Novel Megaplasmid Driving NDM-1-Mediated Carbapenem Resistance in Klebsiella pneumoniae ST1588 in South America.

Carbapenem-resistant Enterobacterales (CRE) is a critical public health problem in South America, where the prevalence of NDM metallo-betalactamases has increased substantially in recent years. In this study, we used whole genome sequencing to characterize a multidrug-resistant (MDR) Klebsiella pneumoniae (UCO-361 strain) clinical isolate from a teaching hospital in Chile. Using long-read (Nanopore) and short-read (Illumina) sequence data, we identified a novel un-typeable megaplasmid (314,976 kb, pNDM-1_UCO-361) carrying the blaNDM-1 carbapenem resistance gene within a Tn3000 transposon. Strikingly, conjugal transfer of pNDM-1_UCO-361 plasmid only occurs at low temperatures with a high frequency of 4.3 × 10-6 transconjugants/receptors at 27 °C. UCO-361 belonged to the ST1588 clone, previously identified in Latin America, and harbored aminoglycoside, extended-spectrum ß-lactamases (ESBLs), carbapenem, and quinolone-resistance determinants. These findings suggest that blaNDM-1-bearing megaplasmids can be adapted to carriage by some K. pneumoniae lineages, whereas its conjugation at low temperatures could contribute to rapid dissemination at the human-environmental interface.

Analysis of payment models applied to federal funding from the perspective of the Brazilian Health System

Objective: This study aims at identifying the payment methods existing in the Unified Health System referring to federal transfers to Primary Health Care (PHC) and Specialized Health Care. Methods: A quantitative and analytical study was carried out, developed in three stages: survey of all types of transfers from the Union; classification of each transfer category according to the types of payment methods and measurement of the participation of each payment methods, according to the financing components analyzed, in relation to the net values transferred. Results: Federal transfers were classified into seven payment methods. For PHC, in 2020, approximately R$ 21.7 billion was calculated, including resources destined for the pandemic, and R$ 20.9 billion without considering resources to face the COVID-19 pandemic. More than 50% of the amounts used were classified as capitation, in both cases. For specialized health care, in 2019, around R$ 48.5 billion were calculated, and in 2020 more than R$ 49.2 billion. For the two years, more than 70% of the funds were allocated to fee for service. Conclusions: This study allowed for an expansion in knowledge about the allocation of resources referring to transfers from the Union to states, the Federal District and municipalities. As the payment methods are related to productivity, access and quality of the health service, knowing and identifying the most appropriate payment methods for each situation contributes to the achievement of the goals and to the mitigation of eventual losses of efficiency in the healthcare systems.

Análise dos modelos de pagamento aplicados ao financiamento federal na perspectiva do Sistema Único de Saúde / Analysis of payment models applied to federal funding from the perspective of the Brazilian Health System

Objetivo: Este estudo tem por objetivo identificar os modelos de pagamento existentes no Sistema Único de Saúde referentes aos repasses federais para a Atenção Primária à Saúde (APS) e a Atenção Especializada à Saúde. Métodos: Foi realizado um estudo quantitativo e analítico, desenvolvido em três etapas: levantamento de todos os tipos de repasse da União; classificação de cada categoria de repasse segundo os tipos de modelos de pagamentos; e mensuração da participação de cada modelo de pagamento, de acordo com os componentes de financiamento analisados, em relação aos valores líquidos repassados. Resultados: Os repasses federais foram classificados em sete modelos de pagamentos. Para a APS, em 2020, foram apurados R$ 21,7 bilhões, aproximadamente, incluindo os recursos destinados para a pandemia, e R$ 20,9 bilhões, sem considerar os recursos para enfrentamento da pandemia de COVID-19. Mais de 50% dos valores empregados foram classificados como capitação, em ambos os casos. Para a Atenção Especializada à Saúde, em 2019, foram computados em torno de R$ 48,5 bilhões e, em 2020, acima de R$ 49,2 bilhões. Para os dois anos, mais de 70% dos recursos foram destinados a pagamentos por procedimento. Conclusões: Este estudo permitiu a ampliação do conhecimento sobre a alocação dos recursos referentes aos repasses da União para estados, Distrito Federal e municípios. Como os modelos de pagamentos estão relacionados com a produtividade, acesso e qualidade do serviço de saúde, conhecer as formas de pagamento e identificar a mais adequada para cada situação contribui para o alcance das metas e para a mitigação de eventuais perdas de eficiência nos sistemas de saúde.

