Antibiotic susceptibility and fosfomycin resistance characterization in a cohort of children older than 6 years of age with urinary tract infection.

Fosfomycin tromethamol (FT) was reintroduced as an option for the treatment of low urinary tract infection (UTI) in children. In this study, we described the antibiotic sensitivity and mechanisms of resistance to fosfomycin in isolates from children older than 6 years with UTI. Urine culture and antibiotic susceptibility study were performed. In fosfomycin resistant strains, PCR for fos, blaCTX-M was performed followed by classification by phylogenetic group and sequencetyping. Escherichia coli was the most frequent etiological agent (89.2%). The susceptibility percentages were: fosfomycin 97.9%; amoxicillin-clavulanate 92.7%; cefuroxime and ceftriaxone 99%; nitrofurantoin 94.4%. An E. coli strain (ST69, phylogenetic group D) was resistant to fosfomycin (MIC 256mg/l) and carried the blaCTX-M-14 and fosA3 genes in a 45kb IncN-type plasmid. This is the first report of E. coli ST69 with blaCTX-M-14/fosA3 of human origin.

First characterization of K. pneumoniae ST11 clinical isolates harboring blaKPC-3 in Latin America.

Antimicrobial resistance due to carbapenemase production in Enterobacteriaceae clinical isolates is a global threat. Klebsiellapneumoniae harboring the blaKPC gene is one of the major concerns in hospital settings in Latin America. The aim of this study was to characterize the antibiotic resistance mechanisms and to typify four carbapenem-resistant K. pneumoniae clinical isolates from the city of Manizales, Colombia. We identified blaKPC-3 in all four isolates by polymerase chain reaction and subsequent sequencing. 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-3 gene was mobilized in a 100-kb IncFIB conjugative plasmid with vagCD toxin-antitoxin system. This work reports multiple resistance genes in blaKPC-producing K. pneumoniae and the first occurrence of ST11 clinical isolates harboring blaKPC-3 in Latin America.

Characterization of the first blaCTX-M-14/ermB-carrying IncI1 plasmid from Latin America.

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.

Outbreak of Pseudomonas aeruginosa High-Risk Clone ST309 Serotype O11 Featuring blaPER-1 and qnrVC6.

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.

Great Plasticity in a Great Pathogen: Capsular Types, Virulence Factors and Biofilm Formation in ESBL-Producing Klebsiella pneumoniae from Pediatric Infections in Uruguay.

Klebsiella pneumoniae is widely recognized as an opportunistic hospital and community pathogen. It is one of the priority microorganisms included in the ESKAPE group, and its antibiotic resistance related to extended-spectrum ß-lactamases (ESBL) is a global public health concern. The multi-drug resistance (MDR) phenotype, in combination with pathogenicity factors, could enhance the ability of this pathogen to cause clinical infections. The aim of this study was to characterize pathogenicity factors and biofilm formation in ESBL-producing K. pneumoniae from pediatric clinical infections. Capsular types, virulence factors, and sequence types were characterized by PCR. Biofilm formation was determined by a semiquantitative microtiter technique. MDR phenotype and statistical analysis were performed. The K24 capsular type (27%), virulence factors related to iron uptake fyuA (35%) and kfuBC (27%), and sequence types ST14 (18%) and ST45 (18%) were the most frequently detected. Most of the strains were biofilm producers: weak (22%), moderate (22%), or strong (12%). In 62% of the strains, an MDR phenotype was detected. Strains with K24 capsular type showed an association with ST45 and the presence of fyuA; strains with kfuBC showed an association with moderate or strong biofilm production and belonging to ST14. Weak or no biofilm producers were associated with the absence of kfuBC. The MDR phenotype was associated with the main ESBL gene, blaCTX-M-15. The high plasticity of K. pneumoniae to acquire an MDR phenotype, in combination with the factors exposed in this report, could make it even more difficult to achieve a good clinical outcome with the available therapeutics.

[Treatment failure due to acquisition of antibiotic resistance: report of two clinical cases]. / Fallo terapéutico por resistencia antibacteriana intra-tratamiento, a propósito de dos casos clínicos.

OBJECTIVE: We describe two cases of treatment failure due to intra-treatment acquisition of antibiotic resistant microorganisms with the aim of highlighting the possible molecular mechanisms by which treatment failure occurred. PATIENTS AND METHODS: We analyzed the clinical histories and the isolates obtained from 2 patients, one with a urinary tract infection (UTI) by E. coli, initially treated with cefuroxim (to which the isolate was susceptible), and another with osteoarthritis (OA) treated initially with meropenem plus vancomycin, developing K. pneumoniae susceptible to meropenem. During treatment, in both patients, resistant microorganisms were isolated, and empirical therapy was modified, initially with ceftriaxone and afterwards meropenem in case 1, and adding amikacin in case 2. Both strains (per patient) were compared by PFGE and resistance genes were sought by PCR. RESULTS: Regarding the UTI, the initial strain acquired an IncFIB SHV-5-producing plasmid. In the OA case, the initial susceptible strain was substituted by a CTX-M-9 and AadB-AadA2-Aac(6')Ib-producing K. pneumoniae.

