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Pseudomonas aeruginosa is a leading cause of hospital-acquired infections worldwide. Biofilm production, antibiotic resistance, and a wide range of virulence factors contribute to their persistence in nosocomial environments. We describe an outbreak caused by a multidrug-resistant P. aeruginosa strain in an ICU. Antibiotic susceptibility was determined and blaPER-1 and qnrVC were amplified via PCR. Clonality was determined using PFGE and biofilm formation was studied with a static model. A combination of antibiotics was assessed on both planktonic cells and biofilms. WGS was performed on five isolates. All isolates were clonally related, resistant to ceftazidime, cefepime, amikacin, and ceftolozane-tazobactam, and harbored blaPER-1; 11/19 possessed qnrVC. Meropenem and ciprofloxacin reduced the biofilm biomass; however, the response to antibiotic combinations with rifampicin was different between planktonic cells and biofilms. WGS revealed that the isolates belonged to ST309 and serotype O11. blaPER-1 and qnrVC6 were associated with a tandem of ISCR1 as part of a complex class one integron, with aac(6')-Il and ltrA as gene cassettes. The structure was associated upstream and downstream with Tn4662 and flanked by direct repeats, suggesting its horizontal mobilization capability as a composite transposon. ST309 is considered an emerging high-risk clone that should be monitored in the Americas.

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Antibiotic resistance is an alarming problem throughout the world and carbapenem-resistant Pseudomonas aeruginosa has been cataloged as critical in the World Health Organization list of microorganisms in urgent need for the development of new antimicrobials. In this work, we describe two novel resistance regions responsible for conferring a multidrug resistance phenotype to two clinical isolates of P. aeruginosa (Pa873 and Pa6415) obtained from patients hospitalized in the ICU of University Hospital of Uruguay. Bacterial identification and antibiotic susceptibility tests were performed using MALDI-TOF and the Vitek 2 system, respectively. WGS was performed for both isolates using Oxford Nanopore Technologies and Illumina and processed by means of hybrid assembly. Both isolates were resistant to ceftazidime, cefepime, piperacillin-tazobactam, aztreonam, and imipenem. Strain Pa6415 also showed resistance to ciprofloxacin. Both strains displayed MICs below the susceptibility breakpoint for CAZ-AVI plus 4 mg/L of aztreonam as well as cefiderocol. Both resistance regions are flanked by the left and right inverted repeats of ISPa40 in two small regions spanning 39.3 and 35.6 kb, for Pa6415 and Pa873, respectively. The resistance region of Pa6415 includes TnaphA6, and the new Tn7516 consists of IRi, In899, qacEΔ1-sul1-ISCR1, qnrVC6-ISCR1-blaPER-1-qacEΔ1-sul1, araJ-like, IS481-like tnpA, ISPa17, and IRR. On the other hand, the resistance region of Pa873 includes Tnaph6 and the new Tn7517 (IRi, In899, qacEΔ1-sul1, ISCR1-blaPER-1-qacEΔ1-sul1, araJ-like, IS481-like tnpA, ISPa17, and IRR). It is necessary to monitor the emergence of genetic structures that threaten to invalidate the available therapeutic resources.

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Antimicrobial resistance is a critical issue that is no longer restricted to hospital settings but also represents a growing problem involving intensive animal production systems. In this study, we performed a microbiological and molecular investigation of priority pathogens carrying transferable resistance genes to critical antimicrobials in 1-day-old chickens imported from Brazil to Uruguay. Bacterial identification was performed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, and antibiotic susceptibility was determined by Sensititre. Antimicrobial resistance genes were sought by PCR, and clonality was assessed by pulsed-field gel electrophoresis (PFGE). Four multidrug-resistant (MDR) representative strains were sequenced by an Illumina and/or Oxford Nanopore Technologies device. Twenty-eight MDR isolates were identified as Escherichia coli (n = 14), Enterobacter cloacae (n = 11), or Klebsiella pneumoniae (n = 3). While resistance to oxyiminocephalosporins was due to blaCTX-M-2, blaCTX-M-8, blaCTX-M-15, blaCTX-M-55, and blaCMY-2, plasmid-mediated quinolone resistance was associated with the qnrB19, qnrE1, and qnrB2 genes. Finally, resistance to aminoglycosides and fosfomycin was due to the presence of 16S rRNA methyltransferase rmtG and fosA-type genes, respectively. Short- and long-read genome sequencing of E. cloacae strain ODC_Eclo3 revealed the presence of IncQ/rmtG (pUR-EC3.1; 7,400 bp), IncHI2A/mcr-9.1/blaCTX-M-2 (pUR-EC3.2, ST16 [pMLST; 408,436 bp), and IncN2/qnrB19/aacC3/aph(3″)-Ib (pUR-EC3.3) resistance plasmids. Strikingly, the blaCTX-M-2 gene was carried by a novel Tn1696-like composite transposon designated Tn7337. In summary, we report that imported 1-day-old chicks can act as Trojan horses for the hidden spread of WHO critical-priority MDR pathogens harboring mcr-9, rmtG, and extended-spectrum ß-lactamase genes in poultry farms, which is a critical issue from a One Health perspective. IMPORTANCE Antimicrobial resistance is considered a significant problem for global health, including within the concept of One Health; therefore, the food chain connects human health and animal health directly. In this work, we searched for microorganisms resistant to antibiotics considered critical for human health in intestinal microbiota of 1-day-old baby chicks imported to Uruguay from Brazil. We describe genes for resistance to antibiotics whose use the WHO has indicated to "watch" or "reserve" (AWaRe classification), such as rmtG and mcr9.1, which confer resistance to all the aminoglycosides and colistin, respectively, among other genes, and their presence in new mobile genetic elements that favor its dissemination. The sustained entry of these microorganisms evades the sanitary measures implemented by the countries and production establishments to reduce the selection of resistant microorganisms. These silently imported resistant microorganisms could explain a considerable part of the antimicrobial resistance problems found in the production stages of the system.

