Detection of KPC enzyme by MALDI-TOF MS from bacteria impregnated in filter paper.

The main mechanism that causes resistance to carbapenem, one of the most potent antibiotic available, in Enterobacterales bacterial isolates, is due to Klebsiella pneumoniae carbapenemase (KPC) production by the bacterium. KPC is spread worldwide, requiring laboratories to be capable of identifying this enzyme, however some methods can be expensive for small laboratories, especially in developing countries. Therefore, the development of methods with low cost of reagents for the detection of KPC enzyme is necessary. The objective of this study was to evaluate the detection of KPC enzyme by MALDI-TOF MS from inactivated bacteria impregnated in filter paper. A total of 129 Enterobacterales isolates were impregnated in filter paper, and after 7 days at room temperature, they were subjected to a protein extraction protocol and spectra acquisition, in triplicates, by MALDI-TOF MS. The spectra were evaluated and KPC was identified according to the presence of a peak of 28,712.62 ± 27.80 m/z. Considering the presence of the KPC peak in at least one spectrum of the triplicates, this method presented 60.8% sensitivity and 96.4% specificity. However, considering the presence of KPC peak in at least two spectra of the triplicate, a specificity of 100% was achieved. The detection of KPC enzyme from inactivated bacteria impregnated in filter paper can be used as a method to confirm the presence of KPC, which could be very significant for small laboratories with limited resources.

Validation and calibration of a novel GEM biosensor for specific detection of Cd2+, Zn2+, and Pb2.

BACKGROUND: In this study, we designed a novel genetic circuit sensitive to Cd2+, Zn2+ and Pb2+ by mimicking the CadA/CadR operon system mediated heavy metal homeostasis mechanism of Pseudomonas aeruginosa. The regular DNA motifs on natural operon were reconfigured and coupled with the enhanced Green Fluorescent Protein (eGFP) reporter to develop a novel basic NOT type logic gate CadA/CadR-eGFP to respond metal ions mentioned above. A Genetically Engineered Microbial (GEM)-based biosensor (E.coli-BL21:pJET1.2-CadA/CadR-eGFP) was developed by cloning the chemically synthesised CadA/CadR-eGFP gene circuit into pJET1.2-plasmid and transforming into Escherichia coli (E. coli)-BL21 bacterial cells. RESULTS: The GEM-based biosensor cells indicated the reporter gene expression in the presence of Cd2+, Zn2+ and Pb2+ either singly or in combination. Further, the same biosensor cells calibrated for fluorescent intensity against heavy metal concentration generated linear graphs for Cd2+, Zn2+ and Pb2+ with the R2 values of 0.9809, 0.9761 and 0.9758, respectively as compared to non-specific metals, Fe3+ (0.0373), AsO43- (0.3825) and Ni2+ (0.8498) making our biosensor suitable for the detection of low concentration of the former metal ions in the range of 1-6 ppb. Furthermore, the GEM based biosensor cells were growing naturally within the concentration range of heavy metals, at 37 °C and optimum pH = 7.0 in the medium, resembling the characteristics of wildtype E.coli. CONCLUSION: Finally, the novel GEM based biosensor cells developed in this study can be applied for detection of targeted heavy metals in low concentration ranges (1-6 ppb) at normal bacterial physiological conditions.

Lactobacillus delbrueckii UFV-H2b20 increases IFN-γ production and CD39+CD73+ Treg cell numbers in lungs, and protects mice against experimental allergic asthma.