Objective: This study aims at identifying the payment methods existing in the Unified Health System referring to federal transfers to Primary Health Care (PHC) and Specialized Health Care. Methods: A quantitative and analytical study was carried out, developed in three stages: survey of all types of transfers from the Union; classification of each transfer category according to the types of payment methods and measurement of the participation of each payment methods, according to the financing components analyzed, in relation to the net values transferred. Results: Federal transfers were classified into seven payment methods. For PHC, in 2020, approximately R$ 21.7 billion was calculated, including resources destined for the pandemic, and R$ 20.9 billion without considering resources to face the COVID-19 pandemic. More than 50% of the amounts used were classified as capitation, in both cases. For specialized health care, in 2019, around R$ 48.5 billion were calculated, and in 2020 more than R$ 49.2 billion. For the two years, more than 70% of the funds were allocated to fee for service. Conclusions: This study allowed for an expansion in knowledge about the allocation of resources referring to transfers from the Union to states, the Federal District and municipalities. As the payment methods are related to productivity, access and quality of the health service, knowing and identifying the most appropriate payment methods for each situation contributes to the achievement of the goals and to the mitigation of eventual losses of efficiency in the healthcare systems.

Diversity and Host Relationships of the Mycoparasite Sepedonium (Hypocreales, Ascomycota) in Temperate Central Chile.

We present the first major survey of regional diversity, distribution and host-association of Sepedonium. Whereas the rather scarce worldwide records of this mycoparasitic fungus suggested no specific distribution pattern of most species before, we provide new evidence of endemic and specific host-parasite guilds of Sepedonium in Southern South America, including the description of a new species. The corresponding inventory was performed in temperate central Chile. The regional landscape, a mosaic of exotic timber plantations and remnants of native Nothofagus forests, facilitates a unique combination of endemic and adventitious Boletales hosts. During a two-year survey, 35 Sepedonium strains were isolated and cultured from infected basidiomata of allochthonous Chalciporus piperatus, Paxillus involutus, Rhizopogon spp. and Suillus spp., as well as from the native Boletus loyita, B. loyo, B. putidus and Gastroboletus valdivianus. Taxonomic diagnosis included morphology of conidia and conidiophores, sequences of ITS, RPB2 and EF1 molecular markers and characteristics of in vitro cultures. Phylogenetic reconstructions were performed using Bayesian methods. Four Sepedonium species could be identified and characterized, viz.: S. ampullosporum, S. chrysospermum, S. laevigatum and the newly described species S. loyorum. The most frequent species on introduced Boletales was S. ampullosporum, followed by S. chrysospermum and S. laevigatum. S. loyorum sp. nov. was found exclusively on native boletacean hosts, separated from its closest relative S. chalcipori by micromorphological and molecular attributes. Species descriptions and identification keys are provided. Ecological and biogeographical aspects of endemic and allochthonous symbiotic units consisting of mycoparasite, ectomycorrhizal fungal host and respective mycorrhizal tree are discussed.

Molecular characterization and antibacterial activity of oral antibiotics and copper nanoparticles against endodontic pathogens commonly related to health care-associated infections.

OBJECTIVE: To carry out molecular characterization and determine the antibacterial activity of oral antibiotics and copper nanoparticles (Cu-NPs) against endodontic strains isolated from persistent infections. MATERIALS AND METHODS: Root canal samples from 24 teeth in different patients with persistent endodontic infections were obtained. The isolated strains were identified by biochemical tests and 16S rDNA sequencing. Genotyping was achieved by molecular methods. The antibacterial activity of antibiotics and copper nanostructures was determined by using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values. Furthermore, a time-kill kinetics assay was evaluated. Nonparametric tests (Kruskal-Wallis ANOVA) were performed (p value <0.05). RESULTS: Twenty-one isolated strains were identified. Six isolates of Enterococcus faecalis were grouped into two clusters of three isolates each, two of which were clones. All were clarithromycin-resistant and erythromycin. Eight Pseudomonas putida presented two clusters, two Pseudomonas spp. were not clonal, and all were resistant to the tested antibiotics except tetracycline. Two of five strains of Cutibacterium acnes were clonal, and all were resistant only to metronidazole. The lowest MIC and MBC values were obtained with Cu-NPs. Time-kill kinetics using Cu-NPs showed a significant decrease in all tested species within 4 h and reached 100% in 2 h for C. acnes. CONCLUSION: In this study, in relation to health care-associated infections, endodontic strains of each species isolated at least in one patient were polyclonal. In Pseudomonas spp., at least one clone was shared between patients. E. faecalis and C. acnes strains were susceptible to low Cu-NP concentrations, while Pseudomonas spp. strains were resistant. CLINICAL RELEVANCE: Assessing and keeping track of the susceptibility of clinical strains to antimicrobial compounds is important for the clinical outcome. Based on our results, Cu-NPs could be an alternative for endodontic treatment, in order to avoid selection of resistant strains.

[Carbapenemases produced by Carbapenem-resistant Pseudomonas aeruginosa isolated from hospitals in Chile]. / Carbapenemasas en aislamientos de Pseudomonas aeruginosa resistentes a carbapenémicos aisladas en hospitales de Chile.

BACKGROUND: Carbapenem resistance mediated by carbapenemases in Pseudomonas aeruginosa is an important mechanism; however, loss of porin OprD remains as the most frequent. AIM: To determine the proportion of P. aeruginosa isolates, resistant to imipenem and/or meropenem, producing carbapenemases, the type of enzyme produced and the genetic relationship between the isolates. METHODS: One hundred and thirteen resistant to at least one carbapenem isolates, obtained in 12 hospitals and 9 cities in Chile were studied. Additionally, susceptibility to ceftazidime, amikacin, gentamicin, piperacillin/tazobactam, ciprofloxacin and colistin was determined. Carba NP was performed and in the positive isolates carbapenemase genes were detected by PCR. The isolates were typified by restriction with SpeI and PFGE. RESULTS: Not all isolates produce carbapenemases, and only in 61/113 of them (54%) the blaKPC (32) or blaVIM (29) was amplified. In none of the isolates was found the coharboring of both genes. The pulsotypes indicated no clonal dissemination of the isolates, evidencing an important genetic diversity. CONCLUSIONS: P. aeruginosa isolates producing carbapenemases, obtained in Chilean hospitals carry blaKPC and blaVIM genes and, mostly, are polyclonal. These results emphasize the importance of carrying out epidemiological studies with a greater number of isolates to allow a better understanding of the epidemiology of carbapenemase-producing P. aeruginosa in Chile.

Carbapenemasas en aislamientos de Pseudomonas aeruginosa resistentes a carbapenémicos aisladas en hospitales de Chile

Resumen Introducción: La resistencia a carbapenémicos mediada por carbapenemasas en Pseudomonas aeruginosa es un mecanismo importante; sin embargo, la pérdida de la porina OprD continúa siendo el mecanismo más frecuente. Objetivo: Determinar la proporción de aislados de P. aeruginosa, resistentes a imipenem y/o meropenem, productores de carbapenemasas, el tipo de enzima producida y la relación genética entre los aislados. Material y Métodos: Se incluyó 113 aislados resistentes al menos a un carbapenémico, provenientes de 12 hospitales de 9 ciudades de Chile. Adicionalmente se determinó la susceptibilidad a ceftazidima, amikacina, gentamicina, piperacilina/tazobactam, ciprofloxacina y colistina. Se realizó Carba NP y en los aislados positivos (n: 61) se detectó genes de carbapenemasas por RPC. Los aislados fueron tipificados por restricción con SpeI y PFGE. Resultados: No todos los aislados presentan carbapenemasas, y sólo en 61/113 de ellos (54%) se amplificó blaKPC (32) o blaVIM (29). En ninguno de los aislados se encontró co-portación de ambos genes. Los pulsotipos indican que no hay diseminación clonal de los aislados, evidenciando una importante diversidad genética. Conclusiones: Los aislados de P. aeruginosa productores de carbapenemasas, obtenidos en hospitales de Chile, portan genes blaKPC y blaVIM y, en su mayoría, son policlonales. Estos resultados ponen énfasis en la importancia de realizar estudios epidemiológicos con mayor número de aislados que permitan conocer mejor la epidemiología de P. aeruginosa productoras de carbapenemasas en Chile.