Novel Resistance Regions Carrying TnaphA6, blaVIM-2, and blaPER-1, Embedded in an ISPa40-Derived Transposon from Two Multi-Resistant Pseudomonas aeruginosa Clinical Isolates.

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.

First human isolate of Salmonella enterica serotype Enteritidis harboring blaCTX-M-14 in South America.

We studied a clinical isolate of Salmonella enterica serotype Enteritidis showing resistance to oxyiminocephalosporins. PCR analysis confirmed the presence of bla(CTX-M-14) linked to IS903 in a 95-kb IncI1 conjugative plasmid. Such a plasmid is maintained on account of the presence of a pndAC addiction system. Multilocus sequence typing (MLST) analysis indicated that the strain belongs to ST11. This is the first report of bla(CTX-M-14) in Salmonella Enteritidis of human origin in South America.

Extended-spectrum ß-lactamases and plasmid-mediated quinolone resistance in enterobacterial clinical isolates in the paediatric hospital of Uruguay.

OBJECTIVES: To analyse the prevalence of resistance to ß-lactams and plasmid-mediated quinolone resistance in Enterobacteriaceae in the paediatric hospital of Uruguay. METHODS: A total of 368 enterobacterial isolates collected between 1 May and 30 November 2009 were studied for the presence of extended-spectrum ß-lactamases (ESBLs), qnr alleles and aac(6')Ib by phenotypic and molecular methods. The genomic context and transferability of ß-lactamase and qnr genes were examined by PCR and conjugation, respectively. RESULTS: The proportion of inpatients having an infection caused by ESBL-producing enterobacteria was 0.23% (16/7073) in paediatrics wards, 0.64‰ (3/4696) in the neonatology department and 0.03‰ (1/32 557) in the emergency department. ESBL-carrying enterobacteria constituted a total of 21.6% (16/74), 13% (3/23) and 0.37% (1/271) when samples were obtained from paediatrics wards, the neonatology department and the emergency department, respectively. Overall, CTX-M-2 (n = 7), CTX-M-9 (n = 3), CTX-M-8 (n = 2), CTX-M-15 (n = 1), SHV-5 (n = 5) and SHV-2 (n = 2) ß-lactamases were detected. Thirteen out of 20 ESBL-producing isolates also carried the aac(6')Ib gene, and the cr variant was detected in one of them. qnr alleles were detected in four isolates comprising two qnrA1 genes, a qnrB8-like variant and a new qnrB gene showing 26 amino acid differences from QnrB1. CONCLUSIONS: The proportion of ESBL-producing enterobacteria in Uruguay's paediatric hospital during the study period was 2.3 per 1000 hospitalized patients. The number of different microorganisms detected, as well as the various EBSLs, suggests the occurrence of sporadic episodes instead of nosocomial outbreaks. Nevertheless, the presence of new resistance genes reinforces the necessity for permanent surveillance programmes.

In Vivo High Plasticity of Multi-Drug Resistant ST258 Klebsiella pneumoniae.

Carbapenemase production in Enterobacterales clinical isolates is a global threat. Multi-drug resistant Klebsiella pneumoniae harboring carbapenemases are a major concern among the hospital settings in Latin America. Aim: The aim of this study was to analyze the genetic relatedness between three isolates of K. pneumoniae recovered from one patient in the same bacteriological round on the same day, which exhibited different susceptibility profiles to carbapenems (CP) and to colistin (Col). Isolates' profiles were as follows (susceptible-S/resistant-R): CPS/ColR, CPR/ColR, and CPR/ColS. Pulse-field gel electrophoresis, multilocus sequence typing, and whole genome sequencing were performed. Conjugation assays were carried out and PCR determination in transconjugants (Tcs) was made for: blaCTX-M-groups, blaNDM, blaKPC, blaTEM, qnr alleles, aac(6')Ib-cr, ermB, and plasmid incompatibility groups (Inc). Results: All isolates belonged to the same clone, to ST258 and harbored blaCTX-M-14, blaCTX-M-15, qnrA1, qnrB1, aac(6')Ib-cr, and wzi154 (capsule-locus KL107). One isolate had additional wzi gene, wzi109 (capsule-locus KL36). In CPR isolates, the pattern was explained for blaNDM-1 or blaNDM-1/blaKPC-2 presence, and in ColR for IS5-like element insertion in mgrB at different positions. Co-mobilization of blaNDM-1/qnrA1 was associated to a different plasmid Inc (A/C-FII) in both blaNDM-1 donors. Mobilization of blaCTX-M-14 was related to IncI1 in one donor. Conclusion: These findings highlight the potential plasticity of ST258 K. pneumoniae clone. To the best of our knowledge, this is the first description of blaNDM-1/blaKPC-2-producing K. pneumoniae ST258 in Latin America.

