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The increasing prevalence and dissemination of carbapenemase-producing Enterobacterales represent a serious concern for public health. We studied the genetic features of a multidrug-resistant isolate of high-risk clone ST147 Klebsiella pneumoniae coharboring mcr-1 and blaNDM-1 recovered from a human clinical urine sample in 2017 in Peru. Whole-genome sequencing and conjugation assays identified mcr-1 and blaNDM-1 genes on two different conjugative plasmids, which belong to IncI2 and IncFIB/HI1B incompatibility groups, respectively. The presence of blaCTX-M-15 (in the studied isolate, located on the chromosome) and mutations in GyrA S83I and ParC S80I were detected, as expected for ST147. In addition, other β-lactamases (blaTEM-26 and blaOXA-1) and PMQR (qnrE2 and aac(6')-Ib-cr) among several resistance determinants were identified. The coexistence not previously described of these genes in the same high-risk clone is a cause for serious concern that supports the need for implementation of genomic surveillance studies.

Different phenotype-based techniques and molecular tools were used to describe the distribution of different Achromobacter species in patients with cystic fibrosis (CF) in Argentina, and to evaluate their antibiotic resistance profile. Phenotypic identification was performed by conventional biochemical tests, commercial galleries and MALDI-TOF MS. Genetic approaches included the detection of A. xylosoxidans specific marker blaoxa-114, the amplification and sequencing of the 16S rRNA gene, nrdA and blaOXA complete sequence, and MLST analysis. Phenotypic approaches, even MALDI-TOF, rendered inconclusive or misleading results. On the contrary, concordant results were achieved with the nrdA sequencing or sequence type (ST) analysis, and the complete blaOXA sequencing, allowing a reliable discrimination of different Achromobacter species. A. xylosoxidans accounted for 63% of Achromobacter infections and A. ruhlandii accounted for 17%. The remaining species corresponded to A. insuavis, A. dolens, A. marplatensis and A. pulmonis. Antimicrobial susceptibilities were determined by the agar dilution method according to CLSI guidelines. Piperacillin, piperacillin/tazobactam and carbapenems were the most active antibiotics. However, the emergence of carbapenem-resistant isolates was detected. In conclusion, prompt and accurate identification tools were necessary to determine that different Achromobacter species may colonize/infect the airways of patients with CF. Moreover, antimicrobial therapy should be administered based on the susceptibility profile of individual Achromobacter sp. isolates. Se emplearon diversas técnicas fenotípicas y moleculares para describir la distribución de diferentes especies del género Achromobacter en pacientes con fibrosis quística (FQ) en Argentina, y se evaluó el perfil de resistencia a los antibióticos. Se realizó la identificación fenotípica por pruebas bioquímicas convencionales, galerías comerciales y MALDI-TOF MS. El enfoque genético incluyó la detección del marcador especie-específico de A. xylosoxidans blaoxa-114, la amplificación y la secuenciación de los genes ARNr 16S, nrdA y secuencia completa de blaOXA, y el análisis por MLST. Los enfoques fenotípicos, incluso la técnica de MALDI-TOF, proporcionaron resultados no concluyentes o erróneos. Por el contrario, se obtuvieron resultados concordantes entre la secuenciación del gen nrdA o el análisis de secuenciotipos (ST) y la secuenciación completa de blaOXA, lo que permitió una discriminación confiable de las diferentes especies de Achromobacter. A. xylosoxidans representó el 63% de las infecciones por Achromobacter y A. ruhlandii representó el 17%. Las especies restantes correspondieron a A. insuavis, A. dolens, A. marplatensis y A. pulmonis. Se determinó la sensibilidad a antimicrobianos por el método de dilución en agar de acuerdo al CLSI. Los antibióticos más activos fueron piperacilina, piperacilina/tazobactam y carbapenemes. Sin embargo, se detectó la emergencia de aislamientos resistentes a carbapenemes. En conclusión, resultaron necesarias herramientas de identificación rápida y precisas para determinar las diferentes especies del género Achromobacter capaces de colonizar/infectar las vías respiratorias de los pacientes con FQ. Asimismo, la terapia antimicrobiana debería llevarse a cabo en función del perfil de sensibilidad de los aislamientos individuales de Achromobacter spp. Fil: Papalia, Mariana Andrea. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Steffanowski, Carla Giselle. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Traglia, German Matias. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Almuzara, Marisa. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Bioquímica Clínica; Argentina Fil: Martina, Pablo F.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Universidad Nacional de Misiones. Instituto de Biología Subtropical; Argentina Fil: Galanternik, Laura. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina Fil: Vay, Carlos. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Bioquímica Clínica; Argentina Fil: Gutkind, Gabriel Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Cátedra de Microbiología; Argentina Fil: Ramírez, María Soledad. California State University; Estados Unidos Fil: Radice, Marcela Alejandra. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina

Series of 1,3-dibenzyl-1H-4,5,6,7-tetrahydro-1,3-diazepinium and 1,4,5,6,7,8-hexahydro-1,3-diazocinium salts derivatives were efficiently synthesized in excellent yields by dehydrogenation of the corresponding N,N'-dibenzyl aminals employing N-bromosuccinimide (NBS) as dehydrogenating agent under ultrasound irradiation. The present methodology has proven to be simple, efficient and environmentally benign. All novel compounds were identified and characterized by 1H and 13C NMR spectra. The synthesized compounds were screened for their antimicrobial activities.

Changes in Kluyvera taxonomy may clarify each species contribution for recruitment and dissemination of their relevant β-lactamases. The CTX-M-2 subgroup is linked to Kluyvera ascorbata , KLUC to Kluyvera cryocrescens , and CTX-M-25 to Kluyvera georgiana .

In this study, we identified specific carbapenemase-producing isolates applying an easy and rapid protocol for the detection of mature KPC-2 β-lactamase by MALDI-TOF MS from colony and positive blood culture bottles. In addition, we evaluated the correlation of the ~11,109 Da signal as a biomarker associated with KPC-2 production. A collection of 126 well-characterized clinical isolates were evaluated (including 60 KPC-2-producing strains). Presence of KPC-2 was assessed by MALDI-TOF MS on protein extracts. Samples were prepared using the double layer sinapinic acid technique. In order to identify mature KPC-2, raw spectra were analyzed focusing on the range between m/z 25,000–30,000 Da. A single distinctive peak, at approximately m/z 28,544 Da was found in all clinical and control KPC-2-producing strains, and consistently absent in the control groups (ESBL producers and susceptible strains). This peak was detected in all species independently of where the gene bla KPC-2 was embedded. Statistical results showed 100% sensitivity, CI95%: [94.0%; 100%] and 100% specificity, CI95%: [94.6%; 100%], indicating a promising test with a high discriminative power. KPC-2 β-lactamase could be directly detected from both colonies and blood culture bottles. On the other hand, the m/z 11,109 Da signal determinant was only associated with 32% of Klebsiella pneumoniae and Escherichia coli KPC positive isolates. This MALDI-TOF MS methodology has the potential to detect directly the widespread and clinically relevant carbapenemase, KPC-2, in Enterobacterales with a straightforward, low cost process, assuming MALDI-TOF MS is already adopted as the main identification tool, with clear clinical implications on antibiotic stewardship for early infection treatment. Fil: Figueroa Espinosa, Roque Arnulfo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Costa, Agustina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Cejas, Daniela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Barrios, Rubén. No especifíca; Fil: Vay, Carlos Alberto. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Bioquímica Clínica; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina Fil: Radice, Marcela Alejandra. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Gutkind, Gabriel Osvaldo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Di Conza, José Alejandro. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina

Nosocomial infections caused by multidrug-resistant (MDR) Klebsiella pneumoniae are a major health problem worldwide. The aim of this study was to describe NDM-1-producing K. pneumoniae strains causing bacteremia in a tertiary referral hospital in Mexico. MDR K. pneumoniae isolates were screened by polymerase chain reaction for the presence of resistance genes. In resistant isolates, plasmids were identified and conjugation assays were performed. Clonal diversity and the sequence types were determined by pulsed-field gel electrophoresis and multilocus sequence typing. A total of 80 K. pneumoniae isolates were collected from patients with bacteremia over a 1-year period. These isolates showed a level of resistance of 59% (47/80) to aztreonam, 56–60% (45–48/80) to cephalosporins, 54% (43/80) to colistin and 12.5% (10/80) to carbapenems. The carbapenem resistant isolates were blaNDM–1 carriers and negative for blaKPC, blaNDM, blaIMP, blaVIM and blaOXA–48–like carbapenemases genes. Conjugative plasmids IncFIIA and IncF group with sizes of 82–195 kbp were carriers of blaNDM–1, blaCTX–M–15, blaTEM–1, aac(6′)-Ib and/or aac(3′)-IIa. Clonal variability and nine different multilocus sequence types were detected (ST661, ST683, ST1395, ST2706, ST252, ST1198, ST690, ST1535, and ST3368) for the first time in the isolates carrying blaNDM–1 in Mexico. This study demonstrates that blaNDM–1 has remained within this hospital in recent years and suggests that it is currently the most prevalent carbapenemase among K. pneumoniae MDR strains causing bacteremia in Mexico. The horizontal transfer of blaNDM–1 gene through IncF-like plasmids among different clones demonstrates the dissemination pathway of antimicrobial resistance and underscore the need for strong and urgent joint measures to control the spread of NDM-1 carbapenemase in the hospital.

