Study of an Enterococcus faecium strain isolated from an artisanal Mexican cheese, whole-genome sequencing, comparative genomics, and bacteriocin expression.

Enterococci are ubiquitous microorganisms in almost all environments, from the soil we step on to the food we eat. They are frequently found in naturally fermented foods, contributing to ripening through protein, lipid, and sugar metabolism. On the other hand, these organisms are also leading the current antibiotic resistance crisis. In this study, we performed whole-genome sequencing and comparative genomics of an Enterococcus faecium strain isolated from an artisanal Mexican Cotija cheese, namely QD-2. We found clear genomic differences between commensal and pathogenic strains, particularly in their carbohydrate metabolic pathways, resistance to vancomycin and other antibiotics, bacteriocin production, and bacteriophage and CRISPR content. Furthermore, a bacteriocin transcription analysis performed by RT-qPCR revealed that, at the end of the log phase, besides enterocins A and X, two putative bacteriocins not reported previously are also transcribed as a bicistronic operon in E. faecium QD-2, and are expressed 1.5 times higher than enterocin A when cultured in MRS broth.

Supplementation with a Symbiotic Induced Neuroprotection and Improved Memory in Rats with Ischemic Stroke.

After an ischemic stroke, various harmful mechanisms contribute to tissue damage, including the inflammatory response. The increase in pro-inflammatory cytokines has been related to greater damage to the neural tissue and the promotion of neurological alterations, including cognitive impairment. Recent research has shown that the use of prebiotics and/or probiotics counteracts inflammation and improves cognitive function through the production of growth factors, such as brain-derived neurotrophic factor (BDNF), by reducing inflammatory molecules. Therefore, in this study, the effect of the symbiotic inulin and Enterococcus faecium on neuroprotection and memory improvement was evaluated in a rat model of transient middle cerebral artery occlusion (tMCAO). In order to accomplish this, the animals were subjected to ischemia; the experimental group was supplemented with the symbiotic and the control group with the vehicle. The neurological deficit as well as spatial and working memory were evaluated using the Zea Longa scale, Morris water maze, and the eight-arm maze tests, respectively. Infarct size, the levels of BDNF, and tumor necrosis factor-alpha (TNF-α) were also assessed. The results show that supplementation with the symbiotic significantly diminished the neurological deficit and infarct size, improved memory and learning, increased BDNF expression, and reduced TNF-α production. These findings provide new evidence about the therapeutic use of symbiotics for ischemic stroke and open up the possibilities for the design of further studies.

Outbreak of vancomycin-resistant Enterococcus faecium ST1133 in paediatric patients with acute lymphoblastic leukaemia from southern Brazil.

OBJECTIVES: We aimed to investigate an outbreak of vancomycin-resistant Enterococcus faecium (VREfm) in paediatric patients from Hospital Pequeno Príncipe. The susceptibility profile was determined, and whole-genome sequencing (WGS) was used to analyse the genetic context of the strains. METHODS: Five VREfm isolates were recovered from sterile sites and surveillance cultures of two paediatric patients with acute lymphoblastic leukaemia. Species identification was performed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and the minimum inhibitory concentration (MIC) was assessed according to the European Committee for Antimicrobial Susceptibility Testing (EUCAST). WGS was performed to analyse the genetic context of virulence and resistance genes, and in silico multilocus sequence typing was performed to identify the sequence typing of the strains. RESULTS: High-level vancomycin resistance was observed in all isolates (≥256 mg/L). WGS revealed the presence of mobile genetic elements, such as plasmids (rep2, rep11a, repUS15, rep17, and rep18a), insertion sequences, and phages. Multiple resistance genes (aac(6')-aph(2"), dfrG, ermB, and vanA) and virulence genes (acm and efaAfm) were identified. All the isolates were assigned to ST117 (ST1133 - via a novel MLST), an important epidemic lineage associated with nosocomial infections and outbreaks. CONCLUSION: Our results show that the ST117 (ST1133) VREfm isolates are circulating in paediatric patients, which raises a great concern. The development of new drugs as well as the implementation of an antimicrobial stewardship program are necessary for their correct management, limiting the spread of resistance in oncohematological patients.

Enterococcus faecium M7AN10 probiótico em matriz alimentar para cães / Enterococcus faecium M7AN10 probiotic in food matrix for dogs / Enterococcus faecium M7AN10 probiótico en matriz alimentaria para perros