First three Escherichia coli isolates harbouring mcr-1 in Uruguay.

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.

Detection of qnrVC6, within a new genetic context, in an NDM-1-producing Citrobacter freundii clinical isolate from Uruguay.

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 ß-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 108bp downstream from ISCR1 and 45bp 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.

Molecular Characterization of Carbapenem-Resistant Acinetobacter baumannii in the Intensive Care Unit of Uruguay's University Hospital Identifies the First rmtC Gene in the Species.

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.

Antibiotic susceptibility and fosfomycin resistance characterization in a cohort of children older than 6 years of age with urinary tract infection / Sensibilidad antibiótica y caracterización de la resistencia a fosfomicina en una cohorte de niños mayores de 6 años con infección urinaria

Fosfomycin tromethamol (FT) was reintroduced as an option for the treatment of low urinary tract infection (UTI) in children. In this study, we described the antibiotic sensitivity and mechanisms of resistance to fosfomycin in isolates from children older than 6 years with UTI. Urine culture and antibiotic susceptibility study were performed. In fosfomycin resistant strains, PCR for fos, blaCTX-M was performed followed by classification by phylogenetic group and sequencetyping. Escherichia coli was the most frequent etiological agent (89.2%). The susceptibility percentages were: fosfomycin 97.9%; amoxicillin-clavulanate 92.7%; cefuroxime and ceftriaxone 99%; nitrofurantoin 94.4%. An E. coli strain (ST69, phylogenetic group D) was resistant to fosfomycin (MIC 256mg/l) and carried the blaCTX-M-14 and fosA3 genes in a 45kb IncN-type plasmid.

La fosfomicina-trometamol (FT) se reintrodujo como una opción para el tratamiento de la infección del tracto urinario (ITU) baja en niños. En este estudio describimos la sensibilidad antibiótica y los mecanismos de resistencia a FT en aislamientos de niños mayores de 6 anos con ITU. Se realizaron urocultivos y estudios de sensibilidad antibiótica. En las cepas resistentes a fosfomicina se realizó la técnica de PCR para fos, blaCTX-M, y su identificación según su grupo filogenéticoy secuenciotipo. Escherichiacoli fue el agente etiológico más frecuente (89,2%). Los porcentajes de sensibilidad fueron: fosfomicina 97,9%; amoxicilina-clavulánico 92,7%; cefurox-ima y ceftriaxona 99%; nitrofurantoína 94,9%. Una cepa de E. coli (ST69, grupo filogenético D) fue resistente a fosfomicina (CIM 256mg/l) y portaba los genes blaCTX-M-14 y fosA3 en un plás-mido de 45 kb del tipo IncN. Este es el primer reporte de E. coli ST69 con blaCTX-M-14/fosA3 de origen humano.

Analysis of plasmid-mediated quinolone and oxyimino-cephalosporin resistance mechanisms in Uruguayan Salmonella enterica isolates from 2011-2013.

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.

CTX-M-15 in combination with aac(6')-Ib-cr is the most prevalent mechanism of resistance both in Escherichia coli and Klebsiella pneumoniae, including K. pneumoniae ST258, in an ICU in Uruguay.

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.

Extended-spectrum ß-lactamases, transferable quinolone resistance, and virulotyping in extra-intestinal E. coli in Uruguay.

INTRODUCTION: To characterize extended-spectrum ß-lactamases (ESBLs) and plasmid-mediated quinolone resistance (PMQR) genes in Escherichia coli isolates obtained from extra-intestinal samples in three Uruguayan hospitals. METHODOLOGY: Fifty-five ESBL-producing E. coli isolates were studied. Virulence genes, ESBLs, and PMQR genes were detected by polymerase chain reaction. ESBL-producing isolates were compared by pulsed-field gel electrophoresis. Multi-locus sequence typing was also performed on 13 selected isolates. RESULTS: Thirty-seven isolates harbored blaCTX-M-15 (67.3%), eight blaCTX-M-2 (14.6%), five blaCTX-M-14 (9.1%), three carried both blaCTX-M-2 and blaCTX-M-14, one blaCTX-M-9, and one blaCTX-M-8. Among the CTX-M-15 producers, 92% belonged to sequence types ST131 and ST405, and carried aac(6')Ib-cr as well. Isolates harboring blaCTX-M-2, blaCTX-M-14, blaCTX-M-9, or blaCTX-M-8 were found to be genetically unrelated. CONCLUSIONS: The successful dissemination of CTX-M-15-producing E.coli isolates seems to be linked to the spreading of high-risk clones and horizontal gene transfer. A trade-off between carrying more antibiotic resistance and less virulence-related genes could partially account for the evolutionary advantages featured by successful clones.

