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Selection for antibiotic resistance at very low antibiotic concentrations has been demonstrated for individual antibiotics in single species experiments. Furthermore, selection in these focal strains is reduced when taking place in complex microbial community context. However, in the environment, bacteria are rarely exposed to single, but rather complex mixtures of selective agents. Here, we explored how the presence of a second selective agent affects selection dynamics between isogenic pairs of focal E. coli strains, differing exclusively in a single resistance determinant, in the absence and presence of a model wastewater community across a gradient of antibiotics. An additional antibiotic that exclusively affects the model wastewater community, but to which the focal strains are resistant to, was chosen as the second selective agent. This allowed exploring how inhibition alters the community’s ability to reduce selection. In the presence of the community, the selection coefficient at specific antibiotic concentrations was consistently decreased compared to the absence of the community. While pressure through the second antibiotic significantly decreased the activity and diversity of the community, its ability to reduce selection was consistently maintained at levels comparable to those recorded in absence of the second antibiotic. This indicates that the observed effects of community context on selection dynamics are rather based on competitive or protective effects between the focal strains and a small proportion of bacteria within the community, than on general competition for nutrients. These findings have implications for our understanding of the evolution and selection for multi-drug resistant strains.

Antibiotic resistance genes (ARGs) are widely disseminated within microbiomes of the human, animal, and environmental spheres. There is a clear need for global monitoring and risk assessment initiatives to evaluate the risks of ARGs towards human health. Therefore, not only ARG abundances within a given environment, but also their mobility, hence their ability to spread to human pathogenic bacteria needs to be quantified. Consequently, methods to accurately quantify the linkage of ARGs with mobile genetic elements are urgently needed.We developed a novel, sequencing-independent method for assessing ARG mobility by combining multiplexed droplet digital PCR (ddPCR) on DNA sheared into short fragments with statistical analysis. This allows quantifying the physical linkage between ARGs and mobile genetic elements, here demonstrated for the sulfonamide ARG sul1 and the Class1 integron integrase gene intI1. The method’s efficiency is demonstrated using mixtures of model DNA fragments with either linked and unlinked target genes: Linkage of the two target genes can be accurately quantified based on high correlation coefficients between observed and expected values (R2) as well as low mean absolute errors (MAE) for both target genes, sul1 (R2=0.9997, MAE=0.71%, n=24) and intI1 (R2=0.9991, MAE=1.14%, n=24). Furthermore, we demonstrate that the chosen fragmentation length of DNA during shearing allows adjusting the rate of false positives and false negative detection of linkage. The applicability of the developed method for environmental samples is further demonstrated by assessing the mobility of sul1 across a wastewater treatment plant.The presented method allows rapidly obtaining reliable results within hours. It is labor- and costefficient and does not rely on sequencing technologies. Furthermore, it has a high potential to be scaled up to multiple targets. Consequently, it merits consideration to be included within global AMR surveillance initiatives for assessing ARG mobility.Author AbstractAntibiotic resistance represents a major problem in treating bacterial infections and endangers public health. Antibiotic resistant genes (ARGs) can spread among microbes in humans, animals, and the environment, thanks to mobile genetic elements. These are genetic structures involved in the mobility of genetic information and hence microbial traits. Methods that allow simultaneously quantifying the abundance of ARGs together with their association/linkage with mobile genetic elements are fundamental for assessing the risk they pose to human health, as mobility increases their likelihood to spread to human pathogens. Here we developed a novel method that allows to quantify the abundance and the linkage between ARGs and mobile genetic elements. The method relies on droplet digital PCR (ddPCR) technology performed on fragmented environmental DNA (of a chosen size), combined with statistical analysis. We found that the method accurately quantifies the linkage between the two targets in model and environmental DNA. The method is delivering rapid, labor- and cost-efficient results as it does not rely on technology that require prior bioinformatics knowledge and can be included in future monitoring frameworks.

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

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

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.

KPC-2-producing Klebsiella pneumoniae isolates mainly correspond to clonal complex 258 (CC258); however, we describe KPC- 2-producing K. pneumoniae isolates belonging to invasive sequence type 23 (ST23). KPC-2 has scarcely been reported to occur in ST23, and this report describes the first isolation of this pathogen in the Americas. Acquisition of resistant markers in virulent clones could mark an evolutionary step toward the establishment of these clones as major nosocomial pathogens. Fil: Cejas, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Fernández Canigia, Liliana. Hospital Alemán; Argentina Fil: Cruz, Giovanna Rincón. Universidad Industrial Santander; Colombia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Elena, Alan Xavier. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Maldonado, Ivana. Hospital Alemán; 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. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina