Comparative Analysis of Carbapenem-Resistant Acinetobacter baumannii Sequence Types in Southern Brazil: From the First Outbreak (2007-2008) to the Endemic Period (2013-2014).

Acinetobacter baumannii is considered an important pathogen of clinical significance that is responsible for a wide range of nosocomial infections. Carbapenem resistance among A. baumannii isolates has increased dramatically in the Past years because of the emergence and dissemination of specific epidemic clones. We aimed to characterize the population structure of A. baumannii isolates from Porto Alegre city, Southern Brazil, in two distinct periods: during the first carbapenem-resistant A. baumannii (CRAB) outbreak (2007-2008) and 5 years later when the CRAB reached endemic levels (2013-2014). The study included 49 CRAB isolates collected in two periods: 2007-2008 (31 isolates) and 2013-2014 (18 isolates). Multilocus sequence typing (MLST) was performed according to Institute Pasteur, followed by eBURST analysis. PCR was used to detect integrase gene, blaNDM, and oxacillinase genes, and also to detect the ISAba1 element upstream blaOXA-23. The eBURST analysis identified the clonal complexes (CCs) CC15, CC32, CC79, CC216, CC221, and CC464 in the first period (2007-2008) and CC1, CC2, CC15, CC79, and CC162 during the endemic period (2013-2014). Molecular analysis by MLST identified 13 new sequence types. As we found A. baumannii with the blaOXA-23 gene of several CCs, it can be concluded that the increase of CRAB infections are not related to a specific clone. Furthermore, the high-risk CC15 and CC79 were related to the first CRAB outbreak and these CCs persisted up to 2014 in Porto Alegre city. The international clones CC1 and CC2 were observed for the first time in only the second period (2013-2014), alerting to the emergence of these clones in Southern Brazil.

Carbapenem-resistant Acinetobacter baumannii in Brazil: susceptibility profile and diversity of oxacillinases / Acinetobacter baumannii resistente aos carbapenêmicos no Brasil: perfil de suscetibilidade e diversidade de oxacilinases

ABSTRACT Introduction: The Acinetobacter calcoaceticus-baumannii (ABC) complex includes five species, and the A. baumannii is the most important of them because it carries mechanisms of carbapenems resistance, especially the oxacillinases. Objectives: The objectives of this study were to identify the species of the ABC complex, to evaluate the susceptibility profile and to investigate the presence of oxacillinases in carbapenems-resistant isolates from four Brazilian States. Methods: In the study period, 92 isolates from Rio Grande do Sul (RS), Rio de Janeiro (RJ), Paraná (PR) and São Paulo (SP) were collected. The isolates were identified by matrix-assisted laser desorption ionization-time of fight mass spectrometry (MALDI-TOF MS) and sequencing of gyrB gene. Evaluation of susceptibility was performed by disk diffusion and broth microdilution. The presence of oxacillinases was performed by in-house multiplex polymerase chain reaction (PCR). Results: Ninety-one (99%) isolates were identified as A. baumannii by MALDI-TOF and sequencing. The majority of isolates (56; 61%) showed resistance to the six antimicrobial agents tested. Three isolates were resistant to polymyxin B [minimum inhibitory concentration (MIC) ≥ 4 µg/ml). Eighty (87%) isolates were positive to OXA-23-like, and twelve (13%) isolates to OXA-24-like. Conclusion: Our findings confirm the knowledge about the dissemination of the blaOXA-23 gene in Brazil and suggest the recent emergence and spread of blaOXA-24 gene, since it was identified in three of the four sampled states.

RESUMO Introdução: O complexo Acinetobacter calcoaceticus-baumannii (ABC) inclui cinco espécies, sendo A. baumannii a mais importante clinicamente por carrear muitos mecanismos de resistência aos carbapenêmicos, sobretudo as oxacilinases. Objetivos: Os objetivos deste estudo foram identificar as espécies do complexo ABC, avaliar o perfil de suscetibilidade e investigar a presença de oxacilinases em isolados resistentes aos carbapenêmicos provenientes de quatro estados brasileiros. Métodos: No período do estudo, foram coletados 92 isolados do Rio Grande do Sul (RS), do Rio de Janeiro (RJ), do Paraná (PR) e de São Paulo (SP). Os isolados foram identificados por matrix-assisted laser desorption ionization-time of fight mass spectrometry (MALDI-TOF MS) e sequenciamento do gene gyrB. A avaliação da suscetibilidade foi realizada por disco-difusão e microdiluição de caldo. A presença de oxacilinases foi realizada por reação em cadeia da polimerase (PCR) multiplex in house. Resultados: Noventa e um (99%) isolados foram identificados como A. baumannii por MALDI-TOF e pelo sequenciamento. A maioria dos isolados (56; 61%) apresentou resistência aos seis agentes antimicrobianos testados. Três isolados foram resistentes à polimixina B [concentração inibitória mínima (CIM) ≥ 4 µg/ml). Oitenta (87%) isolados foram positivos para OXA-23 e 12, (13%) para OXA-24. Conclusão: Nossos resultados confirmam a disseminação do gene blaOXA-23 no Brasil e sugerem a recente emergência e disseminação do gene blaOXA-24, uma vez que ele foi identificado em três dos quatro estados amostrados.