First report of a clinical isolate of Leclercia adecarboxylata harbouring multiple resistance genes in Uruguay and review of the literature.

Here we report the detection of a Leclercia adecarboxylata strain, isolated from a case of osteomyelitis, harbouring multiple antibiotic resistance genes encoded on a 450-kb IncHI1/HI2 conjugative plasmid (pLa12). The plasmid carried a complex class 1 integron with the genetic array intI1-aac(6')-Ib-cr-blaOXA-1-catB3-arr3-qacEΔ1-sul1-ISCR1; in addition, a blaDHA-1-like allele linked to ampR-qacEΔ1-sul1 as well as blaSHV-12, blaTEM-1 and qnrB4-like genes were found. To the best of our knowledge, this is the first report of L. adecarboxylata harbouring transferable resistance genes to quinolones, chloramphenicol and rifampicin as well as a plasmidic class C ß-lactamase.

Urinary tract infection in Uruguayan children: Aetiology, antimicrobial resistance and uropathogenic Escherichia coli virulotyping.

Uropathogenic Escherichia coli (UPEC) is the most frequent cause of urinary tract infection (UTI). Virulence factors (VFs) of UPEC in children are not well known. Circulating antibiotic resistance mechanisms in the community are increasing. In this study, the aetiological agents of UTI and antibiotic resistance mechanisms of 124 strains isolated from urine cultures from children with community-acquired UTI were determined. Virulotyping of isolated E. coli strains was also described. ß-Lactam, fluoroquinolone and sulfonamide resistance genes as well as integrons were detected by PCR. E. coli phylogenetic groups and 25 VFs were sought by multiplex PCR. E. coli was the most frequent aetiological agent (88.7%), of which 48.2% belonged to phylogenetic group D and 35.5% to group B2. Moreover, 81.8% were considered UPEC and >93% had virulence structures, with kpsMTII, fimH and iutA being the most frequent. Most of the E. coli isolates were susceptible to amoxicillin/clavulanic acid (AMC) (87.3%), nitrofurantoin (97.3%), cefuroxime and third-generation cephalosporins (100%). Resistance levels to oxyimino-cephalosporins were higher in non-E. coli isolates, with circulation of integrons, blaCTX-M-2 and blaCMY-2 detected in the community. Moreover, 8.1% of isolates were resistant to fluoroquinolones, with qnrB found in two isolates. Resistance to trimethoprim/sulfamethoxazole was found in 37.9% of isolates, with 85.5% harbouring sul genes. E. coli isolated from children with UTI presented high rates of VFs. Nitrofurantoin, AMC and cefuroxime would be suitable antibiotics to treat UTI in children. However, the presence of integrons (fundamentally class 1) and circulation of broad-spectrum ß-lactamases in the community makes continuous surveillance necessary.

Identification of the first blaCMY-2 gene in Salmonella enterica serovar Typhimurium isolates obtained from cases of paediatric diarrhoea illness detected in South America.

The objectives of this study were to investigate clinical isolates of Salmonella enterica serovar Typhimurium resistant to ß-lactam antibiotics, to characterise their mechanisms of antibiotic resistance and to evaluate the possible biological cost of expressing resistance genes. Two oxyimino-cephalosporin-resistant Salmonella isolates obtained from children with diarrhoea were characterised. The occurrence of plasmid-encoded blaCMY-2 genes was confirmed by molecular methods and conjugation assays; transcription levels were determined by quantitative real-time PCR (qRT-PCR). The genomic context of the ß-lactamases, replicon type and addiction systems were analysed by PCR. Genomic relatedness of both isolates was studied by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) assays. Growth curves, motility and invasiveness assays in Caco-2 cells were performed to analyse the bacterial fitness of both isolates. Both isolates carried a blaCMY-2-like allele in an IncI plasmid and belonged to the same MLST sequence type (ST19); nevertheless, they showed extensive differences in their PFGE profiles and virulotypes. Isolate STM709 appeared to lack the Salmonella virulence plasmid and displayed less motility and invasiveness in cultured cells than isolate STM910. qRT-PCR showed that isolate STM709 had higher blaCMY-2 mRNA levels compared with STM910. Altogether, the results suggest that a plasmid carrying blaCMY-2 could be disseminating among different clones of S. Typhimurium. Different levels of blaCMY-2 mRNA could have an effect on the fitness of this micro-organism, resulting in lower invasiveness and motility.