Nutraceutical blends predict enhanced health via microbiota reshaping improving cytokines and life quality: a Brazilian double-blind randomized trial.

Nutraceutical interventions supporting microbiota and eliciting clinical improvements in metabolic diseases have grown significantly. Chronic stress, gut dysbiosis, and metainflammation have emerged as key factors intertwined with sleep disorders, consequently exacerbating the decline in quality of life. This study aimed to assess the effects of two nutraceutical formulations containing prebiotics (fructooligosaccharides (FOS), galactooligosaccharides (GOS), yeast ß-glucans), minerals (Mg, Se, Zn), and the herbal medicine Silybum marianum L. Gaertn., Asteraceae (Milk thistle or Silymarin). These formulations, namely NSupple (without silymarin) and NSupple_Silybum (with silymarin) were tested over 180 days in overweight/obese volunteers from Brazil's southeastern region. We accessed fecal gut microbiota by partial 16S rRNA sequences; cytokines expression by CBA; anthropometrics, quality of life and sleep, as well as metabolic and hormonal parameters, at baseline (T0) and 180 days (T180) post-supplementation. Results demonstrated gut microbiota reshaping at phyla, genera, and species level post-supplementation. The Bacteroidetes phylum, Bacteroides, and Prevotella genera were positively modulated especially in the NSupple_Silybum group. Gut microbiota modulation was associated with improved sleep patterns, quality-of-life perception, cytokines expression, and anthropometric parameters post-supplementation. Our findings suggest that the nutraceutical blends positively enhance cardiometabolic and inflammatory markers. Particularly, NSupple_Silybum modulated microbiota composition, underscoring its potential significance in ameliorating metabolic dysregulation. Clinical trial registry number: NCT04810572. 23/03/2021.

Modulation of the gut microbiome and Firmicutes phylum reduction by a nutraceutical blend in the obesity mouse model and overweight humans: A double-blind clinical trial.

Overweight and obesity are closely linked to gut dysbiosis/dysmetabolism and disrupted De-Ritis ratio [aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio], which may contribute to chronic noncommunicable diseases onset. Concurrently, extensive research explores nutraceuticals, and health-enhancing supplements, for disease prevention or treatment. Thus, sedentary overweight volunteers were double-blind randomized into two groups: Novel Nutraceutical_(S) (without silymarin) and Novel Nutraceutical (with silymarin). Experimental formulations were orally administered twice daily over 180 consecutive days. We evaluated fecal gut microbiota, based on partial 16S rRNA sequences, biochemistry and endocrine markers, steatosis biomarker (AST/ALT ratio), and anthropometric parameters. Post-supplementation, only the Novel Nutraceutical group reduced Clostridium clostridioforme (Firmicutes), Firmicutes/Bacteroidetes ratio (F/B ratio), and De-Ritis ratio, while elevating Bacteroides caccae and Bacteroides uniformis (Bacteroidetes) in Brazilian sedentary overweight volunteers after 180 days. In summary, the results presented here allow us to suggest the gut microbiota as the action mechanism of the Novel Nutraceutical promoting metabolic hepatic recovery in obesity/overweight non-drug interventions.

Pipeline validation for the identification of antimicrobial-resistant genes in carbapenem-resistant Klebsiella pneumoniae.

Antimicrobial-resistant Klebsiella pneumoniae is a global threat to healthcare and an important cause of nosocomial infections. Antimicrobial resistance causes prolonged treatment periods, high mortality rates, and economic impacts. Whole Genome Sequencing (WGS) has been used in laboratory diagnosis, but there is limited evidence about pipeline validation to parse generated data. Thus, the present study aimed to validate a bioinformatics pipeline for the identification of antimicrobial resistance genes from carbapenem-resistant K. pneumoniae WGS. Sequences were obtained from a publicly available database, trimmed, de novo assembled, mapped to the K. pneumoniae reference genome, and annotated. Contigs were submitted to different tools for bacterial (Kraken2 and SpeciesFinder) and antimicrobial resistance gene identification (ResFinder and ABRicate). We analyzed 201 K. pneumoniae genomes. In the bacterial identification by Kraken2, all samples were correctly identified, and in SpeciesFinder, 92.54% were correctly identified as K. pneumoniae, 6.96% erroneously as Pseudomonas aeruginosa, and 0.5% erroneously as Citrobacter freundii. ResFinder found a greater number of antimicrobial resistance genes than ABRicate; however, many were identified more than once in the same sample. All tools presented 100% repeatability and reproducibility and > 75% performance in other metrics. Kraken2 was more assertive in recognizing bacterial species, and SpeciesFinder may need improvements.