Abstract Background: Carbapenem resistance mediated by carbapenemases in Pseudomonas aeruginosa is an important mechanism; however, loss of porin OprD remains as the most frequent. Aim: To determine the proportion of P. aeruginosa isolates, resistant to imipenem and/or meropenem, producing carbapenemases, the type of enzyme produced and the genetic relationship between the isolates. Methods: One hundred and thirteen resistant to at least one carbapenem isolates, obtained in 12 hospitals and 9 cities in Chile were studied. Additionally, susceptibility to ceftazidime, amikacin, gentamicin, piperacillin/tazobactam, ciprofloxacin and colistin was determined. Carba NP was performed and in the positive isolates carbapenemase genes were detected by PCR. The isolates were typified by restriction with SpeI and PFGE. Results: Not all isolates produce carbapenemases, and only in 61/113 of them (54%) the blaKPC (32) or blaVIM (29) was amplified. In none of the isolates was found the coharboring of both genes. The pulsotypes indicated no clonal dissemination of the isolates, evidencing an important genetic diversity. Conclusions: P. aeruginosa isolates producing carbapenemases, obtained in Chilean hospitals carry blaKPC and blaVIM genes and, mostly, are polyclonal. These results emphasize the importance of carrying out epidemiological studies with a greater number of isolates to allow a better understanding of the epidemiology of carbapenemase-producing P. aeruginosa in Chile.

Colistin Heteroresistance among Extended Spectrum ß-lactamases-Producing Klebsiella pneumoniae.

Colistin-heteroresistant (CST-HR) Enterobacterales isolates have been identified recently, challenging the clinical laboratories since routine susceptibility tests fail to detect this phenotype. In this work we describe the first CST-HR phenotype in extended-spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae isolates in South America. Additionally, we determine the genomic mechanisms of colistin heteroresistance in these strains. The CST-HR phenotype was analyzed by the population analysis profile (PAP) method, and mutations associated with this phenotype were determined by whole-genome sequencing (WGS) and the local BLAST+ DB tool. As a result, 8/60 isolates were classified as CST-HR according to the PAP method. From WGS, we determined that the CST-HR isolates belong to three different Sequence Types (STs) and four K-loci: ST11 (KL15 and KL81), ST25 (KL2), and ST1161 (KL19). We identified diverse mutations in the two-component regulatory systems PmrAB and PhoPQ, as well as a disruption of the mgrB global regulator mediated by IS1-like and IS-5-like elements, which could confer resistance to CST in CST-HR and ESBL-producing isolates. These are the first descriptions in Chile of CST-HR in ESBL-producing K. pneumoniae isolates. The emergence of these isolates could have a major impact on the effectiveness of colistin as a "last resort" against these isolates, thus jeopardizing current antibiotic alternatives; therefore, it is important to consider the epidemiology of the CST-HR phenotype.

Antibiotic resistance in bacterial isolates from freshwater samples in Fildes Peninsula, King George Island, Antarctica.

Anthropic activity in Antarctica has been increasing considerably in recent years, which could have an important impact on the local microbiota affecting multiple features, including the bacterial resistome. As such, our study focused on determining the antibiotic-resistance patterns and antibiotic-resistance genes of bacteria recovered from freshwater samples collected in areas of Antarctica under different degrees of human influence. Aerobic heterotrophic bacteria were subjected to antibiotic susceptibility testing and PCR. The isolates collected from regions of high human intervention were resistant to several antibiotic groups, and were mainly associated with the presence of genes encoding aminoglycosides-modifying enzymes (AMEs) and extended-spectrum ß-lactamases (ESBLs). Moreover, these isolates were resistant to synthetic and semi-synthetic drugs, in contrast with those recovered from zones with low human intervention, which resulted highly susceptible to antibiotics. On the other hand, we observed that zone A, under human influence, presented a higher richness and diversity of antibiotic-resistance genes (ARGs) in comparison with zones B and C, which have low human activity. Our results suggest that human activity has an impact on the local microbiota, in which strains recovered from zones under anthropic influence were considerably more resistant than those collected from remote regions.