Mobile genetic elements related to carbapenem resistance in Acinetobacter baumannii

Abstract Acinetobacter baumannii is widely recognized as an important pathogen associated with nosocomial infections. The treatment of these infections is often difficult due to the acquisition of resistance genes. A. baumannii presents a high genetic plasticity which allows the accumulation of these resistance determinants leading to multidrug resistance. It is highlighted the importance of the horizontal transfer of resistance genes, through mobile genetic elements and its relationship with increased incidence of multidrug resistant A. baumannii in hospitals. Considering that resistance to carbapenems is very important from the clinical and epidemiological point of view, the aim of this article is to present an overview of the current knowledge about genetic elements related to carbapenem resistance in A. baumannii such as integrons, transposons, resistance islands and insertion sequences.

Emergence of Acinetobacter baumannii ST730 carrying the blaOXA-72 gene in Brazil.

Over the last decade, Acinetobacter baumannii resistant to carbapenems has emerged in many medical centres and has been commonly associated with high morbimortality. In Brazil, this resistance is mainly attributed to the spread of OXA-23-producing clones and, to a lesser extent, to OXA-143-producing clones. Here, we describe, for the first time, two OXA-72-producing A. baumannii isolates in southern Brazil to a broad spectrum of antibiotics, except polymyxin B and tigecycline. Molecular typing by multilocus sequence typing (MLST) demonstrated that both OXA-72-producing isolates belong to a new sequence type (ST), ST730, which was recently identified in OXA-23-producing A. baumannii isolates in São Paulo, Brazil. We demonstrate that the two A. baumannii ST730 isolates carrying blaOXA-72share a common ancestral origin with the blaOXA-23producers in Brazil. This observation reinforces the importance of strain-typing methods in order to clarify the dynamics of the emergence of new clones in a geographic region.

Emergence of Acinetobacter baumannii ST730 carrying the blaOXA-72 gene in Brazil

Over the last decade, Acinetobacter baumannii resistant to carbapenems has emerged in many medical centres and has been commonly associated with high morbimortality. In Brazil, this resistance is mainly attributed to the spread of OXA-23-producing clones and, to a lesser extent, to OXA-143-producing clones. Here, we describe, for the first time, two OXA-72-producing A. baumannii isolates in southern Brazil to a broad spectrum of antibiotics, except polymyxin B and tigecycline. Molecular typing by multilocus sequence typing (MLST) demonstrated that both OXA-72-producing isolates belong to a new sequence type (ST), ST730, which was recently identified in OXA-23-producing A. baumannii isolates in São Paulo, Brazil. We demonstrate that the two A. baumannii ST730 isolates carrying blaOXA-72share a common ancestral origin with the blaOXA-23producers in Brazil. This observation reinforces the importance of strain-typing methods in order to clarify the dynamics of the emergence of new clones in a geographic region.

Mobile genetic elements related to carbapenem resistance in Acinetobacter baumannii.

Acinetobacter baumannii is widely recognized as an important pathogen associated with nosocomial infections. The treatment of these infections is often difficult due to the acquisition of resistance genes. A. baumannii presents a high genetic plasticity which allows the accumulation of these resistance determinants leading to multidrug resistance. It is highlighted the importance of the horizontal transfer of resistance genes, through mobile genetic elements and its relationship with increased incidence of multidrug resistant A. baumannii in hospitals. Considering that resistance to carbapenems is very important from the clinical and epidemiological point of view, the aim of this article is to present an overview of the current knowledge about genetic elements related to carbapenem resistance in A. baumannii such as integrons, transposons, resistance islands and insertion sequences.

Emergence of NDM-1-producing Enterobacteriaceae in Porto Alegre, Brazil.