Elevated D-Dimer as a Marker For Thromboembolic Events in Pediatric Patients With Covid-19: A Systematic Review

Abstract Background: The 2019 Coronavirus disease is known to cause thromboembolic events. There is little information on the severe COVID-19 consequences in children. Objectives: To determine whether elevated D-dimer levels in the pediatric population with COVID-19 are a risk marker for the development of thromboembolic events. If so, D-dimer levels could be used to determine prophylactic anticoagulation measures if needed. Methods: This is a systematic review, performed according to the recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and registered in the International Prospective Register of Systematic Reviews (PROSPERO) database. The last database search update was on December 14, 2021, resulting in 79 documents for analysis. Data were taken from various databases and queried by topic, keyword, or abstract. Results: Of the 79 articles found, only seven were selected for this analysis. Of these articles, only one had thromboembolic events. In the other articles, D-dimer levels were elevated but were considered controversial in terms of predicting events, with no clear association between the magnitude of D-dimer change and the magnitude of thrombosis risk. Conclusions: Although used for adults, D-dimer was not a good parameter for assessing the risk of thromboembolic events in individuals younger than 21 years. The main shortcomings are the fact that D-dimer increases with any type of inflammation and is, therefore, not a specific marker, and that it is elevated in many patients even without the occurrence of thromboembolic events.

Association Between Gut Microbiota and Delirium in Acutely Ill Older Adults.

Our aim was to investigate the association between gut microbiota and delirium occurrence in acutely ill older adults. We included 133 participants 65+ years consecutively admitted to the emergency department of a tertiary university hospital, between September 2019 and March 2020. We excluded candidates with ≥24-hour antibiotic utilization on admission, recent prebiotic or probiotic utilization, artificial nutrition, acute gastrointestinal disorders, severe traumatic brain injury, recent hospitalization, institutionalization, expected discharge ≤48 hours, or admission for end-of-life care. A trained research team followed a standardized interview protocol to collect sociodemographic, clinical, and laboratory data on admission and throughout the hospital stay. Our exposure measures were gut microbiota alpha and beta diversities, taxa relative abundance, and core microbiome. Our primary outcome was delirium, assessed twice daily using the Confusion Assessment Method. Delirium was detected in 38 participants (29%). We analyzed 257 swab samples. After adjusting for potential confounders, we observed that a greater alpha diversity (higher abundance and richness of microorganisms) was associated with a lower risk of delirium, as measured by the Shannon (odds ratio [OR] = 0.77; 95% confidence interval [CI] = 0.60-0.99; p = .042) and Pielou indexes (OR = 0.69; 95% CI = 0.51-0.87; p = .005). Bacterial taxa associated with pro-inflammatory pathways (Enterobacteriaceae) and modulation of relevant neurotransmitters (Serratia: dopamine; Bacteroides, Parabacteroides: GABA) were more common in participants with delirium. Gut microbiota diversity and composition were significantly different in acutely ill hospitalized older adults who experienced delirium. Our work is an original proof-of-concept investigation that lays a foundation for future biomarker studies and potential therapeutic targets for delirium prevention and treatment.

Differences in the gut microbiota of women according to ultra-processed food consumption.

BACKGROUND AND AIMS: High consumption of ultra-processed food (UPF) has been associated with increased risk of obesity and other metabolic diseases, and this dietary pattern seems to be responsible for chronic changes in the gut microbiota. The aim of this study was to assess the associations of UPF with the gut microbiota and obesity-associated biometrics in women. METHODS AND RESULTS: This cross-sectional study examined 59 women. The following parameters were evaluated: food consumption using NOVA classification, anthropometric and metabolic parameters, and gut microbiome by next-generation sequencing. The mean age was 28.0 ± 6.6 years. The mean caloric intake was 1624 ± 531 kcal, of which unprocessed or minimally processed food (G1) accounted for 52.4 ± 13.5%, and UPF accounted for 31.4 ± 13.6%. Leptin levels adjusted for fat mass were negatively associated with G1 and positively associated with UPF. We found 15 species in the gut microbiota that correlated with G1 (3 positively and 12 negatively) and 9 species associated with UPF (5 positively and 4 negatively). CONCLUSION: Higher consumption of UPF was directly associated with leptin resistance, and this study suggests that the consumption of UPF or G1 may affect the composition of the gut microbiota.

Neisseria gonorrhoeae arthritis in a patient with systemic lupus: resistance and virulence profiles.