Probióticos são capazes de melhorar o equilíbrio da microbiota intestinal, trazendo benefícios ao hospedeiro. Atualmente no mercado há poucas opções de alimentos, com probióticos em sua composição, destinados a cães e gatos. Portanto, o objetivo deste trabalho foi desenvolver uma matriz alimentar canina (ração úmida) com o probiótico Enterococcus faecium M7AN10. Para tal, avaliou-se a inocuidade, atividade enzimática, atividade antimicrobiana, potencial probiótico e a viabilidade do microrganismo em matriz alimentar canina. O isolado foi considerado inócuo, pois apresentou ausência de atividade hemolítica e de gelatinase, além de ser suscetível a diversos antimicrobianos. E. faecium M7AN10 apresentou atividade proteolítica e capacidade de produção de exoplissacarídeo. Em relação a atividade antimicrobiana pelo método da estria radial, o isolado inibiu Acinetobacter sp. 1, Corynebacterium sp. 4, Micrococcus luteus 33, Micrococcus luteus 43, Micrococcus sp. 3, Micrococcus sp. 20, Micrococcus sp. 36. Além disso, E. faecium M7AN10 apresentou capacidade de autoagregação de 33,50% e resistiu de forma constante quando submetido ao trato gastrointestinal in vitro em conjunto com Lacticaseibacillus rhamnosus LB 1.5 e Lacticaseibacillus paracasei LB 6.4. O cultivo misto manteve-se viável em matriz alimentar canina durante o período de oito dias. Com base nesses resultados, o isolado E. faecium M7AN10 foi considerada uma bactéria candidata a probiótico que pode vir a ser usada como aditivo em alimento para cães.

Probiotics are capable of improving the balance of the intestinal microbiota, bringing benefits to the host. Currently, on the market, there are few food options with probiotics in their composition intended for dogs and cats. Therefore, this research aimed to develop a canine food matrix (wet food) with the probiotic Enterococcus faecium M7AN10. To this end, the harmlessness, enzymatic activity, antimicrobial activity, probiotic potential, and viability of the microorganism in the canine food matrix were evaluated. The isolate was considered harmless, as it showed no hemolytic and gelatinase activity and was susceptible to several antimicrobials. E. faecium M7AN10 showed proteolytic activity and exopolysaccharide production capacity. Regarding antimicrobial activity using the radial stria method, the isolate inhibited Acinetobacter sp. 1, Corynebacterium sp. 4, Micrococcus luteus 33, Micrococcus luteus 43, Micrococcus sp. 3, Micrococcus sp. 20, Micrococcus sp. 36. Furthermore, E. faecium M7AN10 showed a self-aggregation capacity of 33.50% and resisted consistently when subjected to the gastrointestinal tract in vitro together with Lacticaseibacillus rhamnosus LB 1.5 and Lacticaseibacillus paracasei LB 6.4. The mixed culture remained viable in a canine food matrix over eight days. Based on these results, the isolate E. faecium M7AN10 was considered a candidate bacterium for a probiotic that could be used as an additive in dog food.

Los probióticos son capaces de mejorar el equilibrio de la microbiota intestinal, aportando beneficios al huésped. Actualmente en el mercado existen pocas opciones de alimentos con probióticos en su composición, destinados a perros y gatos. Por lo tanto, el objetivo de este trabajo fue desarrollar una matriz alimentaria canina (comida húmeda) con el probiótico Enterococcus faecium M7AN10. Para ello se evaluó la inocuidad, actividad enzimática, actividad antimicrobiana, potencial probiótico y viabilidad del microorganismo en matriz alimentaria canina. El aislado fue considerado inofensivo, ya que no mostró actividad hemolítica ni gelatinasa, además de ser susceptible a varios antimicrobianos. E. faecium M7AN10 mostró actividad proteolítica y capacidad de producción de exoplisacáridos. En cuanto a la actividad antimicrobiana mediante el método de las estrías radiales, el aislado inhibió a Acinetobacter sp. 1, Corynebacterium sp. 4, Micrococcus luteus 33, Micrococcus luteus 43, Micrococcus sp. 3, Micrococcus sp. 20, Micrococcus sp. 36. Además, E. faecium M7AN10 mostró una capacidad de autoagregación del 33,50% y resistió consistentemente cuando se sometió al tracto gastrointestinal in vitro junto con Lacticaseibacillus rhamnosus LB 1.5 y Lacticaseibacillus paracasei LB 6.4. El cultivo mixto permaneció viable en una matriz de alimento canino durante un período de ocho días. Con base en estos resultados, el aislado E. faecium M7AN10 se consideró una bacteria candidata para un probiótico que podría usarse como aditivo en la comida para perros.

Enterococcus isolated from poultry intestine for potential probiotic use.

Background and Aim: To develop species-specific probiotics for poultry, it is ideal to obtain these probiotic microorganisms directly from the intestinal tract of broiler and egg-laying chicks in production environments to ensure adaptation to actual conditions. This study aimed to isolate lactic acid bacteria (LAB) from the intestinal tract of broiler and egg-laying chicks to determine their probiotic potential. Materials and Methods: Twenty-five Ross-308 broilers and 25 Isa Brown egg-laying chicks were raised until days 42 and 120, respectively; they were housed in an individual poultry building. Lactic acid bacteria were isolated and identified from the small intestine mucus of broiler and layer chicks and then evaluated based on resistance to acidic pH levels, bile salt concentration, and antagonistic activity against wild strains of Escherichia coli and Salmonella spp. selected strains with probiotic potential were identified by polymerase chain reaction and confirmed by rDNA sequencing. Results: One hundred and fifty Gram-positive isolates were obtained; 28% (42) were catalase and oxidase negative and biochemical identification was made by crystal system: 76.2% (32) Enterococcus spp., 16.6% (7) Lactococcus spp., and 7.2% (3) Streptococcus spp.; and evaluated for hemolysin production; tolerance to low pH and bile salts, and antagonistic potential were carried out. Molecular characterization yielded 56% (24) Enterococcus faecium, and 44% (18) Enterococcus faecalis. About 81% (34) of strains were without vancomycin resistance genes criterion. Conclusion: This study isolated and characterized 36 strains of LAB with probiotic qualities, from the intestines of broiler and egg-laying chicks, selecting E. faecium, Enterococcus avium, and Enterococcus casseliflavus, Lactococcus garviae as promising strains for further in vitro and in vivo research.