OBJECTIVES: To evaluate the emergence of New Delhi metallo-ß-lactamase 1 (NDM-1)-producing Enterobacteriaceae isolates in Brazil. METHODS: From April to October 2013, following the detection of the first NDM-1-producing isolate, a surveillance study was performed for the detection of blaNDM-1 among Enterobacteriaceae isolates with reduced susceptibility to carbapenems in 17 hospitals of Porto Alegre, Brazil. Real-time PCR was used to determine the presence of carbapenemase genes, which were further sequenced. Clonal relatedness was assessed by pulsed-field gel electrophoresis (PFGE). RESULTS: A total of 1134 isolates were evaluated. blaNDM-1 was detected in 11 (0.97%) isolates: nine Enterobacter cloacae complex (eight belonging to a single clone recovered from two distinct hospitals and the other strain from a third hospital) and two Morganella morganii (belonging to a single clone recovered from one hospital). Most isolates presented high-level resistance to carbapenems. CONCLUSIONS: NDM-1-producing Enterobacteriaceae have emerged rapidly in the hospitals of the Brazilian city where they were first detected. The emergence of NDM-1 in Brazil is of great concern, since it is a severe threat to antimicrobial therapy against Enterobacteriaceae in this country.

Hydrophobic proteins secreted into the apoplast may contribute to resistance against Phytophthora infestans in potato.

During plant-pathogen interaction, oomycetes secrete effectors into the plant apoplast where they interact with host resistance proteins, which are accumulated after wounding or infection. Previous studies showed that the expression profile of pathogenesis related proteins is proportional to the resistance of different cultivars toward Phytophthora infestans infection. The aim of this work was to analyze the expression pattern of apoplastic hydrophobic proteins (AHPs), after 24 h of wounding or infection, in tubers from two potato cultivars with different resistance to P. infestans, Spunta (susceptible) and Innovator (resistant). Intercellular washing fluid (IWF) was extracted from tubers and chromatographed into a PepRPC™ HR5-5 column in FPLC eluted with a linear gradient of 75% acetonitrile. Then, AHPs were analyzed by SDS-PAGE and identified by MALDI-TOF-MS. Innovator cv. showed a higher basal AHP content compared to Spunta cv. In the latter, infection induced accumulation of patatins and protease inhibitors (PIs), whereas in Innovator cv. no changes in PIs accumulation were observed. In response to P. infestans infection, lipoxygenase, enolase, annexin p34 and glutarredoxin/cyclophilin were accumulated in both cultivars. These results suggest that the AHPs content may be related to the protection against the oomycete and with the degree of potato resistance to pathogens. Additionally, a considerable number of the proteins putatively identified lacked the signal peptide and, being SecretomeP positive, suggest unconventional protein secretion.

High endemic levels of multidrug-resistant Acinetobacter baumannii among hospitals in southern Brazil.

BACKGROUND: Most published data on multidrug-resistant Acinetobacter baumanii (MDR Ab) are derived from outbreaks. We report incidence trends on health care-acquired infections due to MDR Ab over a 12-month period in the city of Porto Alegre in southern Brazil. METHODS: Clinical and epidemiologic data were obtained from the local health care information system of the municipal health department. Polymerase chain reaction was used to detect the presence of the genes bla(OXA-23-like), bla(OXA-24-like), bla(OXA-51), and bla(OXA-58), and repetitive sequence-based polymerase chain reaction and pulsed-field gel electrophoresis were performed for molecular typing. RESULTS: The highest rate of infection (9.0/1,000 inpatient-days) was identified in a trauma hospital. The gene bla(OXA-23-like) was identified in 99.0% of MDR Ab isolates. Eight main clonal groups were identified by molecular typing, and 3 of these were found in all hospitals. CONCLUSION: The presence of 3 clones in all hospitals demonstrates the ability of MDR Ab to spread among hospitals. Moreover, the occurrence of one particular clone (clone 4) throughout the study period suggests its increased ability to cause outbreaks and to remain in the environment. The monitoring of epidemic strains by molecular methods is of paramount importance to prevent or reduce the spread of MDR Ab.

DNA as molecular target of analogous palladium and platinum anti-Trypanosoma cruzi compounds: a comparative study.