Role of wastewater treatment plants on environmental abundance of Antimicrobial Resistance Genes in Chilean rivers.

BACKGROUND: Point sources such as wastewater treatment plants (WWTPs) commonly discharge their effluent into rivers. Their waste may include antibiotic residues, disinfectants, antibiotic resistant bacteria (ARB), and Antimicrobial Resistance Genes (ARG). There is evidence that ARG can be found in the natural environment, but attribution to specific point sources is lacking. OBJECTIVES: The goal of this study was to assess the release and dissemination of ARG from three WWTPs in southern Chile via two pathways: through the river systems, and through wild birds. METHODS: A longitudinal study was conducted, collecting river sediment samples at different distances both upstream and downstream from each WWTP. Wild birds were sampled from around one of the WWTPs once a month for 13 months. A microfluidic q-PCR approach was used to quantify 48 genes covering different molecular mechanisms of resistance, and data was analyzed using ordination methods and linear mixed regression models. RESULTS: There was a statistically significant increase downstream from the WWTPs (p < 0.05) for 17 ARG, but the downstream dissemination through the rivers was not clear. Beta-lactamase genes blaKPC, blaTEM, and blaSHV were the most abundant in birds, with higher abundance of blaSHV in migratory species compared to resident species (p < 0.05). The gene profile was more similar between the migratory and resident bird groups compared to the WWTP gene profile. CONCLUSIONS: While results from this study indicate an influence of WWTPs on ARG abundance in the rivers, the biological significance of this increase and the extent of the WWTPs influence are unclear. In addition, wild birds were found to play a role in disseminating ARG, although association to the specific WWTP could not be ascertained.

Antibiotic Resistance Genes in Freshwater Trout Farms in a Watershed in Chile.

Point sources such as wastewater treatment plants, terrestrial agriculture, and aquaculture may release antibiotic residues, antibiotic resistant bacteria, and antibiotic resistance genes (ARGs) into aquatic ecosystems. However, there is a lack of quantitative studies attributing environmental ARG abundance to specific sources. The goal of this study was to evaluate the role of freshwater trout farms in the release and dissemination of ARGs into the environment. Sediment samples upstream and downstream from five rainbow trout farms were collected over time in southern Chile. A microfluidic quantitative polymerase chain reaction approach was used to quantify an ARG array covering different mechanisms of resistance, and data were analyzed using principal component analysis (PCA) and linear mixed regression models. Surveys were also conducted to obtain information about management practices, including antibiotic use, at the farms. Florfenicol and oxytetracycline were used at these farms, although at different rates. A total of 93 samples were analyzed. In the PCA, , , , , (A), (B), (C), (W), and grouped together. A statistically significant increase in abundance of , , , and several genes was found downstream from the farms compared with upstream sites, and retention ponds had the highest ARG abundance at each site. Antibiotic resistance gene levels returned to baseline at an average distance of 132.7 m downstream from the farms. Although results from this study indicate an influence of trout farms on the presence of ARGs in the immediate environment, the extent of their contribution to ARG dissemination is unknown and deserves further investigation.

Nor-guanacastepene pigments from the Chilean mushroom Cortinarius pyromyxa.

For the first time, the pigment composition of basidiocarps from the Chilean mushroom Cortinarius pyromyxa was studied under various aspects like phylogeny, chemistry and antibiotic activity. A molecular biological study supports the monotypic position of C. pyromyxa in subgenus Myxacium, genus Cortinarius. Four undescribed diterpenoids, named pyromyxones A-D, were isolated from fruiting bodies of C. pyromyxa. Their chemical structures were elucidated based on comprehensive one- and two-dimensional NMR spectroscopic analysis, ESI-HRMS measurements, as well as X-ray crystallography. In addition, the absolute configurations of pyromyxones A-D were established with the aid of JH,H, NOESY spectra and quantum chemical CD calculation. The pyromyxones A-D possess the undescribed nor-guanacastane skeleton. Tested pyromyxones A, B, and D exhibit only weak activity against gram-positive Bacillus subtilis and gram-negative Aliivibrio fischeri as well as the phytopathogenic fungi Botrytis cinerea, Septoria tritici and Phytophthora infestans.