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The aim of this work was to detect Escherichia coli isolates displaying resistance to oxyimino-cephalosporins, quinolones, and colistin in feces from livestock in Uruguay. During 2016-2019, fecal samples from 132 broiler and layer chicken flocks, 100 calves, and 50 pigs, were studied in Uruguay. Samples were cultured on MacConkey Agar plates supplemented with ciprofloxacin, ceftriaxone, or colistin. E. coli isolates were identified by mass spectrometry and antibiotic susceptibility testing was performed by disk diffusion agar method and colistin agar test. Antibiotic resistance genes were detected by polymerase chain reaction and sequencing. The most frequently detected resistance gene was qnrB19, recovered from 87 animals. Regarding plasmid-mediated quinolone resistance genes, qnrS1 was the second in prevalence (23 animals) followed by qnrE1, found in 6 chickens and two calves. Regarding resistance to oxyimino-cephalosporins, 8 different ß-lactamase genes were detected: bla CTX-M-8 and bla CMY-2 were found in 23 and 19 animals, respectively; next, bla CTX-M-2 and bla SHV-12 in 7 animals each, followed by bla CTX-M-14 in 5, bla CTX-M-15 and bla SHV2a in 2, and bla CTX-M-55 in a single animal. Finally, the mcr-1 gene was detected only in 8 pigs from a single farm, and in a chicken. Isolates carrying bla CMY-2 and bla SHV-12 were also found in these animals, including two isolates featuring the bla CMY-2/mcr-1 genotype. To the best of our knowledge, this is the first work in which the search for transferable resistance to highest priority critically important antibiotics for human health is carried out in chickens and pigs chains of production animals in Uruguay.

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OBJECTIVE: This report described the first Escherichia coli (E. coli) isolates harbouring mcr-1 in Uruguay. METHODS: Three E. coli isolates were obtained from blood, urine and rectal swabs from different patients in two hospitals. Extended-spectrum ß-lactamases (ESBL), plasmid-encoded (pAmpC) ß-lactamases, plasmid-mediated quinolone resistance (PMQR) genes, class 1 integrons, and mcr-1, mcr-2 and mcr-3 were sought and characterised in three E. coli isolates. Transfer of resistance determinants was assessed by conjugation. Clonality was analysed by multilocus sequence typing. RESULTS: All isolates were categorised as being colistin-resistant and the mcr-1 gene was detected. Two isolates were also resistant to oxyimino cephalosporins: one on account of blaCMY-2 and the other due to blaCTX-M-15, the latter also harbouring transferable quinolone-resistance genes (aac(6')Ib-cr and qnrB). All mcr-1 genes were transferred by conjugation to recipient strains. The mcr-1-bearing isolates belonged to sequence types ST10, ST93 and ST5442. CONCLUSIONS: ST10 is considered as a high-risk clone worldwide. This type of mcr-1-harbouring clone is a major concern for human and animal health and must be under close surveillance. This study detected the presence of mcr-1 for the first time in Uruguay, albeit in an allodemic manner, associated with different antibiotic-resistance genes and from diverse clinical contexts. Considering that colistin is often the last therapeutic option available for multidrug-resistant Gram-negative bacilli infections, it is important to maximise precautions to avoid dissemination of isolates carrying mcr-1.

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Multidrug-resistant Salmonella enterica isolates are an increasing problem worldwide; nevertheless, the mechanisms responsible for such resistance are rarely well defined. Multidrug-resistant S. enterica serovar Typhimurium isolates ST3224 and ST827 were collected from two patients. The characteristics of both genomes and antimicrobial resistance genes were determined using next-generation sequencing.

