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Objectives We sequenced two IncA/C plasmids harbouring blaCTX-M-2 in Klebsiella pneumoniae clinical isolates and compared their antibiotic resistance islands. Methods Transconjugants were obtained from two clinical K. pneumoniae isolates harbouring blaCTX-M-2. Plasmid DNA from transconjugants underwent short-read whole-genome sequencing, reads were assembled, and gaps were closed by PCR and sequencing. Determination of plasmid replicons, antibiotic resistance genes, identification and characterisation of insertion sequence (IS) elements, and comparison with publicly available plasmid sequences were performed. Results blaCTX-M-2 was located in a complex class 1 integron In35::ISCR1::blaCTX-M-2, inserted in two different transposons designated Tn7057 and Tn7058, that reside in the resistance islands of plasmids pUR-KP0923 and pUR-KP1025, respectively. The general modules of both transposons were In35::ISCR1::blaCTX-M-2–Tn1000-like–Tn2*–ISKpn11-12-13 variable module–ΔTn21. In Tn7057 there was ΔIS10R–catA2 associated with an additional ISKpn13. Both plasmids belonged to IncC type 2 and ST3. pUR-KP0923 was 167 138 bp in length and had a 37 926-bp resistance island at position 4 (RI-4). Plasmid pUR-KP1025 was 168 128 bp with a RI-4 of 36 222 bp. Conclusion This report describes the molecular nature of two transposons (Tn7057 and Tn7058) harbouring blaCTX-M-2 that reside in IncC type 2 ST3 plasmids. These transposons mediate resistance to oxyimino-cephalosporins, gentamicin and, in the case of Tn7057, chloramphenicol. CTX-M-2 is an important extended-spectrum β-lactamase (ESBL) to South American epidemiology. It is remarkable that despite being only two plasmid sequences, the information revealed here could contribute to a better understanding of the resistance islands from IncC type 2 plasmids.

Objectives This study aimed to characterise all carbapenemase-producing enterobacteria (CPE) isolates obtained from an outbreak-free setting in Uruguay. Methods We studied 12 CPE isolated from Hospital de Clinicas between 2012–2016. Bacterial identification and antibiotic susceptibility testing were performed using VITEK®2 and Sensititre or agar dilution, respectively. Antimicrobial resistance genes and mobile genetic elements were identified by PCR and sequencing. Multilocus sequence typing was performed for Klebsiella pneumoniae. Plasmid conjugation was assessed, plasmid size was estimated by S1-PFGE and plasmid incompatibility groups were sought by PCR. Results Among 8364 enterobacteria, 12 CPE were isolated from urine, blood culture, wound, peritoneal fluid and punch samples. NDM-1 was the most prevalent carbapenemase, followed by VIM-2 and KPC-2. All isolates were resistant to gentamicin, cefotaxime, ceftazidime, trimethoprim/sulfamethoxazole, ciprofloxacin and imipenem and were susceptible to fosfomycin. We characterised six class 1 integrons: dfrA12–orfF–aadA2; aacA4–blaOXA-2–orfD; aadB–aadA2; dfrA1; aadB–blaOXA-10–aadA1; and blaVIM-2-dfrA7. An association between various aminoglycoside, β-lactam and fluoroquinolone resistance genes were observed, some of them located in transferable plasmids belonging to incompatibility groups IncC, IncHI1 and IncM1. We described a new composite transposon (assigned Tn6935) including blaNDM-1 flanked by two directly-oriented copies of a Tn3-like element ISKox2-like family transposase. The sequence types of K. pneumoniae isolates were ST11, ST14 and ST661. Conclusions The presence of CPE is sporadic and could be due to measures taken by the Public Health Committee. Nevertheless, the coexistence of several resistance mechanisms and their presence in conjugative plasmids and high-risk clones is worrisome.

Acinetobacter baumannii is a relevant opportunistic pathogen, and one of the main microorganisms responsible for outbreaks in nosocomial infections worldwide. Its pathogenicity is mainly due to its resistance to multiple antibiotics and to its ability to form biofilms on abiotic surfaces. The objective of this study was to characterize the biofilm formation cycle of A. baumannii isolated from a patient in a hospital and compare its antibiotic resistance with the planktonic cells. To study biofilm formation, the classical microtiter assay was used, with crystal violet staining and optical density reading to classify the type of biofilm. Also, the effect of gentamicin and colistin on bacterial biofilm was studied with an extra step of antibiotic addition. For the characterization of the different biofilm formation stages, the strain was grown on a coverslip, and the stain was made with a mixture of fluorophores markers to visualize the biofilm with a confocal laser microscope. It was possible to differentiate the A. baumannii biofilm formation stages. Through these observations, it was possible to estimate the time elapsed between each stage. As the strain was susceptible to colistin and gentamicin, both antibiotics were evaluated after the biofilm was formed. Neither antibiotics showed an effect on the eradication of A. baumannii biofilm.

Azithromycin represents a valid therapeutic option for gastrointestinal and systemic infections caused by multidrug resistant (MDR) diarrheagenic Escherichia coli, Shigella and Salmonella species. However, acquired macrolide resistance in these species has been increasingly described, mostly related to macrolide modifying enzymes encoded by mph- and erm-type genes. In this study, we characterized the first blaCTX-M-14/ermB-carrying IncI1 plasmid from Latin America, which was detected in a MDR E. coli clinical isolate from Uruguay. Plasmid pUR-EC07 was a 105,836-bp IncI1 ST80 plasmid harboring blaCTX-M-14,blaTEM-1 and ermB, and characterized by a backbone region of 92,554 bp, and a multi resistance region (MRR) of 13,282 bp, bounded by two directly-oriented IS26 elements, and inserted upstream of ResD. The MRR comprised two nested IS26-composite transposons (Tn6651 and Tn6652). Tn6651 included the ermBL-ermB operon flanked by two IS26 copies in opposite direction. Tn6652 included blaTEM-1, with an upstream remnant of a Tn3-family transposon, and blaCTX-M-14, located in a genetic context consisting of ISEcp1∆–IS10–blaCTX-M-14–IS903D. To the best of our knowledge, pUR-EC07 represents the first IncI1 plasmid harboring ermB and blaCTX-M-14 described in Latin America. Given the wide dissemination of IncI-type plasmids in Salmonella enterica, an important concern is represented by the potential transfer of similar plasmids to strains resistant to ciprofloxacin, which would result in the loss of the three main therapeutic resources.

Antimicrobial resistance due to carbapenemase production in Enterobacteriaceaeclinical isolates is a global threat. Klebsiella pneumoniae harboring the blaKPCgene is one ofthe major concerns in hospital settings in Latin America.The aim of this study was to characterize the antibiotic resistance mechanisms and to typifyfour carbapenem-resistant K. pneumoniae clinical isolates from the city of Manizales, Colombia.We identified blaKPC-3in all four isolates by polymerase chain reaction and subsequentsequencing. The plasmid-mediated quinolone resistance genes qnrB19-like and aac(6)Ib-cr;fosfomycin resistance gene fosA and an insertion sequence IS5-like in mgrB (colistin resistance)were also detected. Sequence types ST11 with capsular type wzi75, and ST258 with wzi154,were characterized. The blaKPC-3gene was mobilized in a 100-kb IncFIB conjugative plasmidwith vagCD toxin-antitoxin system.This work reports multiple resistance genes in blaKPC-producing K. pneumoniae and the firstoccurrence of ST11 clinical isolates harboring blaKPC-3in Latin America. Resumen La resistencia a antibióticos mediada por la producción de carbapenemasas en aislamientos clínicos de Enterobacteriaceae es una amenaza mundial. Klebsiella pneumoniae portador de blaKPC es uno de los mayores problemas a nivel hospitalario en Latinoamérica. El objetivo de este estudio fue caracterizar los mecanismos de resistencia antibiótica y tipificar cuatro aislamientos clínicos de K. pneumoniae resistentes a carbapenems obtenidos en la ciudad de Manizales, Colombia. Se identificó blaKPC-3 en todos los aislamientos mediante reacción en cadena de polimerasa y secuenciación. También se detectaron los genes de resistencia transferible a quinolonas qnrB19-like y aac(6’)Ib-cr y a fosfomicina fosA, y la secuencia de inserción \S5-like en mgrB (asociada a la resistencia a colistina). Se caracterizaron los secuenciotipos ST11 (cápsula wzi75) y ST258 (cápsula wzi154). Se comprobó que blaKPC-3 fue movilizado por un plásmido conjugativo IncFIB-vagCD de 100kb. En este trabajo se reportan múltiples genes de resistencia en K. pneumoniae productor de blaKPC y se describen por primera vez aislamientos clínicos ST11 productores de blaKPC-3 en Latinoamérica.