Design, Synthesis, and Structure-Activity Relationship Studies of New Quinone Derivatives as Antibacterial Agents.

Resistance to antibacterial agents is a growing global public health problem that reduces the efficacy of available antibacterial agents, leading to increased patient mortality and morbidity. Unfortunately, only 16 antibacterial drugs have been approved by the FDA in the last 10 years, so it is necessary to develop new agents with novel chemical structures and/or mechanisms of action. In response to this, our group takes up the challenge of designing a new family of pyrimidoisoquinolinquinones displaying antimicrobial activities against multidrug-resistant Gram-positive bacteria. Accordingly, the objective of this study was to establish the necessary structural requirements to obtain compounds with high antibacterial activity, along with the parameters controlling antibacterial activity. To achieve this goal, we designed a family of compounds using different strategies for drug design. Forty structural candidates were synthesized and characterized, and antibacterial assays were carried out against high-priority bacterial pathogens. A variety of structural properties were modified, such as hydrophobicity and chain length of functional groups attached to specific carbon positions of the quinone core. All the synthesized compounds inhibited Gram-positive pathogens in concentrations ranging from 0.5 to 64 µg/mL. Two derivatives exhibited minimum inhibitory concentrations of 64 µg/mL against Klebsiella pneumoniae, while compound 28 demonstrated higher potency against MRSA than vancomycin.

Study of Essential Oil Isolated from Achiote (Bixa orellana) Leaves: Chemical Composition, Enantiomeric Distribution and Antimicrobial, Antioxidant and Anticholinesterase Activities.

Bixa orellana is a native and cultivated species of Ecuador commonly known as achiote (annatto), this species is very versatile with a wide variety of uses and applications of its leaves, fruits and seeds. In this study, the chemical composition, enantiomeric distribution and biological activity of essential oil isolated from the leaves of Bixa orellana were determined. Hydrodistillation was used to isolate the essential oil. Gas chromatography coupled with mass spectrometry was used to determine the qualitative composition, a gas chromatograph equipped with a flame ionization detector was used to determine quantitative composition and gas chromatography on an enantioselective column was used to determine enantiomeric distribution. Antibacterial activity was determined using the broth microdilution method, for which we used three Gram-positive cocci bacteria, a Gram-positive bacilli bacterium and three Gram-negative bacilli bacteria. 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic (ABTS) acid radical cation and 2,2-diphenyl-1-picrylhydryl (DPPH) free radical were used as reagents for determining the antioxidant activity of the essential oil. The spectrophotometric method was used to analyze acetylcholinesterase inhibitory effect of the essential oil. The yield of leaves in essential oil was 0.13 ± 0.01% (v/w). A total of 56 chemical compounds, which represent 99.25% of the total composition, were identified in the essential oil. Sesquiterpene hydrocarbons were the most representative group in number of compounds and relative abundance with 31 compounds and 69.06%, respectively. The principal constituents were found to germacrene D (17.87 ± 1.20%), bicyclogermacrene (14.27 ± 0.97%), caryophyllene < (E)- > (8.56 ± 1.24%) and pinene <α-> (6.34 ± 0.13%). Six pairs of enantiomers were identified in the essential oil of Bixa orellana. The essential oil presented strong activity against the Enterococcus faecium (ATCC 27270) with an MIC of 250 µg/mL and weak activity against Enterococcus faecalis (ATCC 19433) and Staphylococcus aureus (ATCC 25923) with an MIC of 1000 µg/mL. The antioxidant activity of the essential oil was strong according to ABTS methods with a SC50 of 61.49 ± 0.04 µg/mL and was moderate in DPPH with a SC50 of 224.24 ± 6,4 µg/mL. Additionally, the essential oil reported moderate anticholinesterase activity with an IC50 of 39.45 ± 1.06 µg/mL.

Complete genome sequence and characterization of a novel Enterococcus faecium with probiotic potential isolated from the gut of Litopenaeus vannamei.