Evolutionary dynamics of carbapenem-resistant Acinetobacter baumannii circulating in Chilean hospitals.

We analyze the evolutionary dynamics of ninety carbapenem-resistant Acinetobacter baumannii (CRAB) isolates collected between 1990 and 2015 in Chile. CRAB were identified at first in an isolate collected in 2005, which harbored the ISAba1-blaOXA-69 arrangement. Later, OXA-58- and OXA-23-producing A. baumannii strains emerged in 2007 and 2009, respectively. This phenomenon was associated with variations in the epidemiology of OXA-type carbapenemases, linked to nosocomial lineages belonging to ST109, ST162, ST15 (CC15) and ST318 (CC15).

Introduction of NDM-1 and OXA-370 from Brazil into Chile in strains of Klebsiella pneumoniae isolated from a single patient.

Carbapenemase-producing Enterobacteriaceae have rapidly disseminated worldwide and can colonize patients in healthcare centers. As in Chile the first isolations of NDM-1 and OXA-370 carbapenemases were related with a patient arriving from Brazil, the genetic relatedness of Klebsiella pneumoniae strains producers of these enzymes and isolated in both countries was assessed. PFGE analyses revealed that the isolates were clonally related, illustrating how travel contributes to the spread of multidrug-resistant microorganisms. In addition, the occurrence of three different carbapenemases in three different K. pneumoniae strains isolated from a single patient is described.

Genetic Platforms of blaCTX-M in Carbapenemase-Producing Strains of K. pneumoniae Isolated in Chile.

Objective: To elucidate whether the genetic platforms of blaCTX-M contribute to the phenotypes of multi-drug-resistance (MDR) in the first carbapenemase-producing K. pneumoniae strains isolated in Chile. Method: Twenty-two carbapenemase-producing K. pneumoniae strains isolated from different Chilean patients and hospitals were studied. Their genetic relatedness was assessed by PFGE and MLST. The levels of antibiotic resistance were evaluated by determining the minimum inhibitory concentration of various antimicrobials. In addition, several antibiotic resistance genes of clinical relevance in Chile were investigated. The prevalence, allelic variants, and genetic platforms of blaCTX-M were determined by PCR and sequencing. Results: Out of the 22 strains studied, 20 carry KPC, one carries NDM-1, and one carries OXA-370. The PFGE analysis showed three clades with a genetic relatedness >85%, two formed by four strains and one by eight strains. The other strains are not genetically related, and a total of 17 different pulse types were detected. Ten different STs were identified, the main ones being ST258 (five strains) and ST1161 (seven strains). The isolates presented different percentages of resistance, and 82% were resistant to all the ß-lactams tested, 91% to ciprofloxacin, 73% to colistin, 59% to gentamicin, 50% to amikacin, and only 9% to tigecycline. All isolates carried blaTEM and blaSHV, whereas 71% carried aac(6')Ib-cr, and 57% one qnr gene (A, B, C, D, or S). The blaCTX-M gene was found in 10 of the isolates (4 blaCTX-M-15 and 6 blaCTX-M-2). The characterization of the platform, in seven selected strains, revealed that the gene is associated with unusual class 1 integrons and insertion sequences such as ISCR1, ISECp1, and IS26. Conclusion: In the first carbapenemase-producing K. pneumoniae strains isolated in Chile the genetic platform of blaCTX-M-2 corresponds to an unusual class 1 integron that can be responsible for the MDR phenotype, whereas the genetic platforms of blaCTX-M-15 are associated with different IS and do not contribute to multi-drug resistance.

The efflux pump inhibitor phenylalanine-arginine ß-naphthylamide (PAßN) increases resistance to carbapenems in Chilean clinical isolates of KPC-producing Klebsiella pneumoniae.