Objectives The objective of this study was to characterise the mechanisms underlying quinolone and oxyimino-cephalosporin resistance in a Citrobacter freundii clinical isolate obtained from the ICU in a university hospital in Uruguay. Methods Citrobacter freundii strain CF638 was isolated from a urine culture. Identification was performed using a VITEK®2 system, and antimicrobial susceptibility was established by MIC determination and disk diffusion assay. Resistance genes and mobile genetic elements were identified by PCR and sequencing. Plasmid transfer was assessed by conjugation and the plasmid size was estimated by S1-PFGE. Plasmid incompatibility (Inc) group and toxin-antitoxin systems were sought by PCR. Results Strain CF638 showed a multidrug-resistant profile, including resistance to carbapenems and quinolones. Transconjugant TcCF638, harbouring an ca. 200-kb IncA/C plasmid, also showed resistance to all β-lactams (except aztreonam) and diminished susceptibility to ciprofloxacin. PCR was positive for blaNDM-1 and qnrVC in CF638 and TcCF638. Two different class 1 integrons were detected (In127 and In907). In127 featured the genetic array aadA2–ltr2. Conversely, complex In907 featured two variable regions (VRs); VR-1 consisted of aadB–blaOXA-10–aadA1cc, whereas VR-2 featured a qnrVC6 gene 108 bp downstream from ISCR1 and 45 bp upstream from qacEΔ1. Expression of qnrVC6 was due to a putative promoter region, detected using the Neural Network Promoter Prediction program. Conclusion To the best of our knowledge, this constitutes the first report of qnrVC within a complex class 1 integron, as well as the first report of the occurrence of such a gene in an NDM-1-producing enterobacterial clinical isolate.

Antimicrobial resistance due to carbapenemase production in Enterobacteriaceae clinical isolates is a global threat. Klebsiellapneumoniae harboring the bla

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 β-lactamases (ESBL), plasmid-encoded (pAmpC) β-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.

Azithromycin represents a valid therapeutic option for gastrointestinal and systemic infections caused by multidrug resistant (MDR) diarrheagenic Escherichia coli, Shigella and Salmonella species. However, acquired macrolide resistance in these species has been increasingly described, mostly related to macrolide modifying enzymes encoded by mph- and erm-type genes. In this study, we characterized the first bla

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.

Summary Objectives The objective of this study was to describe the microbiological characteristics of an extensively drug-resistant (XDR) isolate of Morganella morganii obtained from a patient with sepsis of urinary origin and to describe the patient's clinical characteristics. We further aimed to perform a literature review of the situation in Latin America regarding Gram-negative bacillus (GNB) carriers of New Delhi metallo-β-lactamase (NDM-1) and qnr genes and current reports on the treatment of infections caused by XDR enterobacteria, with particular attention to colistin-resistant isolates. Methods The patient's clinical data were obtained from his medical history. Microbiological identification and susceptibility testing were done using the VITEK 2 Compact System. Resistance genes were detected by PCR and sequencing. Results Blood and urine cultures grew an M. morganii isolate (Mm4232) harboring NDM-1 and qnr D1. The patient was treated successfully with fosfomycin and double doses of meropenem. There are no previous reports of the use of fosfomycin and meropenem to treat infections by XDR enterobacteria harboring NDM-1 carbapenemase. Conclusions This is the first report of qnr D1 in South America. We consider that this report could be helpful to physicians implementing treatments for infections caused by XDR GNB, including colistin–carbapenem-resistant GNB.