In the search for drugs with anti-trypanosome activity, we had previously synthesized two series of platinum and palladium analogous compounds of the formula [M(II)Cl(2)(HL)], where HL were bioactive 5-nitrofuryl or 5-nitroacroleine thiosemicarbazone derivatives. In this work, we thoroughly characterized [M(II)Cl(2)(HL)] complexes interaction with DNA by using different techniques: gel electrophoresis, DNA viscosity measurements, circular dichroism (CD) and atomic force microscopy (AFM). Electrophoresis results showed that all complexes induced a withdrawal of DNA superhelicity demonstrated by a decrease in electrophoretic mobility of supercoiled DNA form. This effect on migration was dependent on dose but also on the nature of both the metal and the ligand. In general, the effect produced by palladium complexes was significantly more intense than that observed for the corresponding platinum analogs. Differences between palladium and platinum complexes were also observed in CD experiments. While palladium complexes induce evident calf thymus (CT)-DNA profile changes compatible with B-DNA to Z-DNA conformational transition, no clear effect was observed for platinum ones. Additionally, AFM studies showed that changes in the shape of plasmid DNA, like supercoiling, kinks and thickness increase resulted more intense for the former. In addition, either Pd or Pt complexes increased the viscosity of CT DNA solutions in a concentration dependent manner. Although the nature of DNA interaction of both series of analogous palladium and platinum complexes seemed to be similar, an explanation for the observed differential intensity of the effect could be related to the known kinetic stability differences between palladium and platinum compounds.

The swaposin-like domain of potato aspartic protease (StAsp-PSI) exerts antimicrobial activity on plant and human pathogens.

Plant-specific insert domain (PSI) is a region of approximately 100 amino acid residues present in most plant aspartic protease (AP) precursors. PSI is not a true saposin domain; it is the exchange of the N- and C-terminal portions of the saposin like domain. Hence, PSI is called a swaposin domain. Here, we report the cloned, heterologous expression and purification of PSI from StAsp 1 (Solanum tuberosum aspartic protease 1), called StAsp-PSI. Results obtained here show that StAsp-PSI is able to kill spores of two potato pathogens in a dose-dependent manner without any deleterious effect on plant cells. As reported for StAPs (S. tuberosum aspartic proteases), the StAsp-PSI ability to kill microbial pathogens is dependent on the direct interaction of the protein with the microbial cell wall/or membrane, leading to increased permeability and lysis. Additionally, we demonstrated that, like proteins of the SAPLIP family, StAsp-PSI and StAPs are cytotoxic to Gram-negative and Gram-positive bacteria in a dose dependent manner. The amino acid residues conserved in SP_B (pulmonary surfactant protein B) and StAsp-PSI could explain the cytotoxic activity exerted by StAsp-PSI and StAPs against Gram-positive bacteria. These results and data previously reported suggest that the presence of the PSI domain in mature StAPs could be related to their antimicrobial activity.

Effect of ruthenium complexation on trypanocidal activity of 5-nitrofuryl containing thiosemicarbazones.

In the search of new therapeutic tools for the treatment of American Trypanosomiasis, the largest parasitic disease burden in the American continent, three series of novel ruthenium complexes of the formula [RuCl(2)(HL)(2)], [RuCl(3)(dmso)(HL)] and [RuCl(PPh(3))(L)(2)] with bioactive 5-nitrofuryl containing thiosemicarbazones as ligands (HL neutral, L monoanionic) were synthesized and characterized. Their in vitro growth inhibition activity against Trypanosoma cruzi and the effect of co-ligands in related physicochemical properties i.e. nitro moiety redox potential, lipophilicity and free radical scavenger capacity were evaluated. Results show that although a loss of activity was observed as a consequence of ruthenium complexation, lipophilicity and free radical scavenger capacity of the obtained complexes could be correlated to their trypanocidal effect.

Roles of glycosylation on the antifungal activity and apoplast accumulation of StAPs (Solanum tuberosum aspartic proteases).

Specific roles of glycosylation appear to be protein-dependent. Plant aspartic proteases (APs) contain two or more consensus N-glycosylation sites; however, the importance of them is not well understood. StAPs (Solanum tuberosum aspartic proteases) are bifunctional proteins with both proteolytic and antimicrobial activities. These proteins are accumulated into the intercellular washing fluid of potato tubers and leaves after wounding or infection. In this paper we investigated the importance of glycosylation on the StAPs apoplast accumulation, biochemical parameters, and fungicidal activity. Assays to evaluate the importance of StAPs glycosylation groups by using glycosylation inhibitors demonstrate that carbohydrate portions are essential to StAPs accumulation into the apoplast of tubers and leaves after wounding or detachment, respectively. Bifunctional activity of StAPs is differentially affected by this post-translational modification. Results obtained show that not significant changes were produced in the physicochemical properties after StAPs deglycosylation (pH and thermal-optimum activity and index of protein surface hydrophobicity). Otherwise, StAPs antifungal activity is affected by deglycosylation. Deglycosylated StAPs (dgStAPs) fungicidal activity is lower than native StAPs at all concentrations and times assayed. In summary, glycosylation has not a significant role on the StAPs conformational structure. However, it is involved in the StAPs subcellular accumulation and antifungal activity suggesting that it could be necessary for StAPs membrane and/or protein interactions and subsequently its biological function(s).