The widespread use of antimicrobial therapy in companion animals could lead to increased levels of resistance. Monitoring these levels is critical to understand not only the zoonotic threat of resistant bacteria but also the interspecies transmission of resistance mechanisms. However, data on resistance levels in companion animals of urban areas in Latin America are lacking. In this retrospective study, we analysed 23,922 isolates recovered from clinical samples of dogs and cats between 2011 and 2017. Growing trends of resistance to fluoroquinolones were observed in most bacterial species of veterinary importance with zoonotic potential (Enterobacterales, Pseudomonas and Staphylococcus). Furthermore, an increase of resistance levels to third-generation cephalosporins was also detected in Enterobacterales. Currently, Pseudomonas aeruginosa, Enterococcus spp. and Streptococcus spp. did not seem to represent a clinical challenge. A high proportion of multidrug-resistant (MDR) Enterobacterales isolated from urine was reported. It is interesting to outline that resistance to amikacin was exceptional. This study might be considered as a baseline for prospective antimicrobial resistance surveillance in companion animals in our region.

Novel variants of OXA-48-type enzymes with the ability to hydrolyze oxyimino-cephalosporins and carbapenems are increasingly reported. Since its first report in 2011, OXA-163 is now extensively spread throughout Argentina, and several variants like OXA-247 have emerged. Here, we characterized a new blaOXA-48-like variant, OXA-438, and we performed a comparative kinetic analysis with the local variants OXA-247 and OXA-163 and the internationally disseminated OXA-48. blaOXA-163, blaOXA-247, and blaOXA-438 were located in a 70 kb IncN2 conjugative plasmid. OXA-438 presented mutations in the vicinity of conserved KTG (214-216), with a 2-aa deletion (R220-I221) and a D224E shift (as in OXA-163) compared to OXA-48. Despite Kpn163 (OXA-163), Kpn247 (OXA-247) and Eco438 (OXA-438) were resistant to meropenem and ertapenem, and the transconjugants (TC) remained susceptible (however, the carbapenems minimum inhibitory concentrations were ≥3 times 2-fold dilutions higher than the acceptor strain). TC163 and Eco48 were resistant to oxyimino-cephalosporins, unlike TC247 and TC438. kcat/Km values for cefotaxime in OXA-163 were slightly higher than the rest of the variants that were accompanied by a lower Km for carbapenems. For OXA-163, OXA-247, and OXA-438, the addition of NaHCO3 improved kcat values for both cefotaxime and ceftazidime; carbapenems kcat/Km values were higher than for oxyimino-cephalosporins. Mutations occurring near the conserved KTG in OXA-247 and OXA-438 are probably responsible for the improved carbapenems hydrolysis and decreased inactivation of oxyimino-cephalosporins compared to OXA-163. Dichroism results suggest that deletions at the β5-β6 loop seem to impact the structural stability of OXA-48 variants. Finally, additional mechanisms are probably involved in the resistance pattern observed in the clinical isolates. Fil: de Belder, Denise Gisele. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas. Área de Antimicrobianos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Ghiglione, Barbara. Instituto de Investigaciones En Bacteriologia y Virologia Molecular (ibavim) ; Facultad de Farmacia y Bioquimica ; Universidad de Buenos Aires; . Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Pasteran, Fernando. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas. Área de Antimicrobianos; Argentina Fil: de Mendieta, Juan Manuel. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas. Área de Antimicrobianos; Argentina Fil: Corso, Alejandra. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas. Área de Antimicrobianos; Argentina Fil: Curto, Lucrecia María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Di Bella, Adriana. Hospital Nacional Profesor Alejandro Posadas; Argentina Fil: Gutkind, Gabriel Osvaldo. Instituto de Investigaciones En Bacteriologia y Virologia Molecular (ibavim) ; Facultad de Farmacia y Bioquimica ; Universidad de Buenos Aires; . Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Gómez, Sonia Alejandra. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas. Área de Antimicrobianos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Power, Pablo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina. Instituto de Investigaciones En Bacteriologia y Virologia Molecular (ibavim) ; Facultad de Farmacia y Bioquimica ; Universidad de Buenos Aires

Serratia fonticola is a human pathogen widely found in the environment, with birds being reported as possible natural hosts. During an epidemiological and genomic surveillance study conducted to monitor the occurrence of extended-spectrum b-lactamase (ESBL)-producing Enterobacterales in South American wild birds, we identified an ESBL-positive S. fonticola in a fecal sample collected from a Hudsonian Whimbrel, during its non-breeding range on the Pacific Coast of Chile. Whole genome sequencing analysis and in silico modeling revealed a novel variant of the class A ESBLs FONA family, designated FONA-7, which shows 96.28% amino acid identity with FONA-6; with amino acid substitutions occurring in the signal peptide sequence (Thr22/Ser), and in the mature protein (Ser39/Asn and Thr227/Ile). This finding denotes that migratory birds can be potential vectors for the transboundary spread of ESBL-producing bacteria, creating a further theoretical riskfor the origin of novel plasmid-encoded b-lactamases. Fil: Fuentes Castillo, Danny. Universidade de Sao Paulo; Brasil Fil: Power, Pablo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Cerdeira, Louise. Universidade de Sao Paulo; Brasil Fil: Cardenas-Arias, Adriana. Universidade de Sao Paulo; Brasil Fil: Moura, Quézia. Universidade de Sao Paulo; Brasil Fil: Oliveira, Flavio A.. Universidade Estadual de Campinas; Brasil Fil: Levy, Carlos E.. Universidade Estadual de Campinas; Brasil Fil: Gutkind, Gabriel Osvaldo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Catão-Dias, José L.. Universidade de Sao Paulo; Brasil Fil: Lincopan, Nilton. Universidade de Sao Paulo; Brasil

In the last decade Achromobacter spp. has been associated with chronic colonization in patients with cystic fibrosis (CF). Although Achromobacter xylosoxidans is the most frequent species recovered within this genus, other species such as A. ruhlandii have also been reported in these patients. Descriptions of mobile elements are scarce in Achromobacter and none of them have been originated in A. ruhlandii. The aim of this study was to report the full characterization of a plasmid which was maintained in four clonally related A. ruhlandii isolates. Between 2013 and 2015, nine A. ruhlandii isolates were recovered from a pediatric patient with CF at a hospital in Buenos Aires. Four selected clonally related isolates were sequenced by Illumina MiSeq, annotated using RAST and manually curated. The presence of a unique plasmid of 34096-bp and 50 CDS was observed in the four isolates, displaying only 1 nucleotide substitution translated into one amino acid change among them. These plasmids have a class 1 integron containing the aac-(6′)-Ib gene, a mercury resistance operon region and the relE/stbE toxin/antitoxin system. Plasmids showed 79% similarity and 99% identity with pmatvim-7 from Pseudomonas aeruginosa. This is the first full description and characterization of a plasmid from A. ruhlandii which was maintained over time.

