 from gulls), 86 isolates (41 from effluents, 45 from gulls) showed identical antibiotic resistance phenotype and genotype and were further analysed for clonal relatedness using pulsed-field gel electrophoresis (PFGE). Despite the overall high diversity of the isolates, 21 isolates from both sources showed similar PFGE profiles. Isolates belonging to epidemiologically important sequence types (ST131, 15 isolates; ST23, three isolates) were subjected to whole-genome sequencing. Subsequent phylogenetic analysis did not reveal any close clonal relationship between the isolates from the effluents and gulls' nestlings with the closest strains showing 90 SNPs difference. Although our study did not provide direct evidence of transmission of antibiotic-resistant E. coli to wild gulls via treated wastewaters, we observed gull chicks as carriers of diverse multi-resistant E. coli, including high-risk clones, posing risk of further bacterial contamination of the surrounding environment.

• Klíčová slova
• Enterobacterales, Environment, Whole-genome sequencing, Wild birds,
• 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

Wild birds including raptors can act as vectors of clinically relevant bacteria with antibiotic resistance. The aim of this study was to investigate the occurrence of antibiotic-resistant Escherichia coli in black kites (Milvus migrans) inhabiting localities in proximity to human-influenced environments in southwestern Siberia and investigate their virulence and plasmid contents. A total of 51 E. coli isolates mostly with multidrug resistance (MDR) profiles were obtained from cloacal swabs of 35 (64%, n = 55) kites. Genomic analyses of 36 whole genome sequenced E. coli isolates showed: (i) high prevalence and diversity of their antibiotic resistance genes (ARGs) and common association with ESBL/AmpC production (27/36, 75%), (ii) carriage of mcr-1 for colistin resistance on IncI2 plasmids in kites residing in proximity of two large cities, (iii) frequent association with class one integrase (IntI1, 22/36, 61%), and (iv) presence of sequence types (STs) linked to avian-pathogenic (APEC) and extra-intestinal pathogenic E. coli (ExPEC). Notably, numerous isolates had significant virulence content. One E. coli with APEC-associated ST354 carried qnrE1 encoding fluoroquinolone resistance on IncHI2-ST3 plasmid, the first detection of such a gene in E. coli from wildlife. Our results implicate black kites in southwestern Siberia as reservoirs for antibiotic-resistant E. coli. It also highlights the existing link between proximity of wildlife to human activities and their carriage of MDR bacteria including pathogenic STs with significant and clinically relevant antibiotic resistance determinants. IMPORTANCE Migratory birds have the potential to acquire and disperse clinically relevant antibiotic-resistant bacteria (ARB) and their associated antibiotic resistance genes (ARGs) through vast geographical regions. The opportunistic feeding behavior associated with some raptors including black kites and the growing anthropogenic influence on their natural habitats increase the transmission risk of multidrug resistance (MDR) and pathogenic bacteria from human and agricultural sources into the environment and wildlife. Thus, monitoring studies investigating antibiotic resistance in raptors may provide essential data that facilitate understanding the fate and evolution of ARB and ARGs in the environment and possible health risks for humans and animals associated with the acquisition of these resistance determinants by wildlife.

• Klíčová slova
• APEC, Escherichia coli, ExPEC, IncHI2, IncI2, Milvus migrans, colistin resistance, mcr-1, qnrE1, wildlife,
• MeSH
• antagonisté receptorů pro angiotenzin MeSH
• antibakteriální látky farmakologie   MeSH
• divoká zvířata MeSH
• Escherichia coli * MeSH
• inhibitory ACE MeSH
• lidé MeSH
• mnohočetná bakteriální léková rezistence genetika   MeSH
• proteiny z Escherichia coli * genetika   MeSH
• ptáci mikrobiologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Sibiř MeSH
• Názvy látek
• antagonisté receptorů pro angiotenzin MeSH
• antibakteriální látky MeSH
• inhibitory ACE MeSH
• proteiny z Escherichia coli * MeSH

INTRODUCTION: Hospitals and wastewater are recognized hot spots for the selection and dissemination of antibiotic-resistant bacteria to the environment, but the total participation of hospitals in the spread of nosocomial pathogens to municipal wastewater treatment plants (WWTPs) and adjacent rivers had not previously been revealed. METHODS: We used a combination of culturing and whole-genome sequencing to explore the transmission routes of Escherichia coli from hospitalized patients suffering from urinary tract infections (UTI) via wastewater to the environment. Samples were collected in two periods in three locations (A, B, and C) and cultured on selective antibiotic-enhanced plates. RESULTS: In total, 408 E. coli isolates were obtained from patients with UTI (n=81), raw hospital sewage (n=73), WWTPs inflow (n=96)/outflow (n=106), and river upstream (n=21)/downstream (n=31) of WWTPs. The majority of the isolates produced extended-spectrum beta-lactamase (ESBL), mainly CTX-M-15, and showed multidrug resistance (MDR) profiles. Seven carbapenemase-producing isolates with GES-5 or OXA-244 were obtained in two locations from wastewater and river samples. Isolates were assigned to 74 different sequence types (ST), with the predominance of ST131 (n=80) found in all sources including rivers. Extraintestinal pathogenic lineages frequently found in hospital sewage (ST10, ST38, and ST69) were also found in river water. Despite generally high genetic diversity, phylogenetic analysis of ST10, ST295, and ST744 showed highly related isolates (SNP 0-18) from different sources, providing the evidence for the transmission of resistant strains through WWTPs to surface waters. DISCUSSION: Results of this study suggest that 1) UTI share a minor participation in hospitals wastewaters; 2) a high diversity of STs and phylogenetic groups in municipal wastewaters derive from the urban influence rather than hospitals; and 3) pathogenic lineages and bacteria with emerging resistance genotypes associated with hospitals spread into surface waters. Our study highlights the contribution of hospital and municipal wastewater to the transmission of ESBL- and carbapenemase-producing E. coli with MDR profiles to the environment.

• Klíčová slova
• Escherichia coli, antibiotic resistance, beta-lactamases, wastewater, whole-genome sequencing,
• MeSH
• antibakteriální látky farmakologie   MeSH
• beta-laktamasy genetika   MeSH
• Escherichia coli genetika   MeSH
• fylogeneze MeSH
• infekce močového ústrojí * mikrobiologie   MeSH
• infekce vyvolané Escherichia coli * mikrobiologie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• multilokusová sekvenční typizace MeSH
• nemocnice MeSH
• odpadní voda MeSH
• odpadní vody mikrobiologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• beta-laktamasy MeSH
• odpadní voda MeSH
• odpadní vody MeSH