Asthma is a disorder characterized by airflow obstruction, inflammation, declining airway function, bronchial hyperresponsiveness and tissue remodelling. Probiotics are defined as "live microorganisms that, when administered in adequate amounts, confer a health benefit on the host". The use of probiotics is becoming increasingly studied and recent evidence has suggested that it may provide therapeutic benefits in asthma and other diseases. Lactobacillus delbrueckii UFV-H2b20 fulfils all the requirements to be classified as probiotic. Previous studies have already shown the ability of L. delbrueckii UFV-H2b20 to stimulate the immune system. Our objective was to evaluate the protective effects of L. delbrueckii UFV-H2b20 in experimental allergic asthma. We used a murine model of ovalbumin-induced allergic airway inflammation to mimic allergic asthma. Oral treatment with L. delbrueckii UFV-H2b20 improves respiratory parameters and inhibits the inflammatory response in the lungs by decreasing the numbers of inflammatory monocytes, eosinophils and alveolar macrophages, as well as IgE levels. Treatment increased the IFN-γ/IL-4 cytokine ratio. Levels of IL-10 in the lungs were also increased in treated animals. Our results also showed that the probiotic administration increases the number of CD39+CD73+ T regulatory lymphocytes in the lung, suggesting a role for purinergic signals in the regulation of inflammation promoted by the treatment. Understanding the mechanisms of modulation of the immune system by probiotics could allow the development of probiotic preparations that are safe and have a direct action. Our results suggest that oral administration of L. delbrueckii UFV-H2b20 could be helpful to treat chronic inflammatory airway diseases, such as asthma.

Uso y seguridad de levosimendan en una unidad de cuidados intermedios: revisión de la experiencia de 3 años / Use and safety of Levosimendan in an Intermediate Care Unit: 3-year experience review

Introduction: Levosimendan is an inodilator with positive inotropiceffect whose demonstration of hemodynamic and clinical benefits hasnot always been consistent. The most recent meta-analyzes show stronger evidence of it, especially in some subgroups. The objective wasto evaluate the experience in the use of levosimendan, characterizingthe mode of prescription, the target population, clinical benefits andadverse effects.Materials and Methodologies: All patients who took Levosimendan in anIntermediate Care Unit during three full years were included. Generalclinical and analytical parameters, co-morbidities and characteristics ofhospitalization were obtained, as well as readmissions up to 6 months.Results: There were 39 events. Thirteen admissions were scheduled.Only 4 patients tolerated the maximum recommended levosimendanspeed. All completed 12.5 mg of levosimendan, 10 of which requiredaminergic support. In-hospital mortality was 15.4%. For all the patientswho died, admission was urgent.Conclusions: No patient with scheduled admission required aminergic support or died during hospitalization. It is not possible to inferwhether it would be possible to perform the same dose in a shorterperiod of time, even because of the small number that tolerated themaximum speed. Results of ongoing studies may help assess safetyand propose selection criteria for patients suitable for day hospitaladministration. Particularly in patients with advanced HF, intermittentand repeated administration, as occurred in this study, is a promising option. However, there are still important gaps, namely which isthe ideal cumulative dose and the frequency with which it shouldbe performed.

Introducción: El levosimendan es un sensibilizador de calcio con efectoinotrópico positivo cuya demostración de beneficios hemodinámicos yclínicos no siempre ha sido consistente. Los metanálisis más recientesmuestran pruebas más contundentes de ello, especialmente en algunossubgrupos. El objetivo fue evaluar la experiencia en el uso de levosimendan, caracterizando el modo de prescripción, la población, los beneficiosclínicos y los efectos adversos.Materiales y Metodologías: Se incluyeron todos los pacientes que tomaron Levosimendan en una Unidad de Cuidados Intermedios durante tresaños. Se obtuvieron parámetros clínicos y analíticos generales, comorbilidades y características de la hospitalización, así como reingresos hastalos 6 meses.Resultados: Hubo 39 eventos. Se programaron trece ingresos. Solo 4 pacientes toleraron la velocidad máxima recomendada de levosimendan.Todos completaron 12,5 mg de levosimendan, 10 de los cuales requirieron apoyo aminérgico. La mortalidad hospitalaria fue del 15,4%. Paratodos los pacientes que fallecieron, el ingreso fue urgente.Conclusiones: Ningún paciente con ingreso programado requirió apoyoaminérgico ni falleció durante la hospitalización. No es posible inferirsi sería posible realizar la misma dosis en un período de tiempo máscorto, incluso por el pequeño número que toleró la velocidad máxima.Los resultados de los estudios en curso pueden ayudar a evaluar la seguridad y proponer criterios de selección para pacientes adecuados para laadministración en un hospital de día. Particularmente en pacientes conIC avanzada, la administración intermitente y repetida, como ocurrió eneste estudio, es una opción prometedora. Sin embargo, existen lagunasimportantes, a saber, cuál es la dosis acumulativa ideal y la frecuenciacon la que debe realizarse.