In this study, we describe a case report of gonococcal arthritis in a Systemic Lupus Erythematosus patient. Although several mechanisms favor disseminated gonococcal infection (DGI) in patients immunosuppressed by SLE, this association is rarely reported in literature. We performed whole genome sequencing (WGS) of the etiologic agent involved and molecular analysis using a global collection of Neisseria gonorrhoeae strains. Ours is the only sample derived from synovial fluid identified in this collection, the others being from the usual anatomical sites. Antimicrobial susceptibility was determined by disk diffusion and Etest, and WGS was conducted to determine multilocus sequence typing profiles, group isolates based on core genome single nucleotide polymorphisms (SNP), and identify virulence genes and antimicrobial resistance determinants. The N. gonorrhoeae samples in the global collection were highly heterogeneous. The SNP tree had a total 19,532 SNPs in 320 samples. Our sample displayed resistance to ciprofloxacin (MIC = 2 µg/mL) and tetracycline (zone diameter = 0 mm) belonged to ST 1588 and was not closely related to any isolate in the global collection of N. gonorrhoeae strains. The isolate had genetic features related to beta-lactam, tetracycline and quinolone resistance. Seventy-one virulence genes were identified in our sample, belonging to the following classes: adherence, efflux pump, immune modulator, invasion, iron uptake, protease and stress adaptation. Moreover, no virulence genes for immune evasion and toxin were identified.

Case Report: Cutaneous Leishmaniasis in a Rheumatoid Arthritis Patient Receiving Methotrexate.

The immunosuppressive effect of methotrexate has rarely been associated with reactivation of cutaneous leishmaniasis. Here we present a case of a cutaneous leishmaniasis patient with atypical clinical symptoms without splenomegaly but with cutaneous manifestations after treatment of rheumatoid arthritis with methotrexate and blood recovery of the parasite. Next-generation sequencing was used to identify Leishmania infantum chagasi in the patient's blood sample.

Synbiotic Supplementation Modulates Gut Microbiota, Regulates ß-Catenin Expression and Prevents Weight Gain in ob/ob Mice: Preliminary Findings.

BACKGROUND: Obesity is one of the main health problems in the world today, and dysbiosis seems to be one of the factors involved. The aim of this study was to examine the impact of synbiotic supplementation on obesity and the microbiota in ob/ob mice. Twenty animals were divided into four groups: obese treated (OT), obese control (OC), lean treated (LT) and lean control (LC). All animals received a standard diet for 8 weeks. The treated groups received a synbiotic (Simbioflora-Invictus Farmanutrição Ltd., Sao Paulo, Brazil) in water, while the nontreated groups received only water. After 8 weeks, all animals were sacrificed, and gut tissue and stool samples were collected for mRNA isolation and microbiota analysis, respectively. ß-Catenin, occludin, cadherin and zonulin in the gut tissue were analyzed via RT-qPCR. Microbiome DNA was extracted from stool samples and sequenced using an Ion PGM Torrent platform. RESULTS: Synbiotic supplementation reduced body weight gain in the OT group compared with the OC group (p = 0.0398) and was associated with an increase in Enterobacteriaceae (p = 0.005) and a decrease in Cyanobacteria (p = 0.047), Clostridiaceae (p = 0.026), Turicibacterales (p = 0.005) and Coprococcus (p = 0.047). On the other hand, a significant reduction in Sutterella (p = 0.009) and Turicibacter (p = 0.005) bacteria was observed in the LT group compared to the LC group. Alpha and beta diversities were different among all treated groups. ß-Catenin gene expression was significantly decreased in the gut tissue of the OT group (p ≤ 0.0001) compared to the other groups. No changes were observed in occludin, cadherin or zonulin gene expression in the gut tissue. CONCLUSIONS: Synbiotic supplementation prevents excessive weight gain, modulates the gut microbiota, and reduces ß-catenin expression in ob/ob mice.

Sal rosa do Himalaia: determinação do teor de iodo, pesquisa de corantes e avaliação da rotulagem / Himalayan pink salt: iodine, colorants research and labeling evaluation

Introdução: O sal rosa do Himalaia tem se destacado no mercado brasileiro por sua coloração atrativa, além dos apelos ao consumidor que o destacam como um produto "mais natural" e com grande diversidade de minerais por ser um sal de rocha. O teor de iodo no sal rosa consumido no país ainda é pouco discutido, mas vital para manter sob controle os distúrbios por deficiência de iodo (DDI).  Objetivo: Avaliar amostras de sal rosa do Himalaia quanto aos teores de iodo, à presença de corantes e à rotulagem. Método: Foram analisadas 71 amostras em 13 cidades do estado de São Paulo para determinação de iodo adicionado na forma de iodato, prova qualitativa para corantes artificiais e avaliação da rotulagem com base na legislação brasileira. Resultados: Um elevado índice de  insatisfatoriedade dos teores de iodo foi encontrado (56%) e um percentual ainda maior nos sais comercializados a granel (74%). Verificou-se que, dentre as amostras insatisfatórias, o maior percentual de inadequação (28%) foi a ausência de iodo, colocando a população consumidora deste produto em risco para as DDI. A irregularidade de rotulagem mais encontrada foi a ausência da declaração da adição de iodo (46%), com contradições entre a declaração no rótulo e a efetiva presença avaliada analiticamente. Foram verificadas expressões de qualidade não previstas (27%) ou superlativas (14%), assim como alegações não comprovadas por estudos científicos, como a redução de 60% de sódio e a presença de 84 minerais. Nenhuma amostra apresentou adição de corante. Conclusões: O sal rosa do Himalaia analisado apresentou irregularidades importantes para a saúde da população, em especial quanto ao teor de iodo, mas também não conformidades de rotulagem que comprometem o acesso à informação correta sobre o produto.

