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.

• Keywords
• Enterobacterales, Environment, Whole-genome sequencing, Wild birds,
• Publication type
• Journal Article MeSH

The study aimed to investigate (i) the occurrence of third-generation cephalosporins and/or carbapenems non-sensitive Enterobacterales in Pavia surface and groundwaters, (ii) their resistance determinants, and (iii) the clonal features of the most relevant strains. During May 13 and 14, 2019, n = 18 water samples from n = 12 sampling sites in the urban/peri-urban area of Pavia (Po Plain, Northern Italy) have been evaluated. At first, hydrochemical analysis and bacterial plate counts were carried out on all the water samples. One milliliter of each water sample was then screened on both MacConkey agar (MC) added with cefotaxime (1 mg/L; 2 mg/L) and MC plus meropenem (0.25 mg/L; 4 mg/L). Species identification and antimicrobial susceptibilities were assessed by MicroScan autoSCAN-4. Double Disk Synergy (DD) test, CT103XL microarray, acc(6')-Ib-cr, qnrS, blaCTX-M-/MOX-/VEB-/OXA-type genes targeted PCR and sequencing, Pulsed-Field Gel Electrophoresis (PFGE), MultiLocus Sequence Typing (MLST), and Whole-Genome Sequencing on selected strains were performed. A total of n = 30 isolates grown on β-lactams enriched MC: Escherichia coli (n = 21; 70%), Klebsiella spp. (n = 5; 16.6%), Citrobacter freundii (n = 2; 6.7%), and Kluyvera intermedia (n = 2; 6.7%). All E. coli and K. pneumoniae were ESβL-producers by DD. The 66.6, 38.0, and 19.0% of E. coli were ciprofloxacin/levofloxacin, trimethoprim-sulfamethoxazole, and gentamicin resistant (EUCAST 2019 breakpoints), respectively. A blaCTX-M-type determinant was identified in E. coli (n = 20/21; 95.2%) and K. pneumoniae (n = 2/3; 66.7%). The remaining E. coli was blaVEB-1 and blaMOX-2 genes positive. The aac(6')-Ib-cr determinant was found in n = 7 E. coli and n = 1 K. pneumoniae, while qnrS was found in n = 1 E. coli and n = 2 K. pneumoniae. PFGE showed clonal heterogeneity among ESβL-E. coli. Two out of four E. coli detected as blaOXA-244-positive, belonged to the pandemic ST131. One XDR K. pneumoniae from a stream sample, detected as blaKPC-2 positive, resulted of ST258. The epidemiological impact of blaOXA-244 ST131 E. coli and blaKPC-2 ST258 K. pneumoniae presence in surface waters of an urban area in Northern Italy must not be underestimated.

• Keywords
• ESβLs, Enterobacterales, Italy, OXA-244, ST131 Escherichia coli, ST258 Klebsiella pneumoniae, carbapenemases, river and ground waters,
• Publication type
• Journal Article MeSH

The aim of the present study is to describe the ongoing spread of the KPC-producing strains, which is evolving to an epidemic in Czech hospitals. During the period of 2018-2019, a total of 108 KPC-producing Enterobacterales were recovered from 20 hospitals. Analysis of long-read sequencing data revealed the presence of several types of blaKPC-carrying plasmids; 19 out of 25 blaKPC-carrying plasmids could be assigned to R (n = 12), N (n = 5), C (n = 1) and P6 (n = 1) incompatibility (Inc) groups. Five of the remaining blaKPC-carrying plasmids were multireplicon, while one plasmid couldn't be typed. Additionally, phylogenetic analysis confirmed the spread of blaKPC-carrying plasmids among different clones of diverse Enterobacterales species. Our findings demonstrated that the increased prevalence of KPC-producing isolates was due to plasmids spreading among different species. In some districts, the local dissemination of IncR and IncN plasmids was observed. Additionally, the ongoing evolution of blaKPC-carrying plasmids, through genetic rearrangements, favours the preservation and further dissemination of these mobile genetic elements. Therefore, the situation should be monitored, and immediate infection control should be implemented in hospitals reporting KPC-producing strains.

• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Drug Resistance, Bacterial * MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• beta-Lactamases metabolism   MeSH
• Epidemics MeSH
• Klebsiella Infections epidemiology   microbiology   MeSH
• Klebsiella pneumoniae isolation & purification   metabolism   MeSH
• Humans MeSH
• Hospitals statistics & numerical data   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic epidemiology   MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Bacterial Proteins MeSH
• beta-Lactamases MeSH
• carbapenemase MeSH Browser

Antimicrobial consumption in veterinary medicine has led to the spread of multi drug-resistance in clinically important bacteria, with the companion animals and their environment involved as emerging reservoirs. While CTX-M-15 and CMY-2 acquired β-lactamases have been widely detected in the bacterial population of companion and breeding animals in European area, DHA-1 enzymes have been rarely reported in veterinary medicine. The aim of the study was to characterize the Escherichia coli associated with mortality of a litter of Bulldog puppies in a breeding kennel located in Pesaro area, Central Italy. The E. coli strains O39 serotype were resistant to 3rd/4th generation cephalosporins, chloramphenicol, aminoglycosides, trimethoprim-sulfamethoxazole, and ciprofloxacin, retaining susceptibility to carbapenems, colistin, fosfomycin, and levofloxacin (by Microscan Autoscan4, EUCAST clinical breakpoints). Pulse field gel electrophoreses (PFGE-XbaI) on five E. coli strains revealed the presence of a single profile. Whole genome sequencing (WGS) analysis revealed a complex resistome, harboring bla TEM-1b, bla CTX-M-15, bla OXA-1, aph(6)-Ib, aac(6')Ib-cr, aac(3)-Ila, aph(6)-Id, aadA1, qnrB1, sul2, catA1, catB3, tetA, and dfrA14 genes located on a 302597 bp IncHI2/HI2A plasmid. Moreover, bla DHA-1, qnrB4, mph(A), sul1, and dfrA17 determinants were carried on an 83,429 bp IncFII plasmid. A bla CMY-2 determinant was carried on a 90,249 bp IncI1 plasmid. Two IncX1 and IncX4 plasmids without antimicrobial resistance genes were also detected. The presence of lpfA, iss, astA, and gad virulence factors was highlighted. This is the first report in Italy on an invasive infection in eight 2-weeks old dogs caused by the same MDR E. coli O39 bla CTX-M-15, bla CMY-2, bla DHA-1, and aac(6')-Ib-cr positive strain. The above MDR E. coli clone caused the death of the entire litter, despite amoxicillin-clavulanate and enrofloxacin administration. The tank for storage of the water used to prepare the milk-based meal for the litter was the suspected reservoir.

• Keywords
• CMY-2, CTX-M-15, DHA-1, E. coli, plasmids, whole genome sequencing,
• Publication type
• Journal Article MeSH

The aim of our study was to determine complete nucleotide sequence of mcr-1-carrying plasmids from Enterobacterales isolates recovered from domestic and imported raw retailed meat and compare them with plasmids available at the GenBank sequence database. A set of 16 plasmids originating from Escherichia coli (n = 13), Klebsiella pneumoniae (n = 2), and Citrobacter braakii (n = 1) were analyzed. In our previous study, data from whole genome sequencing showed that mcr-1 gene was located on plasmids of different incompatibility groups (IncHI2, IncI2, and IncX4). The IncI2 (n = 3) and IncX4 (n = 8) plasmids harbored mcr-1.1 gene only, whereas IncHI2 sequence type 4 plasmids (n = 5) carried large multidrug resistance (MDR) regions. MDR regions of IncHI2 plasmids included additional antimicrobial resistance genes conferring resistance to β-lactams (bla TEM-1), aminoglycosides [aadA1, aadA2, and aph(6)-Id], macrolides [mef (B)], tetracycline (tetA, tetR), and sulphonamides (sul1, sul2, and sul3). Likewise, IncHI2 plasmids carried several insertion sequences including IS1, IS3, IS26, IS1326, and ISApl1. In conclusion, our findings confirmed the involvement of IncX4, IncI2, and IncHI2 plasmids in the dissemination of mcr-1.1 gene in several environmental niches, as in samples of retail meat originating from different geographical regions. In contrast to IncX4 and IncI2, IncHI2 plasmids were more diverse and carried additional genes for resistance to heavy metals and multiple antimicrobials.

• Keywords
• IncHI2, IncI2, IncX4, antimicrobial resistance, colistin, meat,
• Publication type
• Journal Article MeSH