Escherichia coli carrying blaNDM-1 obtained from a migratory penguin (Spheniscus magellanicus) in the Brazilian seacoast.

The reservoirs for NDM-producing Enterobacterales are increasing, not only in hospitals, but also in the environment and in the community, challenging the therapeutic efficacy of carbapenems. We aimed to characterize an isolate of Escherichia coli harboring the blaNDM-1 gene recovered from the bloodstream of a penguin (Spheniscus magellanicus) in Southern Brazil. A total of 74 bacterial isolates recovered from arterial blood samples from dead birds were submitted to species identification and antibiotic susceptibility evaluation. One isolate presented resistance to carbapenems (E. coli 89PenNDM) and proved to harbor the blaNDM-1 gene by multiplex high-resolution melting real-time PCR (PCR-HRM). Conjugation experiments indicated that the blaNDM-1 was transmissible to E. coli J53. Whole genome sequencing (WGS) confirmed the presence of the blaNDM-1 gene in a conjugative plasmid (IncA/C2 plasmid) in both the E. coli 89PenNDM and its transconjugants. The isolate was classified as ST 156 and many other resistance genes (e.g., sul1, sul,2, strA, floR, tet(A)) were identified, all carried in the same IncA/C2 plasmid. This is the first report of blaNDM-1-producing E. coli isolated from a penguin in the Brazilian seacoast. The presence of a carbapenemase gene in wildlife animals is of concern as they may become reservoirs of multidrug-resistant bacteria and disseminate them to the environment.

Yellow grease in sheep diets: intake and digestibility / [Óleo residual de fritura em dietas para ovinos: consumo e digestibilidade]

This study aimed to assess the effects of yellow grease supplementation on the intake, digestibility, and nitrogen balance in sheep. Twenty Santa Inês lambs with a mean age of 95 ± 10 d and body weight of 19.29 ± 3.17kg were evaluated in a completely randomized design. The diets were supplemented with oil at concentrations of 0, 20, 40, 60, and 80 gkg-1 of dry matter (DM) of the concentrate. The diets were based on roughage and concentrate (50:50). The experimental period lasted 19 d and included 14 adaptation days and five collection days for the total supplied diet, orts, feces, and urine. Supplementation with yellow grease had no significant effect on the intake of DM, crude protein (CP), neutral detergent fiber (NDF), or non-fiber carbohydrates (NFC). However, the ether extract (EE) intake increased linearly with supplementation of yellow grease. Moreover, no effect was observed for DM, CP, NDF, and NFC digestibility and nitrogen balance. EE digestibility increased linearly with the yellow grease dietary supplementation. Thus, sheep dietary supplementation with yellow grease may be used at a level of up to 80 gkg-1 of DM of concentrate without impairing nutrient intake and digestibility.(AU)

Objetivou-se, com o estudo, avaliar os efeitos do óleo residual de fritura, em dietas para ovinos, sob o consumo, a digestibilidade e o balanço de nitrogênio. Foram utilizados 20 cordeiros Santa Inês, com idade de 95 ± 10 dias e peso corporal de 19,29 ± 3,17kg, em delineamento inteiramente ao acaso. As dietas continham óleo de fritura nas concentrações de 0; 20; 40; 60 e 80gkg-1 da matéria seca (MS) do concentrado. As dietas tinham relação volumoso:concentrado de 50:50. O período experimental foi de 19 dias, incluindo 14 dias em adaptação e cinco dias de coleta do fornecido, das sobras, das fezes e da urina. A suplementação com óleo de fritura não alterou o consumo de MS, proteína bruta (PB), matéria orgânica (MO), fibra em detergente neutro (FDN) e carboidratos não fibrosos (CNF). Entretanto, o consumo de extrato etéreo (EE) aumentou com a inclusão do óleo. Não foi observado efeito na digestibilidade da MS, da PB, da FDN, dos CNF e no balanço de nitrogênio. A digestibilidade do EE aumentou com a inclusão do óleo. Assim, a inclusão de óleo de fritura em dietas para ovinos pode ser utilizada em até 80gkg-1 da MS do concentrado, sem limitar ingestão e digestibilidade dos nutrientes.(AU)

Increased frequency of blaNDM in a tertiary care hospital in southern Brazil.

Resistance to carbapenems due to metallo-beta-lactamase NDM-1 was first described in Brazil in 2013. To date, only a few scattered reports of the prevalence of NDM-1 in the country have been reported, and most of them indicated a very low prevalence of this metalloenzyme. In the present study, we report a steady increase in the frequency of NDM among Enterobacterales resistant to carbapenems in a tertiary care hospital in southern Brazil. Carbapenemase genes were evaluated by multiplex real-time polymerase chain using high-resolution melting analysis among 3501 isolates of 8 different species of Enterobacterales recovered from January 2015 to May 2020. The blaKPC-like was identified in 3003 isolates (85.8%) and the blaNDM-like was the second most common gene (351 isolates-10%). There was a steady increase in frequency of blaNDM-like, from 4.2% in 2015 to 24% in 2020. The increase of blaNDM frequency raises an important matter as novel therapeutic options are currently very limited for the treatment of patients infected by bacteria carrying the blaNDM.

Regulation of macrophage subsets and cytokine production in leishmaniasis.

Macrophages are host cells for parasites of the genus Leishmania where they multiply inside parasitophorous vacuoles. Paradoxically, macrophages are also the cells responsible for killing or controlling parasite growth, if appropriately activated. In this review, we will cover the patterns of macrophage activation and the mechanisms used by the parasite to circumvent being killed. We will highlight the impacts of the vector bite on macrophage activation. Finally, we will discuss the ontogeny of macrophages that are infected by Leishmania spp.

Transarterial Treatment of Cranial Dural Arteriovenous Fistulas: The Role of Transarterial and Transvenous Balloon-Assisted Embolization.

BACKGROUND AND PURPOSE: Treatment of dural arteriovenous fistulas can be performed by transarterial or transvenous accesses. For those fistulas located at a dural sinus wall, obliteration of the sinus might lead to a substantial risk of complications if the occluded sinus impairs normal venous drainage. For those fistulas with direct leptomeningeal venous drainage, navigation to reach the arteriovenous shunting point of a leptomeningeal vein is usually technically demanding. We report the outcomes of patients with dural AVFs treated by transarterial injection of liquid embolic agents assisted by transarterial double-lumen balloon catheters and/or transvenous balloon catheters. MATERIALS AND METHODS: This was a retrospective, 3-center study including patients with dural AVFs treated with a balloon-assisted technique in at least 1 treatment session. Angiographic follow-up was performed at 6 months. Clinical assessment was performed at admission and discharge and was reassessed at 30-day and 6-month follow-ups. RESULTS: Forty-one patients with 43 dural AVFs were treated. Thirty-four fistulas were located at a dural sinus wall. Treatment was performed using only a transarterial approach in 42 fistulas. Only 1 session was needed for complete obliteration of the fistula in 86% of the patients. Immediate complete angiographic occlusion was achieved in 39 fistulas. Of the 41 controlled fistulas, 40 (97.6%) were completely occluded at 6 months. Thirty-nine fistulas (95.1%) were cured without any report of major neurologic events or death during follow-up. CONCLUSIONS: Transarterial balloon-assisted treatment of dural AVFs with or without transvenous balloon protection was shown to be safe and effective.

Polymyxin B broth disk elution: a feasible and accurate methodology to determine polymyxin B susceptibility in Enterobacterales.

Determination of polymyxins susceptibility by clinical laboratories is a nightmare, mainly because of physicochemical properties of the drug. Elution tests have already been proposed for colistin, but not for polymyxin B. We aimed to evaluate accuracy of Polymyxin B broth disk elution (PBDE) to determine the susceptibility to this drug. We evaluated 196 Enterobacterales (45.9% polymyxin B-resistant). PBDE was done in 15-mL cation-adjusted Mueller-Hinton broth where one polymyxin B disk (300 U) was eluted (2 µg/mL). BMD was performed as reference method. Categorical Agreement (CA), Major Error (ME) and Very Major Error (VME) were 99.5%, 0% and 1.11% (one false-negative K. pneumoniae MIC 4 µg/mL), respectively. As some institutions preferably use polymyxin B over colistin and in some countries colistin are not commercially available, to specifically evaluate polymyxin B is important. PBDE proved to be a cheap and easy to perform methodology to evaluate susceptibility to polymyxin B among Enterobacterales.

Novel Cassette Assay To Quantify the Outer Membrane Permeability of Five ß-Lactams Simultaneously in Carbapenem-Resistant Klebsiella pneumoniae and Enterobacter cloacae.

Poor penetration through the outer membrane (OM) of Gram-negative bacteria is a major barrier of antibiotic development. While ß-lactam antibiotics are commonly used against Klebsiella pneumoniae and Enterobacter cloacae, there are limited data on OM permeability especially in K. pneumoniae Here, we developed a novel cassette assay, which can simultaneously quantify the OM permeability to five ß-lactams in carbapenem-resistant K. pneumoniae and E. cloacae Both clinical isolates harbored a blaKPC-2 and several other ß-lactamases. The OM permeability of each antibiotic was studied separately ("discrete assay") and simultaneously ("cassette assay") by determining the degradation of extracellular ß-lactam concentrations via multiplex liquid chromatography-tandem mass spectrometry analyses. Our K. pneumoniae isolate was polymyxin resistant, whereas the E. cloacae was polymyxin susceptible. Imipenem penetrated the OM at least 7-fold faster than meropenem for both isolates. Imipenem penetrated E. cloacae at least 258-fold faster and K. pneumoniae 150-fold faster compared to aztreonam, cefepime, and ceftazidime. For our ß-lactams, OM permeability was substantially higher in the E. cloacae compared to the K. pneumoniae isolate (except for aztreonam). This correlated with a higher OmpC porin production in E. cloacae, as determined by proteomics. The cassette and discrete assays showed comparable results, suggesting limited or no competition during influx through OM porins. This cassette assay allowed us, for the first time, to efficiently quantify the OM permeability of multiple ß-lactams in carbapenem-resistant K. pneumoniae and E. cloacae Characterizing the OM permeability presents a critical contribution to combating the antimicrobial resistance crisis and enables us to rationally optimize the use of ß-lactam antibiotics.IMPORTANCE Antimicrobial resistance is causing a global human health crisis and is affecting all antibiotic classes. While ß-lactams have been commonly used against susceptible isolates of Klebsiella pneumoniae and Enterobacter cloacae, carbapenem-resistant isolates are spreading worldwide and pose substantial clinical challenges. Rapid penetration of ß-lactams leads to high drug concentrations at their periplasmic target sites, allowing ß-lactams to more completely inactivate their target receptors. Despite this, there are limited tangible data on the permeability of ß-lactams through the outer membranes of many Gram-negative pathogens. This study presents a novel, cassette assay, which can simultaneously characterize the permeability of five ß-lactams in multidrug-resistant clinical isolates. We show that carbapenems, and especially imipenem, penetrate the outer membrane of K. pneumoniae and E. cloacae substantially faster than noncarbapenem ß-lactams. The ability to efficiently characterize the outer membrane permeability is critical to optimize the use of ß-lactams and combat carbapenem-resistant isolates.

High-performance method to detection of Klebsiella pneumoniae Carbapenemase in Enterobacterales by LC-MS/MS.

Carbapenem-resistant Enterobacterales (CREs) have been recognized as an important threat to global health. CRE cause the majority of the difficult-to-treat infections in health-care settings and are associated with high mortality. Klebsiella pneumoniae carbapenemase (KPC)-producing CREs, in particular Klebsiella pneumoniae, are globally disseminated and responsible for a large number of outbreaks. Development of rapid methods for KPC detection can provide great clinical and epidemiological benefits to prevent KPC dissemination. The aim of this study was to standardize and validate a LC-MS/MS method to detect KPC. This method was also tested against a broad variety of species, including CRE with other carbapenemase genes and the recently reported mcr-1. For validation, 111 isolates with reduced susceptibility to carbapenems were selected (49 KPC-positive and 62 KPC-negative). The presence of four tryptic peptides related to the KPC enzyme was evaluated, and the identification of at least two of them classified the isolate as "KPC-positive." The LTLGSALAAPQR and LALEGLGVNGQ peptides were both detected in 47 of 49 isolates with the blaKPC gene. The other two peptides, GFLAAAVLAR and APIVLAVYTR, were detected in 46 and 19 isolates with the blaKPC gene, respectively. The method correctly classified 47 of 49 KPC-positive and all KPC-negative isolates yielding 96.07% of sensitivity and 100% of specificity. In conclusion, our results demonstrate that the KPC peptide markers were robustly detected by the method which presented high sensitivity and full specificity and therefore can be used as a reliable method to identify this resistance mechanism.

Apparent selectivity of sheep in deferred marandu palisadegrass pastures with variable initial heights / Seletividade aparente de ovinos em pastos de capim-marandu diferidos com alturas iniciais variáveis

The objective of this study was to evaluate the apparent selectivity of sheep in marandu palisadegrass (Urochloa brizantha cv. Marandu) pastures with four heights at the beginning of the deferment period (15, 25, 35 and 45cm). The deferment period was 92 days and started on 03/21/2014. Evaluations occurred in the beginning (first week), middle (45th day) and end (92nd day) of the grazing period, in winter (06/21/2014 to 09/21/2014). Deferred pastures with 15 and 25cm presented lower forage mass (FM), but higher live leaf (LL) percentage in FM than deferred pastures with 35 and 45cm. The live stem percentage in the FM and the apparent selectivity index (ASI) of the LL were superior in the deferred pasture with 45cm. The dead stem (DS) percentage in the grazing simulation (GS) and the ASI of this morphological component were lower in the pasture with 15cm, compared to the deferred pasture with 45cm. The FM and the LL percentages in FM and in the GS sample decreased, while the DS percentages in FM and in GS sample increased with the grazing period. Marandu palisadegrass with 15cm at beginning of the deferment period improves the morphology of the deferred pasture. Selective grazing is difficult during the grazing period.(AU)

Objetivou-se avaliar a seletividade aparente de ovinos em pastos de capim-marandu (Urochloa brizantha cv. Marandu) com quatro alturas no início do diferimento (15, 25, 35 e 45cm). O período de diferimento foi de 92 dias e iniciou em 21/03/2014. As avaliações ocorreram no início (primeira semana), meio (45° dia) e fim (92° dia) do período de pastejo, no inverno (21/06/2014 a 21/09/2014). Os pastos diferidos com 15 e 25cm apresentaram menor massa de forragem (MF), mas maior percentual de folha viva (FV) na MF do que os pastos diferidos com 35 e 45cm. O percentual de colmo vivo na MF e o índice de seletividade aparente (ISA) da FV foram superiores no pasto diferido com 45cm. O percentual de colmo morto (CM) na simulação de pastejo (SP) e o ISA desse componente morfológico foram menores no pasto diferido com 15cm, em comparação ao diferido com 45cm. A MF e os percentuais de FV na MF e na amostra de SP se reduziram, enquanto os percentuais de CM na MF e na amostra de SP aumentaram com o período de pastejo. O capim-marandu com 15cm no início do período de diferimento melhora a morfologia do pasto diferido. O pastejo seletivo é dificultado no decorrer do período de pastejo.(AU)

Low level of polymyxin resistance among nonclonal mcr-1-positive Escherichia coli from human sources in Brazil.

We report 26 human isolates of mcr-1-positive Escherichia coli, most of them (65.4%) with a polymyxin B MIC of 2 mg/L. Seventeen out of the 24 mcr-1-positive E. coli proved to be nonclonal by rep-PCR which strengthens the hypothesis of environmental or animal origin of these strains and reinforces the one health context of antimicrobial resistance.

Dissemination of blaOXA-370 is mediated by IncX plasmids and the Tn6435 transposon.

In Enterobacteriaceae, the blaOXA-48-like genes have been identified on plasmids in different regions of the world. The OXA-370 is a plasmid-encoded OXA-48-like enzyme reported in two distinct regions of Brazil. Recently, we demonstrate that the blaOXA-370 gene is disseminated among several Enterobacteriaceae species and clones, indicating a high potential for dissemination. In this work, we described for the first time the complete nucleotide sequence of six plasmids harboring the blaOXA-370 gene. Complete DNA sequencing using the Illumina platform and annotation of the plasmids showed that they belonged to incompatibility groups IncX and had in average 70 kbp. The blaOXA-370 gene is located in a composite transposon containing four genes encoding transposases, named Tn6435. In this study, highly similar plasmids were detected in different Enterobacteriaceae genera.

KPC-producing Klebsiella pneumoniae bloodstream isolates from Brazilian hospitals: What (still) remains active?

OBJECTIVES: This study assessed susceptibility to polymyxin B (PMB) and alternative antimicrobials, with focus on aminoglycosides and tigecycline, according to different breakpoints in KPC-producing Klebsiella pneumoniae (KPC-Kp) bloodstream isolates from Brazilian hospitals. METHODS: Bloodstream K. pneumoniae isolates non-susceptible to any of the three carbapenems (meropenem, imipenem or ertapenem) from four Brazilian tertiary-care hospitals were selected. Antimicrobial susceptibility was determined and interpreted according to distinct breakpoints. Twenty-nine PMB-resistant KPC-Kp isolates were selected for molecular typing. RESULTS: A total of 158 KPC-Kp were analysed. MIC50/90 values for PMB were 0.25/16mg/L; 40 isolates (25.3%) were resistant to PMB. MIC50/90 values for meropenem were 32/≥256mg/L; no isolates were susceptible to meropenem according to CLSI, but 10 isolates were intermediate using EUCAST breakpoints (1, MIC=4mg/L; 9, MIC=8mg/L). MIC50/90 values for tigecycline were 2/8mg/L; 53 (33.5%) and 94 (59.5%) isolates were susceptible according to EUCAST and FDA breakpoints, respectively. MIC50/90 values were 32/≥64mg/L for amikacin and ≥16/≥16mg/L for gentamicin; 48 (30.4%), 28 (17.7%) and 16 (10.1%) were susceptible to amikacin according to CLSI, EUCAST and USCAST, respectively, but susceptibility rates to gentamicin were <7.0%. Eighteen distinct clonal profiles were identified among 29 PMB-resistant isolates by DNA macrorestriction. Most clones belonged to CC11. CONCLUSION: Elevated rates of PMB-resistant KPC-Kp bloodstream infections were found in four Brazilian hospitals, mostly of polyclonal origin. Alternative antimicrobials with the highest in vitro activity were tigecycline and amikacin, although susceptibility rates significantly decreased using criteria with stricter breakpoints (e.g. EUCAST, USCAST).

Molecular investigation of isolates from a multistate polymicrobial outbreak associated with contaminated total parenteral nutrition in Brazil.

BACKGROUND: Between November 2013 and June 2014, 56 cases of bacteremia (15 deaths) associated with the use of Total Parenteral Nutrition (TPN) and/or calcium gluconate (CG) were reported in four Brazilian states. METHODS: We analyzed 73 bacterial isolates from four states: 45 from blood, 25 from TPN and three from CG, originally identified as Acinetobacter baumannii, Rhizobium radiobacter, Pantoea sp. or Enterobacteriaceae using molecular methods. RESULTS: The first two bacterial species were confirmed while the third group of species could not be identified using standard identification protocols. These isolates were subsequently identified by Multi-Locus Sequence Analysis as Phytobacter diazotrophicus, a species related to strains from similar outbreaks in the United States in the 1970's. Within each species, TPN and blood isolates proved to be clonal, whereas the R. radiobacter isolates retrieved from CG were found to be unrelated. CONCLUSION: This is the first report of a three-species outbreak caused by TPN contaminated with A. baumannii, R. radiobacter and P. diazotrophicus. The concomitant presence of clonal A. baumannii and P. diazotrophicus isolates in several TPN and blood samples, as well as the case of one patient, where all three different species were isolated simultaneously, suggest that the outbreak may be ascribed to a discrete contamination of TPN. In addition, this study highlights the clinical relevance of P. diazotrophicus, which has been involved in outbreaks in the past, but was often misidentified as P. agglomerans.

In vitro antimicrobial activity of imipenem plus amikacin or polymyxin B against carbapenem-resistant Pseudomonas aeruginosa isolates.

The aim of this study was to evaluate the antimicrobial activity of imipenem plus amikacin or polymyxin B against six carbapenem-resistant P. aeruginosa by time-kill assay. Synergism was observed in two isolates for combinations with amikacin. For combinations with polymyxin B, synergism and antagonism also occurred in two isolates.