Litopenaeus vannamei, the Pacific whiteleg shrimp, is one of the most marketable species in aquaculture worldwide. However, it is susceptible to different infections causing considerable losses in production each year. Consequently, using prebiotics that promotes the proliferation of beneficial bacteria and strengthen the immune system is a current strategy for disease control. In this study, we isolated two strains of E. faecium from the gut of L. vannamei fed with agavin-supplemented diets. These isolates showed antibacterial activity against Vibrio parahaemolyticus, Vibrio harveyi and Vibrio alginolyticus, most likely due to peptidoglycan hydrolase (PGH) activity. Furthermore, we sequenced the genome of one isolate. As a result, we observed three proteins related to the production of bacteriocins, a relevant trait for selecting probiotic strains since they can inhibit the invasion of potential pathogens. Additionally, the genome annotation showed genes related to the production of essential nutrients for the host. It lacked two of the most common factors associated with virulence in Enterococcus pathogenic strains (esp and hyl). Thus, this host-probiotic-derived strain has potential application not only in shrimp health but also in alternative aquatic environments, as it is adapted to coexist within the gut shrimp microbiota, independently of the diet.

Bacteriemia por Enterococcus faeciumresistente a daptomicina / Daptomycin-resistant enterococcus faecium bacteremia

La aparición de cepas de enterococos resistentes a daptomicina es un tema de preocupación clínica y epidemiológica en años recientes. Se presenta el caso de un paciente de 50 años con antecedente de artritis reumatoide e inmunosupresión crónica hospitalizado en contexto de neumonía viral por COVID-19, con sobreinfección bacteriana y choque séptico, en quien se documentó en tres oportunidades diferentes aislamientos de Enterococcus faecium vancomicino-resistente VAN A y B con falla terapéutica a daptomicina, por deterioro clínico y persistencia de hemocultivos positivos. Se inició manejo con linezolid con control de la infección, negativización de hemocultivos y evolución clínica satisfactoria. Se realiza reporte del presente caso para dar a conocer la aparición de enterococos resistentes a daptomicina, la cual es una creciente preocupación epidemiológica, con el fin de realizar identificación temprana, prevenir falla terapéutica y poder conocer la epidemiología local

n recent years, the emergence of daptomycin-resistant enterococcus strains is a growing clinical and epidemio-logical topic of concern. We report the case of a 50 year old patient with an antecedent of rheumatoid arthritis and chronic immunosuppression hospitalized in the con-text of COVID-19 pneumonia with bacterial co-infection and septic shock in which a three different moments an isolate of a "vancomycin-resistant enterococcus faecium"(VRE) Van A and B that presented therapeutic failure with daptomycin was documented after clinical deterioration and persistence of positive blood cultures. Linezolid was initiated, with clinical recovery and negativization of blood cultures following the change in antibiotic treatment. This case is reported in order to expose the ever growing con-cern of daptomycin-resistant enterococcus strains in or-der to prevent therapeutic failure, make early identifica-tion and get to know the local epidemiological status.

Effects of feeding different probiotic types on metabolic, performance, and carcass responses of Bos indicus feedlot cattle offered a high-concentrate diet.

Two experiments were designed to evaluate the effects of different probiotic combinations on rumen fermentation characteristics, performance, and carcass characteristics of feedlot Bos indicus beef bulls offered a high-concentrate diet. In experiment 1, 30 rumen-fistulated Nellore steers were blocked by initial body weight (BW = 350 ± 35.0 kg) and within blocks (n = 10), animals were randomly assigned to receive: 1) high-concentrate diet without probiotic supplementation (n = 10; CONT), 2) CONT plus 1 g per head of a probiotic mixture containing three strains of Enterococcus faecium and one strain of Saccharomyces cerevisiae (3.5 × 109 CFU/g; n = 10; EFSC), and 3) CONT plus 2 g per head of a probiotic mixture containing Bacillus licheniformis and Bacillus subtilis (3.2 × 109 CFU/g; n = 10; BLBS). The experimental period lasted 35 d, being 28 d of adaptation and 7 d of sampling. From day 34 to day 35 of the experimental period, ruminal fluid and fecal samples were collected every 3 h, starting immediately before feeding (0 h) for rumen fermentation characteristics and apparent nutrient digestibility analysis, respectively. In experiment 2, 240 Nellore bulls were ranked by initial shrunk BW (375 ± 35.1 kg), assigned to pens (n = 4 bulls per pen), and pens randomly assigned to receive the same treatments as in experiment 1 (n = 20 pens per treatment). Regardless of treatment, all bulls received the same step-up and finishing diets throughout the experimental period, which lasted 115 d. In both experiments, data were analyzed as orthogonal contrasts to partition-specific treatment effects: 1) probiotic effect: CONT vs. PROB and 2) probiotic type: EFSC vs. BLBS (SAS Software Inc.). In experiment 1, no contrast effects were observed on nutrient intake, overall nutrient digestibility, and rumen fermentation analyses (P ≥ 0.13). Nonetheless, supplementation of probiotics, regardless of type (P = 0.59), reduced mean acetate:propionate ratio and rumen ammonia-N concentration vs. CONT (P ≤ 0.05). In experiment 2, no significant effects were observed for final BW and dry matter intake (P ≥ 0.12), but average daily gain and feed efficiency tended to improve (P ≤ 0.10) when probiotics were offered to the animals. Probiotic supplementation or type of probiotic did not affect carcass traits (P ≥ 0.22). In summary, supplementation of probiotics containing a mixture of E. faecium and S. cerevisiae or a mixture of B. licheniformis and B. subtilis reduced rumen acetate:propionate ratio and rumen ammonia-N levels and tended to improve the performance of feedlot cattle offered a high-concentrate diet.