OBJECTIVES: KPC-producing strains present a wide range of carbapenem minimum inhibitory concentrations (MICs). This variation may be due to differential expression of blaKPC and porin genes, efflux pump activity and the production of extended-spectrum ß-lactamases and/or AmpC ß-lactamases. The aim of this study was to determine the role of efflux pumps inhibited by phenylalanine-arginine ß-naphthylamide (PAßN) in resistance to carbapenems in Chilean clinical isolates of blaKPC-harbouring Klebsiella pneumoniae. METHODS: MICs were determined by the agar dilution method for imipenem, meropenem, ertapenem and ciprofloxacin in the presence and absence of PAßN (25mg/L) in 17 carbapenem-resistant KPC-producing K. pneumoniae strains. Outer protein membrane (OMP) profiles were determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Expression levels of the ompK35 and ompK36 genes were also determined by real-time quantitative reverse transcription PCR (qRT-PCR). RESULTS: No contribution of PAßN-inhibited efflux pumps to carbapenem resistance was found, unlike ciprofloxacin resistance. However, a ≥4-fold increase in the MIC of at least one carbapenem was observed in 13 isolates in the presence of PAßN. Additionally, decreased gene expression of ompK35 and ompK36 in the presence of PAßN was detected, however no obvious differences in porin band intensity were observed by SDS-PAGE. CONCLUSIONS: The presence of PAßN resulted in an increase in carbapenem MICs unrelated to efflux pump inhibition, and a decrease in the expression of ompK35 and ompK36 genes without an obvious difference in OMP profiles observed by SDS-PAGE. Therefore, additional factors are responsible for the increase in carbapenem MIC in the presence of PAßN.

KPC: Klebsiella pneumoniae carbapenemasa, principal carbapenemasa en enterobacterias / KPC: Klebsiella pneumoniae carbapenemase, main carbapenemase in Enterobacteriaceae

Resumen En la actualidad, la diseminación de enterobacterias productoras de carbapenemasas se considera un grave problema en clínica debido al fracaso en el tratamiento de las infecciones que ellas producen. Entre las carbapenemasas, la enzima KPC se ha diseminado mundialmente y ha sido identificada en las principales especies de enterobacterias relacionadas con infecciones asociadas a la atención en salud, con claro predominio de Klebsiella pneumoniae a nivel mundial. El gen blaKPC es transportado, principalmente, por el transposón Tn4401, detectado en diversas especies de enterobacterias con distintos secuencio-tipo (ST) y diferente origen geográfico. Adicionalmente, se han descrito nuevas plataformas genéticas que se distinguen del Tn4401 original debido a inserciones y deleciones de otros genes. Los plásmidos que albergan el gen blaKPC pueden ser del tipo conjugativo y no conjugativo movilizable, y además contener otros determinantes genéticos de resistencia. Las cepas productoras de KPC pueden presentar diversos niveles de resistencia a los carbapenémicos, debido a la participación de mecanismos adicionales como diferente grado de expresión de porinas y bombas de expulsión asociados con la producción de β-lactamasas de espectro extendido y/o AmpC. Sin embargo, las carbapenemasas, con KPC como la enzima más frecuente, otorgan grados de resistencia más elevados.

The dissemination of carbapenemase-producing Enterobacteriaceae is currently considered a serious clinical problem due to the failure in the treatment of infections produced by them. Among the carbapenemases, the enzyme KPC has spread worldwide and has been identified in the main enterobacterial species related with healthcareassociated infections, although Klebsiella pneumoniae is the predominant specie. The blaKPC gene is transported, mainly by the transposon Tn4401, detected in various enterobacterial species of different sequence types (ST) and geographical origin. In addition, new genetic platforms that are distinguished, from Tn4401 because of insertions or deletions of other genes have been described. Plasmids containing the blaKPC gene can be conjugative and mobilizable non-conjugative plasmids, and can carry other genetic determinants of resistance. The KPC-producing strains may have different levels of resistance to carbapenems, due to the involvement of additional mechanisms such as different expression levels of porins and efflux pumps associated with the production of extended spectrum β-lactamases and/or AmpC. However, the carbapenemases, with KPC as the most common enzyme, provide higher levels of resistance.