Introduction: Metallo-â-lactamases (MBL) producing Pseudomonas aeruginosa isolates have been well characterised. Quionolones are commonly used in the treatment of carbapenem resistant P. aeruginosa infections; however, data about PMQR in this species is scarce. The objective was to report the simultaneous presence of qnrS and blaVIM-11 in P. aeruginosa, and to characterize the qnrS harbouring plasmid. Methods: Thirty-eight carbapenem resistant P. aeruginosa isolates were recovered at a hospital in Buenos Aires during 2012. Screening for MBL was assessed by double disc synergy test using EDTA and carbapenem discs. Plasmid DNA extraction was performed by a method using phenol44 chloroform. PCR followed by sequencing was carried out to determine each MBL and PMQR allele. PCR-BseGI-RFLP was performed to detect aac-(6?)-Ib-cr. The gyrA-QRDR was sequenced in those PMQR harbouring isolates. Plasmid incompatibility groups and addiction systems were characterised by PCR. The PMQR carrying plasmid was sequenced using Illumina technology, annotated using RAST and manually curated.Results: Eleven/38 isolates were VIM producers (blaVIM-2 and blaVIM-11) while 1/38 harboured blaIMP-13. One isolate harboured blaVIM-11 and the PMQR qnrS1, however, both markers were located in different plasmids. The qnrS1 harbouring plasmid (pP6qnrS1) was 117.945 bp in size, presented 154 CDS and corresponded to IncR group. In addition to qnrS1, it harboured several aminoglycoside resistancemarkers. Despite being pP6qnrS1 non-conjugative, it presented an OriT which makes it possible for this plasmid to be transferable.Conclusions: This is the first report on P. aeruginosa carrying both blaVIM-11 and qnrS1, plus the first detection of an IncR plasmid in Argentina. Las quinolonas son comúnmente utilizadas en el tratamiento de las infecciones producidas por Pseudomonas aeruginosa resistentes a carbapenems (PARC); aun así, la información sobre la resistencia a quinolonas mediada por plásmidos (PMQR) en esta especie es escasa. El objetivo de este trabajo fue reportar la presencia simultánea de los genes qnrS y blaVIM-11 en PARC y caracterizar el plásmido portador de qnrS. Durante 2012 se recuperaron 38 PARC en un hospital de Buenos Aires. El tamizaje para detectar producción de metalo-beta-lactamasas (MBL) se llevó a cabo mediante sinergia de doble disco utilizando EDTA y carbapenems. El ADN plasmídico fue extraído utilizando fenolcloroformo. Para determinar los alelos de los genes implicados en la síntesis de MBL y de PMQR, se llevó a cabo PCR-secuenciación. Para la detección de aac-(6 )-Ib-cr se realizó PCR-BseGIRFLP. En aquellos aislamientos portadores de PMQR se secuenció el gen gyrA. Los grupos de incompatibilidad y sistemas de adicción fueron caracterizados por PCR. El plásmido portador de PMQR fue secuenciado completamente y curado manualmente. De 38 aislamientos, 11 fueron productores de VIM (blaVIM-2 y blaVIM-11), mientras que uno contenía blaIMP-13. Si bien un aislamiento fue portador de blaVIM-11 y de qnrS1, dichos marcadores se encontraban en distintos plásmidos. El plásmido portador de qnrS1 (pP6qnrS1) presentó un tamano˜ de 117.945 pb y 154 secuencias codificantes (CDS); este correspondió al grupo de incompatibilidad IncR. Además de qnrS1, el plásmido portaba diversos marcadores de resistencia a aminoglucósidos. Aun cuando pP6qnrS1 no resultó conjugativo, presentó un oriT, de modo que posiblemente sea transferible. Este es el primer informe acerca de PARC portadora de blaVIM-11 y de qnrS1 en simultáneo, además, es la primera descripción de un plásmido IncR en Argentina. Fil: Elena, Alan Xavier. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Quinteros, Mirta. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas "Dr. Francisco Javier Muñiz"; Argentina Fil: Di Conza, José Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina Fil: Gutkind, Gabriel Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina Fil: Cejas, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina Fil: Radice, Marcela Alejandra. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

The aim of this work was to evaluate an easy-to-perform assay basedupon inhibition of mobile colistin resistance (MCR) activity by EDTA. We included 92nonrelated isolates of Enterobacteriaceae (74 Escherichia coli, 17 Klebsiella pneumoniae,and 1 Serratia marcescens). Our proposed method is based on a modificationof the colistin agar-spot screening test (CAST), a plate containing 3 g/ml colistin,by adding an extra plate of colistin agar-spot supplemented with EDTA (eCAST).Bacterial growth was evaluated after 24 h of incubation at 35°C. All the colistinresistantisolates showed development on the CAST plates. Colistin-resistant K. pneumoniaewithout mcr-1 and S. marcescens also grew on the eCAST plates. In contrast,colistin-resistant MCR-producing E. coli was not able to grow in eCAST plates. Thecombined CAST/eCAST test could provide a simple and easy-to-perform method todifferentiate MCR-producing Enterobacteriaceae from those in which colistin resistanceis mediated by chromosomal mechanisms. Fil: Gonzales Escalante, Edgar. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina Fil: Yauri Condor, Katherine. Universidad Nacional Mayor de San Marcos; Perú Fil: Di Conza, José Alejandro. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Gutkind, Gabriel Osvaldo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina

Different MALDI-TOF MS databases were evaluated for the identification of Achromobacter species. The in-house and extended database generated in this study rendered more accurate identification (58/64 and 57/64 isolates,respectively) in comparison with the Bruker commercial database (42/64 isolates), especially in those infrequent species that are not available or poorly represented. Fil: Papalia, Mariana Andrea. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Figueroa Espinosa, Roque Arnulfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Steffanowski, Carla Giselle. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Barberis, Claudia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Almuzara, Marisa. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Barrios, Rubén. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Vay, Carlos. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Gutkind, Gabriel Osvaldo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Di Conza, José Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Radice, Marcela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina

This paper describes our findings regarding the accumulation of motile bacteria at the rear of a confined obstacle and the physical description of the mechanisms at play. We found that the modification of flow due to the presence of the obstacle produces vorticity that favor the diffusion of bacteria towards the downstream stagnation point. By testing different flow rates, we determined the range in which bacteria accumulate. More interestingly, we observe that hydrodynamic interaction between the bacteria and the top and bottom surface of the microfluidic chip maintain the bacteria in the region where the flow velocity is lower than their own velocity. In the case of non-motile bacteria, this effect is not observed because bacteria follow the streamlines as passive tracers do.

The objectives of this study were to determine the kinetic parameters of purified recombinant BlaMab and BlaMmas by spectrophotometry, analyze the genetic environment of the blaMab and blaMmas genes in both species by polymerase chain reaction and sequencing, furthermore, in silico models of both enzymes in complex with imipenem were obtained by modeling tools. Our results showed that BlaMab and BlaMmas have a similar hydrolysis behavior, displaying high catalytic efficiencies toward penams, cephalothin, and nitrocefin; none of the enzymes are well inhibited by clavulanate. BlaMmas hydrolyzes imipenem at higher efficiency than cefotaxime and aztreonam. BlaMab and BlaMmas showed that their closest structural homologs are KPC-2 and SFC-1, which correlate to the mild carbapenemase activity toward imipenem observed at least for BlaMmas. They also seem to differ from other class A β-lactamases by the presence of a more flexible Ω loop, which could impact in the hydrolysis efficiency against some antibiotics. A -35 consensus sequence (TCGACA) and embedded at the 3' end of MAB_2874, which may constitute the blaMab and blaMmas promoter. Our results suggest that the resistance mechanisms in fast-growing mycobacteria could be probably evolving toward the production of β-lactamases that have improved catalytic efficiencies against some of the drugs commonly used for the treatment of mycobacterial infections, endangering the use of important drugs like the carbapenems.

The diazabicyclooctane (DBO) avibactam (AVI) reversibly inactivates most serine-lactamases. Previous investigations showed that inhibition constants of AVI toward class A PER-2 are reminiscent of values observed for class C and D -lactamases (i.e., k2/K of 103 M1 s1) but lower than other class A -lactamases (i.e., k2/K 104 to 105 M1 s1). Herein, biochemical and structural studies were conducted with PER-2 and AVI to explore these differences. Furthermore, biochemical studies on Arg220 and Thr237 variants with AVI were conducted to gain deeper insight into the mechanism of PER-2 inactivation. The main biochemical and structural observations revealed the following: (i) both amino-acid substitutions in Arg220 and the rich hydrophobic content in the active site hinder the binding of catalytic waters and acylation, impairing AVI inhibition; (ii) movement of Ser130 upon binding of AVI favors the formation of a hydrogen bond with the sulfate group of AVI; and (iii) the Thr237Ala substitution alters the AVI inhibition constants. The acylation constant (k2/K) of PER-2 by AVI is primarily influenced by stabilizing hydrogen bonds involving AVI and important residues such as Thr237 and Arg220. (Variants in Arg220 demonstrate a dramatic reduction in k2/K.) We also observed that displacement of Ser130 side chain impairs AVI acylation, an observation not made in other extended-spectrum -lactamases (ESBLs). Comparatively, relebactam combined with a -lactam is more potent against Escherichia coli producing PER-2 variants than -lactam–AVI combinations. Our findings provide a rationale for evaluating the utility of the currently available DBO inhibitors against unique ESBLs like PER-2 and anticipate the effectiveness of these inhibitors toward variants that may eventually be selected upon AVI usage. Fil: Ruggiero, Melina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina Fil: Papp Wallace, Krisztina M.. Case Western Reserve University. School of Medicine; Estados Unidos. Louis Stokes Cleveland VA Medical Center; Estados Unidos Fil: Brunetti, Florencia Lourdes. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Barnes, Melissa D.. Case Western Reserve University. School of Medicine; Estados Unidos. Louis Stokes Cleveland VA Medical Center; Estados Unidos Fil: Bonomo, Robert A.. Louis Stokes Cleveland VA Medical Center; Estados Unidos. Case Western Reserve University. School of Medicine; Estados Unidos. Center for Antimicrobial Resistance and Epidemiology; Estados Unidos Fil: Gutkind, Gabriel Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina Fil: Klinke, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Plataforma Argentina de Biología Estructural y Metabolómica; Argentina Fil: Power, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina

Objectives: To assess the epidemiological features of 76 Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp) isolates recovered from three hospitals in Buenos Aires, Argentina, during 2015–2017. Methods: Antimicrobial susceptibilities were determined according to CLSI Clinical and Laboratoy Standards guidelines. Molecular typing of KPC-Kp was performed by pulsed-field gel electrophoresis (PFGE)-Xbal and multilocus sequence typing. Plasmid encoded genes involved in carbapenem, fosfomycin and colistin resistance were detected by polymerase chain reaction (PCR) and sequencing. Also, mgrB inactivation was investigated in those colistin-resistant isolates. Genetic platforms involved in horizontal spread of blaKPC were investigated by PCR mapping. Results: Besides β-lactams, high resistance rates were observed for gentamycin, quinolones and trimethoprim-sulfamethoxazole. KPC-Kp sequence type (ST)258 corresponded to 26% of the isolates, while 42% corresponded to ST25. The other isolates were distributed in a diversity of lineages such as ST11 (10.5%), ST392 (10.5%), ST307, ST13, ST101, ST15 and ST551. blaKPC-2 was detected in 75 of 76 isolates, and one ST307 isolate harboured blaKPC-3. Tn4401 was identified as the genetic platform for blaKPC in epidemic lineages such as ST258 and ST307. However, in ST25 and ST392, which are usually not related to blaKPC, a blaKPC-bearing non-Tn4401 element was identified. Alterations in mgrB were detected in seven of 11 colistin-resistant isolates. Conclusions: Despite previous reports in Argentina, ST258 is no longer the absolute clone among KPC-Kp isolates. In the present study, dissemination of more virulent lineages such as the hypermucoviscous ST25 was detected. The emergence of the high-risk clone ST307 and occurrence of blaKPC-3 was noticed for the first time in this region. Fil: Cejas, Daniela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Elena, Alan Xavier. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Nuñez, Daiana Guevara. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina Fil: Platero, Priscila Sevillano. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina. Universidad del Salvador; Argentina Fil: De Paulis, Adriana. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina Fil: Magariños, Francisco. Gobierno de la Ciudad de Buenos Aires. Hospital de Agudos "D. F. Santojanni"; Argentina Fil: Alfonso, Claudia. Gobierno de la Ciudad de Buenos Aires. Hospital de Agudos "D. F. Santojanni"; Argentina Fil: Berger, María Alejandra. Hospital Aleman; Argentina Fil: Fernández Canigia, Liliana. Hospital Aleman; Argentina Fil: Gutkind, Gabriel Osvaldo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Radice, Marcela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina

The aim of this study was to investigate the presence of class 1 integrons in a collection of Shiga toxin-producing Escherichia coli (STEC) from different origins and to characterize pheno- and genotypically the antimicrobial resistance associated to them. A collection of 649 isolates were screened for the class 1 integrase gene (intI1) by Polymerase chain reaction The variable region of class 1 integrons was amplified and sequenced. Positive strains were evaluated for the presence of antimicrobial resistance genes with microarray and for antimicrobial susceptibility by the disk diffusion method. Seven out of 649 STEC strains some to serogroups, O26, O103 and O130 isolated from cattle, chicken burger, farm environment and pigs were identified as positive for intl1. Different arrangements of gene cassettes were detected in the variable region of class 1 integron: dfrA16, aadA23 and dfrA1-aadA1. In almost all strains, phenotypic resistance to streptomycin, tetracycline, trimethoprim/sulfamethoxazole, and sulfisoxazole was observed. Microarray analyses showed that most of the isolates carried four or more antimicrobial resistance markers and STEC strains were categorized as Multridrug-resistant. Although antimicrobials are not usually used in the treatment of STEC infections, the presence of Multridrug-resistant in isolates collected from farm and food represents a risk for animal and human health.

Introduction: To characterize extended-spectrum β-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. Fil: Vignoli, Rafael. Universidad de la República; Uruguay Fil: García Fulgueiras, Virginia. Universidad de la República; Uruguay Fil: Cordeiro, Nicolás F.. Universidad de la República; Uruguay Fil: Bado, Inés. Universidad de la República; Uruguay Fil: Seija, Verónica. Universidad de la República; Uruguay. Hospital Pasteur de Montevideo; Uruguay Fil: Aguerrebere, Paula. Universidad de la República; Uruguay Fil: Laguna, Gabriel. Universidad de la República; Uruguay Fil: Araújo, Lucía. Universidad de la República; Uruguay Fil: Bazet, Cristina. Universidad de la República; Uruguay Fil: Gutkind, Gabriel Osvaldo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Chabalgoity Rodríguez, José Alejandro. Universidad de la República; Uruguay

Extended-spectrum β-lactamase (ESBL), plasmid-mediated AmpC (pAmpC) and MCR-1 phosphoethanolamine transferase enzymes have been pointed out as the main plasmid-mediated mechanisms of resistance to third generation cephalosporins (TGC) and colistin, respectively, and are currently considered a major concern both in human and veterinary medicine. Little data on these resistance determinants prevalence in companion animal infections is available. The aim of this study was to determine the resistance profile of Escherichia coli isolated from pet infections, in Argentina, and to characterize the resistance mechanisms to TGC, as well as the presence of the plasmid-borne colistin resistance gene, mcr-1. A total of 54 E. coli isolates were collected from clinical samples in dogs and cats; from them, 20/54 (37%, CI95: [24%; 51%]) displayed resistance to TGC. In this regard, thirteen pAmpC-producing isolates were positive for blaCMY-2 genes, whereas seven ESBL- producers harboured blaCTX-M-2 (n = 4), blaCTX-M-15 (n = 2) and blaCTX-M-14 (n = 1) genes. One E. coli strain (V80), isolated from a canine urinary tract infection, showed resistance to colistin (MIC = 8 μg/ml) and whole-genome sequencing analysis revealed co-occurrence of mcr-1.1, blaCTX-M-2, aadA1, ant(2′')-Ia, catA1 and sul1 genes; the former being carried by a 60,587-bp IncI2 plasmid, previously reported in human colistin-resistant E. coli. E. coli V80 belonged to ST770 and the highly virulent phylogenetic group B2. In general, most of these multidrug-resistant isolates belonged to the phylogenetic group F (11/20) and to a lesser extent B2 (5/20), B1 (2/20), D (1/20) and E (1/20). In summary, CMY- and CTX-M-type β-lactamases may constitute the main TGC resistance mechanism in E. coli isolated from pet infections in Argentina, whereas dissemination of colistin resistance mechanism MCR-1 in the human-animal interface has been mediated by IncI2 plasmids.

The emergence and dissemination of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) is a worrisome public health issue compromising the treatment and outcome of infections caused by this pathogen. We performed a detailed virulome and resistome analysis of representative KPC- and/or CTX-M-producing K. pneumoniae belonging to clonal group (CG) 258 (sequence types ST11, ST258, ST340, ST437), circulating in Argentina, Brazil, Chile, Colombia and Peru; with further evaluation of the virulence behavior using the Galleria mellonella infection model. Genomic analysis of K. pneumoniae strains recovered from the human-animal-environment interface revealed a wide resistome characterized by the presence of genes and mutations conferring resistance to human and veterinary antibiotics, quaternary ammonium compounds (QACs) and heavy metals. Plasmid Inc typing revealed the presence of a wide diversity of replicon types with IncF, IncN, IncR and Col-like being frequently detected. Moreover, KPC-2-producing K. pneumoniae belonging to ST11 (KL-64 andKL-105) and ST340 (KL-15) carried multiple variants of distinct yersiniabactin siderophore (ybt) and/or genotoxic colibactin (clb) genes. In this regard, ICEKp3, ICEKp4 and ICEKp12 were identified in strains belonging to ST11 and ST340, recovered from Argentina, Brazil, Chile and Colombia; whereas ybt 17 and a novel ybt sequence type (YbST346) were identified together with clb in ICEKp10 structures from ST11 and ST258, from Brazil and Colombia, respectively. K. pneumoniae ST11 (ICEKp10/YbST346 and ICEKp4/ybt 10) strains killed 100% of wax moth larvae, in a similar way to hypervirulent K1/ST23 strain (ybt- and clb-negative) carrying the pLVPK-like plasmid, indicating enhanced virulence. In summary, our results indicate that yersiniabactin, colibactin and an expanded resistome have contributed to enhanced virulence and persistence of KPC-2-producing K. pneumoniae CG258 in South America. Therefore, active surveillance of hospital-associated lineages of K. pneumoniae should not only focus on clonal origin and antimicrobial resistance, but also on the virulence factors ybt and clb.