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Carbapenem-resistant Acinetobacter baumannii (CRAB) infections are an increasing concern in intensive care units (ICUs) worldwide. The combination of carbapenemases and 16S rRNA-methyltransferases (16S-RMTases) further reduces the therapeutic options. OXA-carbapenemase/A. baumannii clone tandems in Latin America have already been described; however, no information exists in this region regarding the occurrence of 16S-RMTases in this microorganism. In addition, the epidemiology of A. baumannii in ICUs and its associated resistance profiles are poorly understood. Our objectives were as follows: to study the clonal relationship and antibiotic resistance profiles of clinical and digestive colonizing A. baumannii isolates in an ICU, to characterize the circulating carbapenemases, and to detect 16S-RMTases. Patients admitted between August 2010 and July 2011 with a clinically predicted hospital stay > 48 hr were included. Pharyngeal and rectal swabs were obtained during the first fortnight after hospitalization. Resistance profiles were determined with MicroScan® and VITEK2 system. Carbapenemases and 16S-RMTases were identified by PCR and sequencing, and clonality was assessed by pulsed-field gel electrophoresis and multilocus sequence typing. Sixty-nine patients were studied and 63 were diagnosed with bacterial infections. Among these, 29 were CRAB isolates; 49 A. baumannii were isolated as digestive colonizers. These 78 isolates were clustered in 7 pulsetypes, mostly belonging to ST79. The only carbapenemase genes detected were blaOXA-51 (n = 78), blaOXA-23 (n = 62), and blaOXA-58 (n = 3). Interestingly, two clinical isolates harbored the rmtC 16S-RMTase gene. To the best of our knowledge, this is the first description of the presence of rmtC in A. baumannii.

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The objectives of this study were (i) to determine the extended-spectrum ß-lactamase-producing Escherichia coli and Klebsiella pneumoniae (ESBL-EcKp) clones circulating in an intensive care unit (ICU) in Uruguay between August 2010 and July 2011, (ii) to characterise the ESBL and plasmid-mediated quinolone resistance (PMQR) genes of the studied isolates and (iii) to determine the virulotype of the clinical isolates. Clinical and gut-colonising ESBL-EcKp from ICU patients were studied. Bacterial identification and antibiotic susceptibility determination were performed using a VITEK(®)2 system. Detection of ESBL, KPC and PMQR genes was performed by PCR and sequencing. Clonality was assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). In total, 54 ESBL-EcKp isolates (40 K. pneumoniae and 14 E. coli), with or without PMQR genes, were recovered from 30 of 68 inpatients. Forty-seven isolates were CTX-M-15-producers (36 as a single ESBL and 11 together with CTX-M-14). In addition, four isolates produced CTX-M-14, two produced CTX-M-2 and one produced SHV-5. No carbapenemases were detected either in E. coli or K. pneumoniae isolates. Among the ESBL-producing isolates, 42 also harboured PMQR genes: 27 aac(6')-Ib-cr; 14 aac(6')-Ib-cr and qnrB; and a single isolate carrying only qnrB. K. pneumoniae ST258, ST48 and ST16 and E. coli ST10 and ST405 were detected in 46/54 isolates, including 9 clinical isolates. In conclusion, non-KPC-producing K. pneumoniae ST258 harbouring different ESBL and PMQR genes was the main clone disseminated in the ICU. Extensive surveillance measures must be implemented to prevent the emergence of acquired plasmid-encoded blaKPC by ST258 K. pneumoniae.

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This study characterised the mechanisms of fluoroquinolone and oxyimino-cephalosporin resistance in human Salmonella enterica isolates in Uruguay. Salmonella enterica isolates were collected from 2011-2013 and were selected based on non-susceptibility to ciprofloxacin and/or oxyimino-cephalosporins. The disk diffusion assay was performed for various antibiotics, and the ciprofloxacin minimum inhibitory concentration (MIC) was determined following CLSI guidelines. Genetic relatedness was determined following PulseNet protocols. Extended-spectrum ß-lactamases, ampC alleles and plasmid-mediated quinolone resistance were characterised by PCR and sequencing. Plasmid analyses were carried out by conjugation or transformation assays, and plasmid-encoded genes were identified by PCR. Mutations in the quinolone resistance-determining region of gyrases were sought by PCR and sequencing. Among 579 isolates, 105 (18.4%) ciprofloxacin-non-susceptible (CIP-NS) isolates, 9 (1.6%) oxyimino-cephalosporin-resistant isolates and 2 (0.3%) isolates resistant to both antibiotic families were detected. Thirteen isolates carried qnrB alleles (twelve qnrB19 and one qnrB2), four carried blaCTX-M-8, two blaCTX-M-14, two blaSHV-2 and three blaCMY-2-like genes. No correlation was found between mutations in gyrases and ciprofloxacin MICs. Several co-circulating clones of S. enterica ssp. enterica serovar Typhimurium were detected; conversely, S. enterica ssp. enterica serovar Enteritidis corresponded mainly to a single circulating clone. Nine (75%) of twelve of CIP-NS extraintestinal isolates shared the same pulsotype with intestinal isolates. During the study period, the frequency of CIP-NS isolates increased, albeit with ciprofloxacin MICs of 0.125-0.5mg/L. Detection of the same quinolone-resistant clones recovered both from intestinal and extraintestinal samples highlights the significance of epidemiological surveillance of antibiotic susceptibility for every human Salmonella isolate.

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