To undertake a prospective analysis of the occurrence of colistin-resistant and extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales colonizing pigs at two farms in Portugal, and to evaluate the putative correlations with usage of different antibiotics. Materials and methods: One hundred and two faecal samples recovered from two different Portuguese pig farms were screened for polymyxin-resistant and ESBL-positive Enterobacterales. The authors had undertaken a study at one of the farms previously, but the use of colistin has since been banned; zinc oxide and amoxicillin are used as prophylactic and curative drugs, respectively, at this farm. The other farm included in this study used zinc oxide alone. Results: Ninety-three ESBL-producing isolates (62 Escherichia coli , 29 Klebsiella pneumoniae , one Enterobacter aerogenes and one Enterobacter cloacae ) and 17 colistin-resistant isolates (12 E. coli , four K. pneumoniae and one E. cloacae ) were recovered. Among the ESBL producers, the majority (84%) produced CTX-M-15, while the others produced CTX-M-1 or CTX-M-9. Many different strain and plasmid backgrounds were identified, ruling out a massive dissemination of one major clone. In total, 17 colistin-resistant isolates were recovered, all from the first farm. All produced MCR-1, corresponding to 12 E. coli (10 clones) and three K. pneumoniae (two clones). The MCR-1 producers were all recovered from the farm where colistin had been used 2 years previously. Conclusion:This study showed a surprisingly high rate of CTX-M-15 producers at two Portuguese pig farms. A link was found between antibiotic selective pressure (ß-lactam or polymyxin) and the corresponding resistance rate.

Background. The aim of the present study was to prospectively evaluate the prevalence of intestinal carriage of colistin-resistant and extended-spectrum β-lactamase (ESBL)-producing Enterobacterales among pigs from a Swiss farm attending an animal health and antibiotic stewardship program and to determine the associated mechanisms of resistance. Materials/Methods. Eighty-one fecal samples were recovered and screened for either β-lactam-resistant, colistin-resistant, or aminoglycoside-resistant Enterobacterales, using respective screening media. All recovered isolates were tested for antimicrobial susceptibility and their clonal relationship (PFGE and MLST). Plasmid typing was performed by plasmid-based replicon typing (PBRT). Resistance genes were searched by PCR and sequencing. Results. A total of 38 ESBL-producing Escherichia coli and a single ESBL-producing Enterobacter cloacae were recovered from 81 pigs, corresponding to a prevalence of 50%, no other β-lactamase producer being identified. Among the 38 ESBL-producing E. coli, all belonged to sequence type (ST) ST10, except two ST34 and ST744 isolates. Among the ST10-blaCTX-M-1 isolates, three subclones (n = 22, n = 13, and n = 1, respectively) were identified according to the PFGE analysis. The most commonly identified IncI1 plasmid harboring the blaCTX-M-1 gene was 143 kb in size and coharbored other resistance genes. Only three colistin-resistant Enterobacterales isolates were recovered, namely two Klebsiella pneumoniae isolates and a single E. cloacae isolate. Screening for the plasmid-borne mcr-1 to mcr-9 genes in these three isolates gave negative results. The two K. pneumoniae isolates were clonally related, belonged to ST76, and harbored a truncated mgrB chromosomal gene being the source of colistin resistance. Conclusion. A high prevalence of fecal carriage of ESBL-producing E. coli was found, being mainly caused by the spread of a clonal lineage within the farm. By contrast, a low prevalence of colistin-resistant Enterobacterales was found.

Aminoglycosides (AGs) in combination with β-lactams play an important role in antimicrobial therapy in severe infections. Pan-resistance to clinically relevant AGs increasingly arises from the production of 16S rRNA methylases (RMTases) that are mostly encoded by plasmids in Gram-negative bacteria. The recent emergence and spread of isolates encoding RMTases is worrisome, considering that they often co-produce extended-spectrum β-lactamases (ESBLs) or carbapenemases. Our study aimed to retrospectively analyze and characterize the association of carbapenem- and aminoglycoside-resistant clinical isolates in Switzerland during a 3.5-year period between January 2017 and June 2020. A total of 103 pan-aminoglycoside- and carbapenem-resistant clinical isolates were recovered at the NARA (Swiss National Reference Center for Emerging Antibiotic Resistance) during the 2017–2020 period. Carbapenemase and RMTase determinants were identified by PCR and sequencing. The characterization of plasmids bearing resistance determinants was performed by a mating-out assay followed by PCR-based replicon typing (PBRT). Clonality of the isolates was investigated by multilocus sequence typing (MLST). Over the 991 Enterobacterales collected at the NARA during this period, 103 (10.4%) of them were resistant to both carbapenems and all aminoglycosides. Among these 103 isolates, 35 isolates produced NDM-like carbapenemases, followed by OXA-48-like (n = 23), KPC-like (n = 21), or no carbapenemase (n = 13), OXA-48-like and NDM-like co-production (n = 7), and VIM-like enzymes (n = 4). The RMTases ArmA, RmtB, RmtC, RmtF, RmtG, and RmtB + RmtF were identified among 51.4%, 13.6%, 4.9%, 24.3%, 1%, and 1%, respectively. Plasmid co-localization of the carbapenemase and the RMTase encoding genes was found among ca. 20% of the isolates. A high diversity was identified in terms of the nature of associations between RMTase and carbapenemase-encoding genes, of incompatibility groups of the corresponding plasmids, and of strain genetic backgrounds, highlighting heterogeneous importations rather than clonal dissemination.

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In order to evaluate whether seagulls living on the Lisbon coastline, Portugal, might be colonized and consequently represent potential spreaders of multidrug-resistant bacteria, a total of 88 gull fecal samples were screened for detection of extended-spectrum β-lactamase (ESBL)- or carbapenemase-producing Enterobacteriaceae for methicillin-resistant Staphylococcus aureus (MRSA) and for vancomycin-resistant Enterococci (VRE). A large proportion of samples yielded carbapenemase- or ESBL-producing Enterobacteriaceae (16% and 55%, respectively), while only two MRSA and two VRE were detected. Mating-out assays followed by PCR and whole-plasmid sequencing allowed to identify carbapenemase and ESBL encoding genes. Among 24 carbapenemase-producing isolates, there were mainly Klebsiella pneumoniae (50%) and Escherichia coli (33%). OXA-181 was the most common carbapenemase identified (54%), followed by OXA-48 (25%) and KPC-2 (17%). Ten different ESBLs were found among 62 ESBL-producing isolates, mainly being CTX-M-type enzymes (87%). Co-occurrence in single samples of multiple ESBL- and carbapenemase producers belonging to different bacterial species was observed in some cases. Seagulls constitute an important source for spreading multidrug-resistant bacteria in the environment and their gut microbiota a formidable microenvironment for transfer of resistance genes within bacterial species.