Objectives: The aim of this study was to characterise OXA-258 variants and other features that may contribute to carbapenem resistance in Achromobacter ruhlandii. Methods: Kinetic parameters for purified OXA-258a and OXA-258b were determined measuring the rate of hydrolysis of a representative group of antimicrobial agents. Whole-genome shotgun sequencing was performed on A. ruhlandii 38 (producing OXA-258a) and A. ruhlandii 319 (producing OXA-258b), and in silico analysis of antimicrobial resistance determinants was conducted. Substrates of the AxyABM efflux pump were investigated by inhibition assays using phenylalanine-arginine β-naphthylamide (PAβN). Outer membrane protein profiles were resolved by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results: Kinetic measurements of purified OXA-258 variants displayed an overall weak catalytic efficiency toward β-lactams. A detectable hydrolysis of imipenem was observed. In silico genomic analysis confirmed the presence of 32 and 35 putative efflux pump-encoding genes in A. ruhlandii strains 38 and 319, respectively. Complete sequences for AxyABM and AxyXY efflux pumps, previously described in Achromobacter xylosoxidans, were detected. Decreases in the MICs for chloramphenicol, nalidixic acid and trimethoprim/sulfamethoxazole were observed in the presence of the inhibitor PAβN, suggesting that these antibiotics are substrates of AxyABM. AxyXY-encoding genes of A. ruhlandii 38 and A. ruhlandii 319 displayed 99% identity. No differences were observed in the outer membrane protein profiles. Conclusions: The contribution of OXA-258 enzymes to the final β-lactam resistance profile may be secondary. Further studies on other putative resistance markers identified in the whole-genome analysis should be conducted to understand the carbapenem resistance observed in A. ruhlandii. Fil: Papalia, Mariana Andrea. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Traglia, German Matias. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Ruggiero, Melina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Almuzara, Marisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Vay, Carlos Alberto. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Gutkind, Gabriel Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Ramírez, María Soledad. California State University; Estados Unidos Fil: Radice, Marcela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina

The natural diversification of CTX-M -lactamases led to the emergence of Asp240Gly variants in the clinic that confer reduced susceptibility to ceftazidime (CAZ). In this study, we compared the impact of this substitution on CAZ and ceftazidime-avibactam (CZA) MICs against isogenic Escherichia coli strains with different porin deficiencies. Our results show a noticeable increase in CAZ resistance in clones expressing Asp240Gly-harboring CTX-M when combined with OmpF porin deficiency. Kinetic analysis revealed that the kcat/Km for CAZ was 5- to 15-fold higher for all Asp240Gly variants but remained 200- to 725-fold lower than that for cefotaxime (CTX). In vitro selection of CAZ-resistant clones yielded nonsusceptible CTX-M producers (MIC of 16 g/ml) only after overnight incubation; the addition of avibactam (AVI) decreased MICs to a susceptible range against these variants. In contrast, the use of CZA as a selective agent did not yield resistant clones. AVI inactivated both CTX-M-12 and CTX-M-96, with an apparent inhibition constant comparable to that of SHV-2 and 1,000-fold greater than that of PER-2 and CMY-2, and k2/K for CTX-M-12 was 24- and 35-fold higher than that for CTX-M-96 and CTX-M-15, respectively. Molecular modeling suggests that AVI interacts similarly with CTX-M-96 and CTX-M-15. We conclude that the impact of Asp240Gly in resistance may arise when other mechanisms are also present (i.e., OmpF deficiency). Additionally, CAZ selection could favor the emergence of CAZ-resistant subpopulations. These results define the role of Asp240 and the impact of the -Gly substitution and allow us to hypothesize that the use of CZA is an effective preventive strategy to delay the development of resistance in this family of extended-spectrum -lactamases. Fil: Ghiglione, Barbara. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Rodríguez, María Margarita. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Curto, Lucrecia María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Brunetti, Florencia Lourdes. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Dropa, Milena. Universidade de Sao Paulo; Brasil Fil: Bonomo, Robert A.. Case Western Reserve University School of Medicine; Estados Unidos. Louis Stokes Cleveland VA Medical Center; Estados Unidos Fil: Power, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Gutkind, Gabriel Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina

The bla PER-2 -harboring plasmid pCf587 (191,541 bp) belongs to lineage IncA/C 1 and is closely related to pRA1. It contains a large resistance island including the bla PER-2 gene between two copies of ISKox2-like elements, the toxin-antitoxin module pemK-pemI, several other resistance genes inserted within a Tn2 transposon, a Tn21-like structure, and a class 1 integron. pCf587 belongs to sequence type 13 (ST13), a new plasmid multilocus sequence typing (pMLST) ST. Fil: Ruggiero, Melina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Cátedra de Microbiología; Argentina Fil: Girlich, Delphine. Université Paris Sud; Francia Fil: Dabos, Laura. Université Paris Sud; Francia Fil: Power, Pablo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Naas, Thierry. Associated French National Reference Center for Antibiotic Resistance “Carbapenemase -producing Enterobacteriaceae; Francia. Université Paris Sud; Francia Fil: Gutkind, Gabriel Osvaldo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Thirty one C. difficile isolates recovered in 2015 were characterized. Nineteen/31 were positive for tcdA/B, among them, 4 isolates were also positive for CDT coding genes. Two/4 cdtA/B positives isolates corresponded to ST 1 resembling BI/NAP1/027/ST 1 strain, while the others corresponded to ST 226 and ST 377. Fil: Cejas, Daniela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ríos Osorio, Néstor Raúl. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Quirós, Rodolfo. Universidad Austral. Hospital Universitario Austral; Argentina Fil: Sadorin, Roxana. Universidad Austral. Hospital Universitario Austral; Argentina Fil: Berger, María Alejandra. Hospital Alemán; Argentina Fil: Gutkind, Gabriel Osvaldo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Fernández Canigia, Liliana. Hospital Alemán; Argentina Fil: Radice, Marcela Alejandra. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Fast typing methods for third generation cephalosporin resistance mechanisms are needed to guide appropriate treatment and prevent potential dissemination events. In this study we used a novel short and fast methodology for the identification of CMY-2 in 50 well characterized clinical isolates of E. coli by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry- MALDI-TOF MS. Samples were prepared using the double layer sinapinic acid technique for detection of intact proteins Comparison among mass spectral profile of different strains between m/z 35,000–45,000 Da showed that two groups of isolates could be differentiated after peak analysis. A single distinctive peak with different intensities, at approximately m/z 39,800 Da was found in all CMY-2 producing strains (transconjugant, transformant and wild type) and consistently absent in the control groups (ESBL producers and susceptible strains). Statistical results showed 100% values for sensitivity and specificity, indicating a perfect test and a high discriminative power. In this study, we demonstrated that MALDI-TOF MS has the potential to detect directly the most clinically relevant acquired AmpC β-lactamase, the CMY-2-enzyme, in E. coli with a less time-consuming process as compared to conventional methods. Our results may constitute the basis for further research to detect other β-lactamases, or even other resistance markers. Fil: Figueroa Espinosa, Roque Arnulfo. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Rumi, María Valeria. Universidad de Buenos Aires; Argentina Fil: Marchisio, Martin Luis. Universidad Nacional del Litoral; Argentina Fil: Cejas, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires; Argentina Fil: Radice, Marcela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires; Argentina Fil: Vay, Carlos Alberto. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina. Universidad de Buenos Aires; Argentina Fil: Barrios, Rubén. Bd Life Sciences; Argentina Fil: Gutkind, Gabriel Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires; Argentina Fil: Di Conza, José Alejandro. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

CfiA (CcrA) metallo-β-lactamase is the main carbapenem resistance mechanism in B. fragilis. From cfiA positive isolates detected in a previous surveillance study, 3 displayed resistance to imipenem while the remaining were susceptible. The aim of this study was to identify the cfiA alleles and to analyze the presence of IS elements in their upstream regions. CfiA-1, CfiA-4, CfiA-13, CfiA-19 and CfiA-22 were detected. IS elements belonging to IS21 family and IS942 group were identified upstream to cfiA in the 3 imipenem resistant isolates. We present an exhaustive analysis of cfiA/CfiA registers in databases, illustrating the inconsistencies in both organization and nomenclature. According to this analysis CfiA family comprises nowadays 15 different CfiA variants coded by 24 cfiA sequences. Curation of CfiA database is mandatory, if not new cfiA admission at GenBank will contribute to make this classification more complex. Fil: Litterio, Mirta R.. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina Fil: Cejas, Daniela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Gutkind, Gabriel Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Radice, Marcela Alejandra. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

CTX-M-type extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae have been increasingly identified in humans and animals, and their potential transmission by contaminated food has been highlighted. In this study, we report for the first time the isolation of multidrug-resistant (MDR) Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis strains harboring blaCTXM-2 or blaCTXM-8 gene variants in chicken meat sold in markets in southeast Brazil. In this regard, the genetic environment of the blaCTX-M-2 gene is composed of a complex class 1 integron and an ISCR1-associated sequence with dfr and/or aadA gene cassettes located within the variable region. In summary, chicken meat may be a reservoir of MDR Enterobacteriaceae harboring blaCTX-M-type genes, which is a public health concern.

Fil: de la Torre, Eulalia. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Tandil. Centro de Investigacion Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigacion Veterinaria de Tandil. Provincia de Buenos Aires. Gobernacion. Comision de Investigaciones Cientificas. Centro de Investigacion Veterinaria de Tandil; Argentina

PER-2 accounts for up to 10% of oxyimino-cephalosporin resistance in Klebsiella pneumoniae and Escherichia coli in Argentina and hydrolyzes both cefotaxime and ceftazidime with high catalytic efficiencies (kcat/Km). Through crystallographic analyses, we recently proposed the existence of a hydrogen bond network connecting Ser70-Gln69-oxyanion water-Thr237-Arg220 that might be important for the activity and inhibition of the enzyme. Mutations at Arg244 in most class A β -lactamases (such as TEM and SHV) reduce susceptibility to mechanism-based inactivators, and Arg220 in PER β -lactamases is equivalent to Arg244. Alterations in the hydrogen bond network of the active site in PER-2, through modifications in key residues such as Arg220 and (to a much lesser extent) Thr237, dramatically impact the overall susceptibility to inactivation, with up to 300-And 500-fold reductions in the rate constant of inactivation (kinact)/Kivalues for clavulanic acid and tazobactam, respectively. Hydrolysis on cephalosporins and aztreonam was also affected, although to different extents compared to with wild-Type PER-2; for cefepime, only an Arg220Gly mutation resulted in a strong reduction in the catalytic efficiency. Mutations at Arg220 entail modifications in the catalytic activity of PER-2 and probably local perturbations in the protein, but not global conformational changes. Therefore, the apparent structural stability of the mutants suggests that these enzymes could be possibly selected in vivo. Fil: Ruggiero, Melina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Curto, Lucrecia María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina Fil: Brunetti, Florencia Lourdes. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Sauvage, Eric. Université de Liège; Bélgica Fil: Galleni, Moreno. Université de Liège; Bélgica Fil: Power, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Gutkind, Gabriel Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina

PER β -lactamases are an emerging family of extended-spectrum β -lactamases (ESBL) found in Gram-negative bacteria. PER β -lactamases are unique among class A enzymes as they possess an inverted omega (?) loop and extended B3 β -strand. These singular structural features are hypothesized to contribute to their hydrolytic profile against oxyimino-cephalosporins (e.g., cefotaxime and ceftazidime). Here, we tested the ability of avibactam (AVI), a novel non- β -lactam β -lactamase inhibitor to inactivate PER-2. Interestingly, the PER-2 inhibition constants (i.e., k2/K = 2 × 103 ±0.1 × 103 M-1 s-1, where k2 is the rate constant for acylation (carbamylation) and K is the equilibrium constant) that were obtained when AVI was tested were reminiscent of values observed testing the inhibition by AVI of class C and D β -lactamases (i.e., k2/K range of =103 M-1s-1) and not class A β -lactamases (i.e., k2/K range, 104 to 105 M-1s-1). Once AVI was bound, a stable complex with PER-2 was observed via mass spectrometry (e.g., 31,389 ± 3 atomic mass units [amu] ¡ 31,604 ± 3 amu for 24 h). Molecular modeling of PER-2 with AVI showed that the carbonyl of AVI was located in the oxyanion hole of the β -lactamase and that the sulfate of AVI formed interactions with the β-lactam carboxylate binding site of the PER-2 β -lactamase (R220 and T237). However, hydrophobic patches near the PER-2 active site (by Ser70 and B3-B4 β -strands) were observed and may affect the binding of necessary catalytic water molecules, thus slowing acylation (k2/K) of AVI onto PER-2. Similar electrostatics and hydrophobicity of the active site were also observed between OXA-48 and PER-2, while CTX-M-15 was more hydrophilic. To demonstrate the ability of AVI to overcome the enhanced cephalosporinase activity of PER-2 β-lactamase, we tested different β -lactam-AVI combinations. By lowering MICs to ≤2 mg/liter, the ceftaroline-AVI combination could represent a favorable therapeutic option against Enterobacteriaceae expressing blaPER-2. Our studies define the inactivation of the PER-2 ESBL by AVI and suggest that the biophysical properties of the active site contribute to determining the efficiency of inactivation. Fil: Ruggiero, Melina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Papp Wallace, Krisztina M.. Louis Stokes Cleveland Department of Veterans Affairs; Estados Unidos. Case Western Reserve University; Estados Unidos Fil: Taracila, Magdalena A.. Louis Stokes Cleveland Department of Veterans Affairs; Estados Unidos. Case Western Reserve University; Estados Unidos Fil: Mojica, Maria F.. Louis Stokes Cleveland Department of Veterans Affairs; Estados Unidos Fil: Bethel, Christopher R.. Louis Stokes Cleveland Department of Veterans Affairs; Estados Unidos Fil: Rudin, Susan D.. Louis Stokes Cleveland Department of Veterans Affairs; Estados Unidos. Case Western Reserve University; Estados Unidos Fil: Zeiser, Elise T.. Louis Stokes Cleveland Department of Veterans Affairs; Estados Unidos Fil: Gutkind, Gabriel Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Bonomo, Robert A.. Louis Stokes Cleveland Department of Veterans Affairs; Estados Unidos. Case Western Reserve University; Estados Unidos Fil: Power, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina

We analyzed the kinetic properties of the metagenomic class B3 β-lactamase LRA-12, and determined its crystallographic structure in order to compare it with prevalent metallo-β-lac-tamases (MBLs) associated with clinical pathogens. We showed that LRA-12 confers extended-spectrum resistance on E. coli when expressed from recombinant clones, and the MIC values for carbapenems were similar to those observed in enterobacteria expressing plasmid-borne MBLs such as VIM, IMP or NDM. This was in agreement with the strong carbapenemase activity displayed by LRA-12, similar to GOB β-lactamases. Among the chelating agents evaluated, dipicolinic acid inhibited the enzyme more strongly than EDTA, which required pre-incubation with the enzyme to achieve measurable inhibition. Structurally, LRA-12 contains the conserved main structural features of di-zinc class B β-lactamases, and presents unique structural signatures that differentiate this enzyme from others within the family: (i) two loops (α3-β7 and β11-α5) that could influence antibiotic entrance and remodeling of the active site cavity; (ii) a voluminous catalytic cavity probably responsible for the high hydrolytic efficiency of the enzyme; (iii) the absence of disulfide bridges; (iv) a unique Gln116 at metal-binding site 1; (v) a methionine residue at position 221that replaces Cys/Ser found in other B3 β-lactamases in a predominantly hydrophobic environment, likely playing a role in protein stability. The structure of LRA-12 indicates that MBLs exist in wild microbial populations in extreme environments, or environments with low anthropic impact, and under the appropriate antibiotic selective pressure could be captured and disseminated to pathogens. Fil: Rodríguez, María Margarita. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Herman, Raphaël. Université de Liège; Bélgica Fil: Ghiglione, Barbara. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Kerff, Frédéric. Université de Liège; Bélgica Fil: D'amico González, Gabriela Elena Noemí. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Bouillenne, Fabrice. Université de Liège; Bélgica Fil: Galleni, Moreno. Université de Liège; Bélgica Fil: Handelsman, Jo. University of Yale; Estados Unidos Fil: Charlier, Paulette. Université de Liège; Bélgica Fil: Gutkind, Gabriel Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Sauvage, Eric. Université de Liège; Bélgica Fil: Power, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina

Se llevó a cabo un relevamiento molecular de la resistencia a antibióticos de importancia clínica en aislamientos recuperados en Cochabamba, Bolivia. Se estudiaron los genes codificantes de β-lactamasas de espectro extendido y de resistencia a quinolonas de localización plasmídica (PMQR) en un total de 101 aislamientos de enterobacterias resistentes a oximinocefalosporinas recuperados en distintos centros de salud, durante 4 meses (2012-2013). En todos ellos se detectó la presencia de cefotaximasas, las CTX-M grupo 1 fueron las más prevalentes (88,1%). La presencia de blaOXA-1 se detectó en el 76,4% de estos aislamientos. Se observó una elevada proporción de aislamientos resistentes a quinolonas. El gen aac(6′)-Ib-cr fue el determinante PMQR más frecuentemente identificado (83%). Además, 6 aislamientos resultaron ser portadores de qnrB. Por otro lado, cabe remarcar que 7 Escherichia coli presentaron qepA1 (6) y oqxAB (1); se documenta así por primera vez la presencia de oqxAB en Bolivia. Este estudio constituye el primer relevamiento de marcadores de resistencia en aislamientos clínicos de enterobacterias en Cochabamba, Bolivia; de este modo se contribuye al conocimiento regional de la situación epidemiológica, la cual presenta un escenario similar al observado en el resto de Latinoamérica. A molecular survey was conducted in Cochabamba, Bolivia, to characterize the mechanism involved in the resistance to clinically relevant antibiotics. Extended Spectrum β-lactamase encoding genes and plasmid-mediated quinolone resistance (PMQR) markers were investigated in a total of 101 oxyimino-cephalosporin-resistant enterobacteria recovered from different health centers during four months (2012?2013). CTX-M enzymes were detected in all isolates, being the CTX-M-1 group the most prevalent (88.1%). The presence of blaOXA-1 was detected in 76.4% of these isolates. A high quinolone resistance rate was observed among the included isolates. The aac(6′)-Ib-cr gene was the most frequent PMQR identified (83.0%). Furthermore, 6 isolates harbored the qnrB gene. Interestingly, qepA1 (6) and oqxAB (1), were detected in 7 Escherichia coli, being the latter the first to be reported in Bolivia. This study constitutes the first molecular survey on resistance markers in clinical enterobacterial isolates in Cochabamba, Bolivia, contributing to the regional knowledge of the epidemiological situation. The molecular epidemiology observed herein resembles the scene reported in South America. Fil: Saba Villarroel, Paola M.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Gutkind, Gabriel Osvaldo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Di Conza, José Alejandro. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Radice, Marcela Alejandra. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