Two experiments were designed to evaluate the effects of different probiotic combinations on rumen fermentation characteristics, performance, and carcass characteristics of feedlot Bos indicus beef bulls offered a high-concentrate diet. The two probiotics consisted of a mixture containing three strains of Enterococcus faecium and one strain of Saccharomyces cerevisiae or a mixture of Bacillus licheniformis and Bacillus subtilis. Supplementation of probiotics, regardless of type, reduced acetate:propionate ratio, and mean rumen ammonia-N concentration and tended to improve the performance of feedlot cattle offered a high-concentrate diet, demonstrating the potential of this technology to be used as a feed additive for beef cattle.

Bacteriocin-like inhibitory substances production by Enterococcus faecium 135 in co-culture with Ligilactobacillus salivarius and Limosilactobacillus reuteri.

The use of lactic acid bacteria (LAB) and probiotic cultures in the breeding of animals such as poultry and swine are quite common. It is known that those strains can produce bacteriocins when grown in pure culture. However, the production of bacteriocin using co-culture of microorganisms has not been much studied so far. The present study contributes with innovation in this area by embracing the production of bacteriocin-like inhibitory substances (BLIS) by a newly isolated strain of Enterococcus faecium 135. Additionally, the co-cultivation of this strain with Ligilactobacillus salivarius and Limosilactobacillus reuteri was also investigated. The antimicrobial activity of the produced BLIS was evaluated against Listeria monocytogenes, Listeria innocua, Salmonella enterica, and Salmonella enterica serovar Typhimurium using two methods: turbidimetric and agar diffusion. In addition, the presence of enterocin genes was also evaluated. The BLIS produced showed a bacteriostatic effect against the bio-indicator strains, and the highest antimicrobial activities expressed by arbitrary units per mL (AU/mL) were obtained against L. monocytogenes in monoculture (12,800 AU/mL), followed by the co-culture of E. faecium with Limosilactobacillus reuteri (400 AU/mL). After concentration with ammonium sulfate, the antimicrobial activity raised to 25,600 AU/mL. Assays to determine the proteinaceous nature of the BLIS showed susceptibility to trypsin and antimicrobial activity until 90 °C. Finally, analysis of the presence of structural genes of enterocins revealed that four enterocin genes were present in E. faecium 135. These results suggest that BLIS produced by E. faecium 135 has potential to be a bacteriocin and, after purification, could potentially be used as an antimicrobial agent in animal breeding.

vHULK, a New Tool for Bacteriophage Host Prediction Based on Annotated Genomic Features and Neural Networks.

Background: The experimental determination of a bacteriophage host is a laborious procedure. Thus, there is a pressing need for reliable computational predictions of bacteriophage hosts. Materials and Methods: We developed the program vHULK for phage host prediction based on 9504 phage genome features, which consider alignment significance scores between predicted proteins and a curated database of viral protein families. The features were fed to a neural network, and two models were trained to predict 77 host genera and 118 host species. Results: In controlled random test sets with 90% redundancy reduction in terms of protein similarity, vHULK obtained on average 83% precision and 79% recall at the genus level, and 71% precision and 67% recall at the species level. The performance of vHULK was compared against three other tools on a test data set with 2153 phage genomes. On this data set, vHULK achieved better performance at both the genus and the species levels than the other tools. Conclusions: Our results suggest that vHULK represents an advance on the state of art in phage host prediction.

Increment Antimicrobial Resistance During the COVID-19 Pandemic: Results from the Invifar Network.

Aim: This study aims to assess the changes in antimicrobial resistance among some critical and high-priority microorganisms collected previously and during the coronavirus disease 2019 (COVID-19) pandemic in Mexico. Methods: We collected antimicrobial susceptibility data for critical and high-priority microorganisms from blood, urine, respiratory samples, and from all specimens, in which the pathogen may be considered a causative agent. Data were stratified and compared for two periods: 2019 versus 2020 and second semester 2019 (prepandemic) versus the second semester 2020 (pandemic). Results: In the analysis of second semester 2019 versus the second semester 2020, in blood samples, increased resistance to oxacillin (15.2% vs. 36.9%), erythromycin (25.7% vs. 42.8%), and clindamycin (24.8% vs. 43.3%) (p ≤ 0.01) was detected for Staphylococcus aureus, to imipenem (13% vs. 23.4%) and meropenem (11.2% vs. 21.4) (p ≤ 0.01), for Klebsiella pneumoniae. In all specimens, increased ampicillin and tetracycline resistance was detected for Enterococcus faecium (p ≤ 0.01). In cefepime, meropenem, levofloxacin, and gentamicin (p ≤ 0.01), resistance was detected for Escherichia coli; and in piperacillin-tazobactam, cefepime, imipenem, meropenem, ciprofloxacin, levofloxacin, and gentamicin (p ≤ 0.01), resistance was detected for Pseudomonas aeruginosa. Conclusion: Antimicrobial resistance increased in Mexico during the COVID-19 pandemic. The increase in oxacillin resistance for S. aureus and carbapenem resistance for K. pneumoniae recovered from blood specimens deserves special attention. In addition, an increase in erythromycin resistance in S. aureus was detected, which may be associated with high azithromycin use. In general, for Acinetobacter baumannii and P. aeruginosa, increasing resistance rates were detected.