Diversity structure of culturable bacteria isolated from the Fildes Peninsula (King George Island, Antarctica): A phylogenetic analysis perspective.

It has been proposed that Antarctic environments select microorganisms with unique biochemical adaptations, based on the tenet 'Everything is everywhere, but, the environment selects' by Baas-Becking. However, this is a hypothesis that has not been extensively evaluated. This study evaluated the fundamental prediction contained in this hypothesis-in the sense that species are structured in the landscape according to their local habitats-, using as study model the phylogenetic diversity of the culturable bacteria of Fildes Peninsula (King George Island, Antarctica). Eighty bacterial strains isolated from 10 different locations in the area, were recovered. Based on phylogenetic analysis of 16S rRNA gene sequences, the isolates were grouped into twenty-six phylotypes distributed in three main clades, of which only six are exclusive to Antarctica. Results showed that phylotypes do not group significantly by habitat type; however, local habitat types had phylogenetic signal, which support the phylogenetic niche conservatism hypothesis and not a selective role of the environment like the Baas-Becking hypothesis suggests. We propose that, more than habitat selection resulting in new local adaptations and diversity, local historical colonization and species sorting (i.e. differences in speciation and extinction rates that arise by interaction of species level traits with the environment) play a fundamental role on the culturable bacterial diversity in Antarctica.

[KPC: Klebsiella pneumoniae carbapenemase, main carbapenemase in Enterobacteriaceae]. / KPC: Klebsiella pneumoniae carbapenemasa, principal carbapenemasa en enterobacterias.

The dissemination of carbapenemase-producing Enterobacteriaceae is currently considered a serious clinical problem due to the failure in the treatment of infections produced by them. Among the carbapenemases, the enzyme KPC has spread worldwide and has been identified in the main enterobacterial species related with healthcareassociated infections, although Klebsiella pneumoniae is the predominant specie. The blaKPC gene is transported, mainly by the transposon Tn4401, detected in various enterobacterial species of different sequence types (ST) and geographical origin. In addition, new genetic platforms that are distinguished, from Tn4401 because of insertions or deletions of other genes have been described. Plasmids containing the blaKPC gene can be conjugative and mobilizable non-conjugative plasmids, and can carry other genetic determinants of resistance. The KPC-producing strains may have different levels of resistance to carbapenems, due to the involvement of additional mechanisms such as different expression levels of porins and efflux pumps associated with the production of extended spectrum ß-lactamases and/or AmpC. However, the carbapenemases, with KPC as the most common enzyme, provide higher levels of resistance.

Molecular typing and genetic environment of the blaKPC gene in Chilean isolates of Klebsiella pneumoniae.

The aim of this work was to determine the genetic environment and transferability of blaKPC as well as the pulsotypes of KPC-producing Klebsiella pneumoniae strains isolated from clinical samples in Chilean hospitals. Seventeen strains, principally isolated in Santiago (the capital of Chile) during the years 2012 and 2013, were included. The genetic environment of blaKPC was elucidated by PCR mapping and sequencing. Molecular typing was performed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Curing and conjugation experiments were performed with six strains of different sequence types (STs) and pulsotypes. Thirteen pulsotypes and six STs, mainly belonging to clonal complex 258, were found. In addition, seven strains belonged to a new ST assigned ST1161. The blaKPC sequence indicated that 16 strains had the KPC-2 variant; in only one strain (UC331) an amino acid change (R6P) was detected, corresponding to a new KPC variant designated KPC-24. Molecular characterisation of the blaKPC genetic environment revealed two distinct platforms, namely variant 1a and the Tn4401a isoform, with the first being the most common (11/17 strains). Mating experiments failed to produce transconjugants; however, loss of blaKPC was achieved by plasmid curing in all assayed strains. In conclusion, in Chilean strains of K. pneumoniae, blaKPC is primarily found associated with the variant 1a and is located in non-transferable plasmids. In addition, this study highlights the description of the new ST1161 and the new KPC-24 variant.