The objectives of this study were to investigate clinical isolates of Salmonella enterica serovar Typhimurium resistant to β-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 bla CMY-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 β-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. © 2013 International Society for Chemotherapy of Infection and Cancer., Comisión Sectorial de Investigación Científica (CSIC, Uruguay)

In this investigation, we isolated from an industrial effluent a bacterial community capable of utilizing p-chloroaniline (PCA) as the sole source of carbon and nitrogen. The isolates were identified as Pseudomonas sp., Achromobacter sp., and Sphingopyxis sp. The bacterial community was employed to set up a continuous system for PCA degradation and ammonium removal. The system consists in two sequential aerobic fixed-bed reactors, for PCA biodegradation and nitrification respectively, and an anoxic reactor for denitrification. Biodegradation process was evaluated by chemical analysis of PCA, chloride and ammonium, and bacterial count. Nitrification and denitrification processes were evaluated by chemical analysis of ammonium, nitrite and nitrate, and nitrifying bacteria count. Bioassays of acute toxicity using the standard organisms Vibrio fischeri, Pseudokirchneriella subcapitata, and Daphnia magna were performed in order to assess detoxification. An average efficiency of 88.5%, expressed as PCA removal, was achieved in the biodegradation process. The maximum PCA removal rate reached 11.7 g/m3 day. PCA degradation was associated with the release of chloride and ammonium. Ammonium was completely removed by the nitrification/denitrification process. Toxicity was not detected after the biodegradation process.

Water from six points from the Riachuelo-Matanza basin was analyzed in order to assess ibuprofen biodegradability. In four of them biodegradation of ibuprofen was proved and degrading bacterial communities were isolated. Biodegradation in each point could not be correlated with sewage pollution. The indigenous bacterial community isolated from the point localized in the La Noria Bridge showed the highest degradative capacity and was selected to perform batch and continuous degradation assays. The partial 16S rRNA gene sequence showed that the community consisted of Comamonas aquatica and Bacillus sp. In batch assays the community was capable of degrading 100 mg L−1 of ibuprofen in 33 h, with a specific growth rate (μ) of 0.21 h−1. The removal of the compound, as determined by High performance liquid chromatography (HPLC), exceeded 99% of the initial concentration, with a 92.3% removal of Chemical Oxygen Demand (COD). In a down-flow fixed-bed continuous reactor, the community shows a removal efficiency of 95.9% of ibuprofen and 92.3% of COD for an average inlet concentration of 110.4 mg. The reactor was kept in operation for 70 days. The maximal removal rate for the compound was 17.4 g m−3 d−1. Scanning electron microscopy was employed to observe biofilm development in the reactor. The ability of the isolated indigenous community can be exploited to improve the treatment of wastewaters containing ibuprofen. Fil: Fortunato, María Susana. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Sanidad, Nutrición, Bromatología y Toxicología. Cátedra de Higiene y Sanidad; Argentina Fil: Fuentes Abril, Nancy Piedad. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Sanidad, Nutrición, Bromatología y Toxicología. Cátedra de Higiene y Sanidad; Argentina Fil: Martinefski, Manuela Romina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica. Cátedra de Química Analítica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Tripodi, Valeria Paula. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Tecnología Farmacéutica; Argentina Fil: Papalia, Mariana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina Fil: Radice, Marcela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina Fil: Gutkind, Gabriel Osvaldo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Gallego, Alfredo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Sanidad, Nutrición, Bromatología y Toxicología. Cátedra de Higiene y Sanidad; Argentina Fil: Korol, Sonia Edith. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Sanidad, Nutrición, Bromatología y Toxicología. Cátedra de Higiene y Sanidad; Argentina

An indigenous bacterial strain capable of utilizing 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole carbon and energy source was isolated from a polluted river in Buenos Aires, Argentina. The strain was identified as Delftia sp. by the 16S rRNA gene sequencing. Aerobic biodegradation assays in batch process were performed using a 2 L volume microfermentor at 28 °C with agitation (200 rpm). The continuous-flow system employed consists in a down-flow fixed-bed reactor, using polyurethane foam cubes as a support for immobilizing bacterial cells. Degradation of 2,4-D was evaluated by UV spectrophotometry, chemical oxygen demand (COD) and gas chromatography (GC). In the batch reactor microbial growth was measured by the plate count method. Biofilm development on polyurethane foam was observed by scanning electronic microscopy. Detoxification was assessed by using seeds toxicity tests. Under batch conditions Delftia sp. was able to degrade 100 and 200 mg L −1 of 2,4-D in 24 and 28 h respectively. Compound and COD removals were 99.9% and 91.3% respectively at an initial 2,4-D concentration of 100 mg L −1 , and 99.0% and 89.4% when 2,4-D concentration was increased to 200 mg L −1 . When assays were performed in the continuous-flow reactor under operating conditions 2,4-D and COD removals of 99.6% and 90.8% were achieved, respectively. Toxicity was neither detected at the end of the batch process nor in the continuous-flow reactor effluent.

Nitroaromatics are widely used for industrial purposes and constitute a group of compounds of environmental concern because of their persistence and toxic properties. Biological processes used for decontamination of nitroaromatic-polluted sources have then attracted worldwide attention. In the present investigation m-nitrophenol (MNP) biodegradation was studied in batch and continuous reactors. A bacterial community able to degrade the compound was first selected from a polluted freshwater stream and the isolates were identified by the analysis of the 16S rRNA gene sequence. The bacterial community was then used in biodegradation assays. Batch experiments were conducted in a 2L aerobic microfermentor at 28°C and with agitation (200rpm). The influence of abiotic factors in the biodegradation process in batch reactors, such as initial concentration of the compound and initial pH of the medium, was also studied. Continuous degradation of MNP was performed in an aerobic up-flow fixed-bed biofilm reactor. The biodegradation process was evaluated by determining MNP and ammonium concentrations and chemical oxygen demand (COD). Detoxification was assessed by Vibrio fischeri and Pseudokirchneriella subcapitata toxicity tests. Under batch conditions the bacterial community was able to degrade 0.72mM of MNP in 32h, with efficiencies higher than 99.9% and 89.0% of MNP and COD removals respectively and with concomitant release of ammonium. When the initial MNP concentration increased to 1.08 and 1.44mM MNP the biodegradation process was accomplished in 40 and 44h, respectively. No biodegradation of the compound was observed at higher concentrations. The community was also able to degrade 0.72mM of the compound at pH 5, 7 and 9. In the continuous process biodegradation efficiency reached 99.5% and 96.8% of MNP and COD removal respectively. The maximum MNP removal rate was 37.9gm-3day-1. Toxicity was not detected after the biodegradation process. Fil: González, Ana Julieta. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Sanidad, Nutrición, Bromatología y Toxicología. Cátedra de Higiene y Sanidad; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Fortunato, María Susana. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Sanidad, Nutrición, Bromatología y Toxicología. Cátedra de Higiene y Sanidad; Argentina Fil: Papalia, Mariana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina Fil: Radice, Marcela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina Fil: Gutkind, Gabriel Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina Fil: Magdaleno, Anahí. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Sanidad, Nutrición, Bromatología y Toxicología. Cátedra de Higiene y Sanidad; Argentina Fil: Gallego, Alfredo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Sanidad, Nutrición, Bromatología y Toxicología. Cátedra de Higiene y Sanidad; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Korol, Sonia Edith. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Sanidad, Nutrición, Bromatología y Toxicología. Cátedra de Higiene y Sanidad; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Resistencia a colistina mediada por plásmido en Escherichia coli recuperadas de aves de corral sanas. Fil: Dominguez, Johana Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina Fil: Figueroa Espinosa, Roque Arnulfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Redondo, Leandro Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina Fil: Cejas, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Gutkind, Gabriel Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Chacana, Pablo Anibal. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Di Conza, José Alejandro. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Fernandez Miyakawa, Mariano Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina

Objectives To analyse the prevalence of resistance to β-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 β-lactamases (ESBLs), qnr alleles and aac(6′)Ib by phenotypic and molecular methods. The genomic context and transferability of β-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) β-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. Fil: García Fulgueiras, Virginia. Universidad de la Republica; Uruguay Fil: Bado, Inés. Universidad de la Republica; Uruguay Fil: Mota, María Inés. Universidad de la Republica; Uruguay. Uruguay. Ministerio de Salud Pública; Uruguay Fil: Robino, Luciana. Universidad de la Republica; Uruguay Fil: Cordeiro, Nicolás F.. Universidad de la Republica; Uruguay Fil: Varela, Adriana. Uruguay. Ministerio de Salud Pública; Uruguay Fil: Algorta, Gabriela. Universidad de la Republica; Uruguay. Uruguay. Ministerio de Salud Pública; Uruguay Fil: Gutkind, Gabriel Osvaldo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Ayala, Juan A.. Universidad Autónoma de Madrid; España. Consejo Superior de Investigaciones Cientificas; España Fil: Vignoli, Rafael. Universidad de la Republica; Uruguay

This is the first report of the presence of qepA1 efflux pump gene in Escherichia coli clinical isolate from Argentina, which was associated to other plasmid-mediated quinolone resistance determinants, such as aac(6′)-Ib-cr and qnrB10 and also quinolone resistance determining regions mutations. Fil: Rincón, Giovanna. Universidad Industrial Santander; Colombia. Universidad de Buenos Aires; Argentina Fil: Radice, Marcela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina Fil: Giovanakis, Marta. Hospital Británico de Buenos Aires; Argentina Fil: Di Conza, José Alejandro. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Cátedra de Microbiología General; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Gutkind, Gabriel Osvaldo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

The Southern green stinkbug (N. viridula) feeds on developing soybean seeds in spite of their strong defenses against herbivory, making this pest one of the most harmful to soybean crops. To test the hypothesis that midgut bacterial community allows stinkbugs to tolerate chemical defenses of soybean developing seeds, we identified and characterized midgut microbiota of stinkbugs collected from soybean crops, different secondary plant hosts or insects at diapause on Eucalyptus trees. Our study demonstrated that while more than 54% of N. viridula adults collected in the field had no detectable bacteria in the V1-V3 midgut ventricles, the guts of the rest of stinkbugs were colonized by non-transient microbiota (NTM) and transient microbiota not present in stinkbugs at diapause. While transient microbiota Bacillus sp., Micrococcus sp., Streptomyces sp., Staphylococcus sp. and others had low abundance, NTM microbiota was represented by Yokenella sp., Pantoea sp. and Enterococcus sp. isolates. We found some isolates that showed in vitro β-glucosidase and raffinase activities plus the ability to degrade isoflavonoids and deactivate soybean protease inhibitors. Our results suggest that the stinkbugs´ NTM microbiota may impact on nutrition, detoxification and deactivation of chemical defenses, and Enterococcus sp., Yokenella sp. and Pantoea sp. strains might help stinkbugs to feed on soybean developing seeds in spite of its chemical defenses. Fil: Medina, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; Argentina Fil: Sardoy, Pedro Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; Argentina Fil: Soria, Marcelo Abel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; Argentina Fil: Vay, Carlos Alberto. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Gutkind, Gabriel Osvaldo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Zavala, Jorge Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; Argentina

Fil: Di Conza, José Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Radice, Marcela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Gutkind, Gabriel Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina

In 1993, a fraction of antibodies (Abs) devoid of L chain was found naturally occurring in the Camelidae. They were found to lack L chains, as well as the first constant heavy-chain domain (CH(1)) and therefore they were named "heavy-chain Abs" (HCAbs). Subsequent studies focused on the functional, structural and biochemical properties of recombinant variable fragments (rVHHs) of HCAbs. It was stated that rVHHs have an augmented capacity to interact with "partially hidden" epitopes, like enzymes active sites, and have an increased stability to thermal and chemical aggression. It has been suggested that these unconventional Abs could represent an evolutionary advantage, being more efficient than conventional Abs to inhibit microbial enzymes, and thus exerting a more protective immune response against pathogens. The present work focuses on the immunobiological role of HCAbs, in their capacity to inhibit microbial enzymes. Two animal models were selected, comprising a model for common vertebrates without HCAbs (rabbits), and a model for vertebrates with both conventional and unconventional Abs (Lama glama). A recombinant bacterial beta-lactamase (CTX-M-2) was selected as the microbial enzymatic antigen. After conventional immunization schedules, neither serum titers nor serum inhibitory capacity showed significant differences when rabbits and llamas were compared. These results indicate that the a priori assumption that the adaptive immune system of camelids could be better "prepared" to respond to bacterial enzymes because of the presence of HCAbs, is not always accurate. Furthermore, when the different llama antibody isotypes and subclasses were purified, it was demonstrated that the inhibitory capacity of total serum was due exclusively to IgG(1). HCAbs not only failed to inhibit CTX-M-2, but instead they activated its enzymatic activity. Altogether, these results indicate that the hypotheses extrapolated from the rVHHs properties need to be revised; the real role of HCAbs in vivo remains unknown, as well as their evolutionary cause.

This study characterised the genetic environment of the chromosomally encoded bla(KLUA-9) gene from a clinical Kluyvera ascorbata isolate and performed a kinetic characterisation of KLUA-9. Purified KLUA-9 showed the highest catalytic efficacies towards benzylpenicillin, ampicillin, piperacillin, first-generation cephalosporins, cefuroxime and cefoperazone; like other 'cefotaximases', it showed a much higher rate of hydrolysis of cefotaxime than ceftazidime, whilst dicloxacillin, cefoxitin and imipenem behaved as poor substrates. A 9kb insert from K. ascorbata was cloned (Escherichia coli KK68C1) and sequenced. bla(KLUA-9) and its 266bp upstream flanking region (almost identical to the integron-associated bla(CTX-M-2)) are preceded by an aspat variant, a ypdABC-like operon and two open reading frames with unknown functions. Unlike ISCR1-associated bla(CTX-M-2) genes, we failed to detect the putative orf513 recombination sites. Instead, we were able to localise the 5bp target sites for insertion of ISEcp1B, suggesting that this element could be responsible for future (or still undetected) mobilisation of bla(KLUA-9) to more efficiently transferred elements.

Diversification of the CTX-M β-lactamases led to the emergence of variants responsible for decreased susceptibility to ceftazidime, like the Asp240Gly-harboring "ceftazidimases". We solved the crystallographic structure of the Asp240Gly variant CTX-M-96 at 1.2 Å and evaluated the role of Asp240 in the activity toward oxyimino-cephalosporins through simulated models and kinetics. There seem to be subtle changes in the conformation of the active site cavity of CTX-M-96, compared to enzyme variants harboring the Asp240, and these small rearrangements could be due to localized shifts in the environment of the β3 strand. According to the crystallographic evidence, CTX-M-96 presents a "compact" active site, which in spite of its reduced cavity seems to allow the proper interaction with oxyimino-cephalosporins, as suggested by simulated models. The term "ceftazidimases" that is currently applied for the Asp240Gly-harboring CTX-M variants should be used carefully. Structural differences between CTX-M harboring the Asp240Gly mutation (and also probably others like those at Pro167) do not seem to be conclusive to determine the "ceftazidimase" behavior observed in vivo, which is in turn partially supported by the mild improvement in the catalytic efficiency toward ceftazidime by CTX-M-96 and similar enzymes, compared to "parental" Asp240-harboring variants. In addition, it is observed that alterations in OmpF expression could act synergistically with CTX-M-96 for yielding clinical resistance toward ceftazidime. We therefore propose that the observed resistance in vivo is due to the sum of synergic mechanisms, and the term "cefotaximases associated with ceftazidime resistance" could be conveniently used to describe CTX-M harboring the Asp240Gly substitution.

A prospective analysis on β-lactam resistance mechanisms and β-lactamase prevalence was conducted on Prevotella intermedia and Prevotella nigrescens recovered from patients with chronic periodontitis and peritonsillar abscesses. Both phenotypic and genotypic methods were performed to characterize the β-lactamases, their coding genes and their genetic contexts. Overall, β-lactamase production was observed in 64% (16/25) P.intermedia and 23.8% (5/21) P.nigrescens (p

Shiga toxin-producing Escherichia coli (STEC) cause hemorrhagic colitis (HC) and hemolytic-uremic syndrome in humans (HUS). Cattle are the main reservoir of STEC and transmission to humans occurs through contaminated food and water. Antibiotics are used in pig production systems to combat disease and improve productivity and play a key role in the dissemination of antibiotic resistance genes to the bacteria. Integrons have been identified in resistant bacteria allowing for the acquisition and dissemination of antibiotic resistance genes. STEC strains isolated from humans and animals have developed antibiotic resistance. In our laboratory, 21 non-157 STEC strains isolated from pigs were analyzed to detect class 1 and 2 integrons by PCR. Eight carried integrons, 7 of them harbored intl2. In another study 545 STEC strains were also analyzed for the presence of intl1 and intl2 . Strains carrying intl1 belonged to isolates from environment (n = 1), chicken hamburger (n = 2), dairy calves (n = 4) and pigs (n = 8). Two strains isolated from pigs harbored intl2 and only one intl1 / intl2 , highlighting the presence of intl2 in pigs. The selection for multiresistant strains may contribute to the emergence of antibiotic resistant pathogens and facilitate the spreading of the mobile resistance elements to other bacteria.