Genetic description of VanD phenotype vanA genotype in vancomycin-resistant Enterococcus faecium isolates from a Bone Marrow Transplantation Unit.

BACKGROUND: Vancomycin-resistant Enterococcus faecium (VREfm) is an important agent of hospital-acquired infection. VanA phenotype is characterized by resistance to high levels of vancomycin and teicoplanin and is encoded by the vanA gene, whereas VanD phenotype is characterized by resistance to vancomycin and susceptibility or intermediate resistance to teicoplanin; however, some isolates carry a VanD phenotype with a vanA genotype, but there are many gaps in the knowledge about the genetic mechanisms behind this pattern. OBJECTIVE: To characterize the genetic structure, clonality, and mobile genetic elements of VRE isolates that display a VanD-vanA phenotype. RESULTS: All vanA VRE-fm isolates displayed minimum inhibitory concentration (MIC) for vancomycin > 32µg/mL and intermediate or susceptible MIC range for teicoplanin (8-16µg/mL). The isolates were not clonal, and whole-genome sequencing analysis showed that they belonged to five different STs (ST478, ST412, ST792, ST896, and ST1393). The absence of some van complex genes were observed in three isolates: Ef5 lacked vanY and vanZ, Ef2 lacked vanY, and Ef9 lacked orf1 and orf2; moreover, another three isolates had inverted positions of orf1, orf2, vanR, and vanS genes. IS1542 was observed in all isolates, whereas IS1216 in only five. Moreover, presence of other hypothetical protein-encoding genes located downstream the vanZ gene were observed in six isolates. CONCLUSION: VRE isolates can display some phenotypes associated to vanA genotype, including VanA and VanB, as well as VanD; however, further studies are needed to understand the exact role of genetic variability, rearrangement of the transposon Tn1546, and presence of insertion elements in isolates with this profile.

Staphylococcus aureus and Enterococcus faecium isolated from pigeon droppings (Columba livia) in the external environment close to hospitals

ABSTRACT Background: Domestic pigeons carry pathogens in their droppings, posing a potential public health problem. Methods: The phenotypic and genotypic antimicrobial resistances of Staphylococcus aureus and Enterococcus faecium in the feces of urban pigeons near hospitals with intensive care units were measured. Results: Twenty-nine samples showed Enterococcus growth, whereas one was positive for S. aureus. The S. aureus isolate was sensitive to the antibiotics tested via antibiogram, however resistance genes were identified. E. faecium isolates showed phenotypic resistance to gentamicin, erythromycin, and ciprofloxacin. Conclusions: Antimicrobial profiles harmful to health were demonstrated in bacterial pathogens isolated from the external environment of hospitals.

Efeitos antimicrobianos do óleo essencial de Lavandula dentata sobre cepas clínicas de Enterococcus faecalis e Enterococcus faecium / Antimicrobial effects of essential oil of Lavandula dentata on clinical strains of Enterococcus faecalis and Enterococcus faecium

O principal foco do tratamento endodôntico de sucesso, é a eliminação total de microrganismo, endotoxinas e biofilme bacteriano do sistema de canais radiculares. Quando há falha nesse processo, as bactérias de maior prevalência são Enterococcus spp. Desta forma, é evidente a necessidade de novas alternativas eficazes para erradicá-los dos canais radiculares. O uso de fitoterápicos antimicrobianos como os óleos essenciais (OEs) de plantas ganhou grande notoriedade na comunidade cientifica como uma alternativa muito satisfatória, além de efeitos antimicrobianos sobre diversas cepas bacterianas e fúngicas apresentam efeitos anti-inflamatório. Assim, o objetivo deste trabalho foi avaliar in vitro os efeitos antimicrobianos do OE de Lavandula dentata sobre cepas clínicas e cepas padrão de Enterococcus faecalis e Enterococcus em culturas planctônicas e em biofilme monotípico. Para isso foi determinada a Concentração Bactericida Mínima (CBM) do óleo essencial de L. dentata pela técnica de microdiluição em placa de 96 poços em caldo BHI com semeadura, segundo Clinical and Laboratory Standards Institute (CLSI), norma M7- A6 (E. faecalis e E. faecium). Para a análise da atividade antibiofilme dos microrganismos foram utilizados o teste colorimétrico de MTT nos períodos de 30 minutos e 24 horas de contato com o OE. Os dados obtidos nos testes in vitro apresentaram distribuição normal e foram analisados pelo teste ANOVA complementado pelo teste de Tukey (significância de 5%). Os resultados obtidos mostraram que o OE de L. dentata apresentou atividade antibacteriana e antibiofilme para todas as cepas testadas, com a CBM do óleo de L. dentata de 32%. Em relação a atividade antibiofilme do OE de L. dentata houve redução da carga bacteriana no período de 30 minutos e de 24 horas, este promoveu redução de até 79,25% no tratamento de 30 minutos e até 74,51% no tratamento de 24 horas. Concluiu-se que o óleo essencial de L. dentata apresentou atividade antibacteriana contra cepas clínicas resistente a antibióticos e cepas padrão de E. faecalis e E. faecium com concentração bactericida mínima de 32%, e atividade antibiofilme com tempo de ação de 30 minutos e 24 horas, com resultados semelhantes a clorexidina na maioria das cepas testadas (AU)

The main focus of successful endodontic treatment is the total elimination of microorganisms, endotoxins and bacterial biofilm from the root canal system. When this process fails, the most prevalent bacteria are Enterococcus spp. Thus, the need for new effective alternatives to eradicate them from root canals is evident. The use of antimicrobial phytotherapics such as essential oils (EOs) from plants has gained great notoriety in the scientific community as a very satisfactory alternative, in addition to antimicrobial effects on several bacterial and fungal strains that have anti-inflammatory effects. Thus, the objective of this work was to evaluate in vitro the antimicrobial effects of Lavandula dentata EO on clinical and standard strains of Enterococcus faecalis and Enterococcus faecium in planktonic cultures and in monotypic biofilm. For this, the Minimum Bactericidal Concentration (MBC) of the essential oil of L. dentata was determined by the microdilution technique in a 96-well plate in BHI broth with seeding, according to the Clinical and Laboratory Standards Institute (CLSI), M7-A6 (E. faecalis and E. faecium). For the analysis of the antibiofilm activity of the microorganisms, the MTT colorimetric test was used in the periods of 30 minutes and 24 hours of contact with the EO. The data obtained in the in vitro tests showed normal distribution and were analyzed by the ANOVA test complemented by the Tukey test (5% significance). The results obtained showed that the EO of L. dentata showed antibacterial and antibiofilm activity for all strains tested, with the CBM of L. dentata oil of 32%. Regarding the antibiofilm activity of the EO of L. dentata, there was a reduction in bacterial load in the period of 30 minutes and 24 hours, which promoted a reduction of up to 79.25% in the 30-minute treatment and up to 74.51% in the 24-hour treatment. hours. It was concluded that the essential oil of L. dentata showed antibacterial activity against antibiotic-resistant clinical strains and standard strains of E. faecalis and E. faecium with a minimum bactericidal concentration of 32%, and antibiofilm activity with an action time of 30 minutes and 24 hours, with results similar to chlorhexidine in most strains tested.K (AU)

Effect of Enterococcus faecium as a Water and/or Feed Additive on the Gut Microbiota, Hematologic and Immunological Parameters, and Resistance Against Francisellosis and Streptococcosis in Nile Tilapia (Oreochromis niloticus).

In the present study, we evaluated the effects of administering Enterococcus faecium in food and/or water on the hematological and immunological parameters, intestinal microbiota, resistance to bacterial diseases (streptococcosis and francisellosis) and growth of Nile tilapia. Before the in vivo experiment, probiotic bacteria isolated from Nile tilapia were selected via inhibition tests. Sequencing, annotation, and assembly of the complete genome of the selected bacteria as well as other tests were performed using bioinformatics tools. Three treatments were implemented: G1 (probiotic feeding), G2 (probiotic in water), and G3 (probiotic in food and water); and a negative control (NC) was also employed. Treatment lasted 38 days, and each group consisted of fish and two repetitions. The fish were divided and infected with Streptococcus agalactiae S13 (serotype Ib) and Francisella orientalis. The G1 group had a higher average final weight gain than the G2, G3, and NC groups. Further, a significant increase in the number of thrombocytes was observed in the groups administered probiotics in the diet (G1 and G3). A statistical difference was observed in the mortality of fish infected with S. agalactiae in the NC compared to the treated groups. Cetobacterium was the 43 most abundant genus in the intestinal microbiota of all groups, including the NC group. E. faecium increased the immunity of fish administered the treatment and decreased the mortality caused by S. agalactiae. As an autochtone probiotic, E. faecium does not interfere with the local ecosystem and thus has a great probiotic potential for Nile tilapia in Brazil.

Orodispersible Film Loaded with Enterococcus faecium CRL183 Presents Anti-Candida albicans Biofilm Activity In Vitro.

BACKGROUND: Probiotic bacteria have been emerging as a trustworthy choice for the prevention and treatment of Candida spp. infections. This study aimed to develop and characterize an orodispersible film (ODF) for delivering the potentially probiotic Enterococcus faecium CRL 183 into the oral cavity, evaluating its in vitro antifungal activity against Candida albicans. METHODS AND RESULTS: The ODF was composed by carboxymethylcellulose, gelatin, and potato starch, and its physical, chemical, and mechanical properties were studied. The probiotic resistance and viability during processing and storage were evaluated as well as its in vitro antifungal activity against C. albicans. The ODFs were thin, resistant, and flexible, with neutral pH and microbiologically safe. The probiotic resisted the ODF obtaining process, demonstrating high viability (>9 log10 CFU·g-1), up to 90 days of storage at room temperature. The Probiotic Film promoted 68.9% of reduction in fungal early biofilm and 91.2% in its mature biofilm compared to the group stimulated with the control film. Those results were confirmed through SEM images. CONCLUSION: The probiotic ODF developed is a promising strategy to prevent oral candidiasis, since it permits the local probiotic delivery, which in turn was able to reduce C. albicans biofilm formation.

Occurrence of multidrug-resistant Enterococcus faecium isolated from environmental samples.

Enterococcus species are present in the microbiota of humans and animals and have also been described in the environment. Among the species, Enterococcus faecium is one of the main pathogens associated with nosocomial infections worldwide. Enterococcus faecium isolates resistant to different classes of antimicrobials have been increasingly reported, including multidrug-resistant (MDR) isolates in environmental sources, which is worrying. Therefore, this study aimed to characterize E. faecium isolates obtained from soil and water samples regarding antimicrobial resistance and virulence determinants. A total 40 E. faecium isolates were recovered from 171 environmental samples. All isolates were classified as MDR, highlighting the resistance to the fluoroquinolones class, linezolid and vancomycin. Furthermore, high-level aminoglycoside resistance and high-level ciprofloxacin resistance were detected in some isolates. Several clinically relevant antimicrobial resistance genes were found, including vanC1, ermB, ermC, mefAE, tetM, tetL, ant(6')-Ia, ant(4')-Ia, aph(3')-IIIa and aac(6')-Ie-aph(2″)-Ia. Three virulence genes were detected among the MDR E. faecium isolates, such as esp, gelE and ace. The results of this study contribute to a better understanding of MDR E. faecium isolates carrying antimicrobial resistance and virulence genes in environmental sources and report for the first time in the world the presence of vanC1-producing E. faecium isolated from soil.

Determinacion de los factores de riesgo en sepsis por Enterococcus vancomicino resistente. Estudio de casos y controles en pacientes criticamente enfermos / Determination of risk factors in sepsis by Enterococcus vancomicyn resistant. Study of cases and control in criticall ill patients

Resumen Introducción: las infecciones causadas por Enterococcus resistente a Vancomicina (EVR) presentan mayor mortalidad en pacientes críticos, asociado a un aumento gradual en este patrón de resistencia, especialmente en el continente americano, por lo cual la adecuada terapia antimicrobiana empírica es fundamental para mejorar los desenlaces. Objetivo: determinar los factores de riesgo asociados al desarrollo de infección por EVR en pacientes sépticos en la Unidad de Cuidados Intensivos (UCI) del Hospital San José en Bogotá, Colombia. Métodos: Estudio descriptivo de casos y controles en pacientes sépticos ingresados a la UCI durante 2016 y 2017. Los casos se definieron como pacientes con infección por EVR y los controles los pacientes con infección por otro germen. Resultados: se incluyeron 32 pacientes con aislamiento de EVR y 96 controles. Los factores de riesgo asociados a infección por EVR fueron: nutrición parenteral (OR 15,7 IC 4,2-71,4), lavado peritoneal (OR 8,9 IC 3,2-24,8), cultivo polimicrobiano (OR 19,9 IC 6,0-83,4). La mortalidad fue 56,2% en casos y 33,3% en controles. Conclusiones: Los factores de riesgo hallados con mayor frecuencia fueron: múltiples lavados peritoneales, nutrición parenteral y cultivos polimicrobianos. Encontramos una correlación significativa en el uso de antibiótico empírico adecuado y la reducción en la mortalidad.

Summary Introduction: infections caused by Vancomycin-resistant Enterococcus (VRE) have higher mortality in critically ill patients, associated with increase in this pattern of resistance, especially in the Americas, which is why adequate empirical antimicrobial therapy is essential to improve outcomes Objective: to determine the risk factors associated with the development of infection by VRE in septic patients in the Intensive Care Unit (ICU) of San José Hospital in Bogotá, Colombia. Methods: Case-control study in septic patients admitted to the ICU during 2016 and 2017. The cases were defined as patients with VRE infection and the controls were patients with infection by another germ. Results: 32 patients with EVR isolation and 96 controls were included. The risk factors associated with infection by EVR were: parenteral nutrition (OR 15.7 IC 4.2-71.4), peritoneal lavage (OR 8.9 IC 3.2-24.8), polymicrobial culture (OR 19,9 IC 6.0-83.4). Mortality was 56.2% in cases and 33.3% in controls. Conclusions: The risk factors found most frequently were: multiple peritoneal lavage, parenteral nutrition and polymicrobial cultures. We found a significant correlation in the use of adequate empirical antibiotic and the reduction in mortality