Introduction: Himalayan pink salt has stood out in the Brazilian market for its attractive colors, in addition to appeals to the consumer that highlight it as a "more natural" product and with a great diversity of minerals as it is a rock salt. The iodine content in pink salt consumed in the country is still little discussed, but vital to keep Iodine Deficiency Disorders (DDIs) under control. Objective: Evaluate Himalayan pink salt samples for iodine contents, presence of artificial colorants and labeling. Method: Seventy-one samples from thirteen cities of the São Paulo State were analyzed for determination of iodine added as iodate, qualitative testing colorants, and labeling evaluation based on Brazilian legislation. Results: A high rate of unsatisfactory iodine content was found (56%), even higher in salts sold in bulk (74%). It was found that among the unsatisfactory samples, the highest percentage of inadequacy (28%) was the absence of iodine, exposing the consumers of this product at risk for DDIs. The most frequent labeling irregularity was the absence of iodine declaration (46%), with contradictions between label declaration and effective presence evaluated analytically. Unforeseen (27%) or superlative (14%) quality expressions were verified, as well as claims not supported by scientific studies, such as 60% reduction in sodium and presence of 84 minerals. No sample showed colorants addition. Conclusions: Himalayan pink salt samples analyzed showed important irregularities for health of the population, especially regarding the iodine content, but also labeling inaccuracies that compromise access to correct information about the product.

Clostridioides difficile from Brazilian hospitals: characterization of virulence genes by whole genome sequencing.

Clostridioides difficile (CD) is the most frequent cause of healthcare related diarrhea and its severity has increased in the last decade by the spread of hypervirulent strains. Most important CD virulence factor is toxin production; however, not only toxins are responsible for Clostridioides virulence. We sequenced 38 strains and analyzed the presence and integrity of 24 virulence (including toxin) genes. We identified 28 toxigenic strains, six also presented the cdt genes. Only six strains didn't present all others genes searched. All absent genes were adhesion related. Understand others CD virulence factors can lead to a best understanding on this matter.

Identification of Leishmania species by next generation sequencing of hsp70 gene.

Leishmaniasis is a major public health problem worldwide. Although next generation sequencing technology has been widely used in the diagnosis of infectious diseases, it has been scarcely applied in identification of Leishmania species. The aim of this study was to compare the efficiency of MinION™ nanopore sequencing and polymerase chain reaction restriction fragment length polymorphism in identifying Leishmania species. Our results showed that the MinION™ sequencer was able to discriminate reference strains and clinical samples with high sensitivity in a cost and time effective manner without the prior need for culture.

Phenotypic and genotypic characteristics of a carbapenem-resistant Serratia marcescens cohort and outbreak: describing an opportunistic pathogen.

Serratia marcescens is an emerging opportunistic pathogen with high genetic diversity. This article describes the microbiological characteristics of isolates and the risk factors for infections caused by carbapenem-resistant S. marcescens. A retrospective study of patients colonized (n=43) and infected (n=20) with carbapenem-resistant S. marcescens over a 3-year period was conducted. Polymerase chain reaction for carbapenemase genes and molecular typing of all available strains was performed. Forty-two isolates were analysed, including three environmental samples identified during an outbreak. Thirty-five carbapenem-resistant S. marcescens carried blaKPC-2, one isolate was blaNDM-positive and four isolates carried blaOXA-101. The genomes were grouped into three clusters with 100% bootstrap; three patterns of mutations on ompC and ompF were found. The strains carried virulence genes related to invasion and haemolysis, and the environmental strains presented fewer mutations on the virulence genes than the clinical strains. Multi-variate analysis showed that previous use of polymyxin (P=0.008) was an independent risk factor for carbapenem-resistant S. marcescens infection. This study highlighted that blaKPC-2 in association with ompC or ompF mutation was the most common mechanism of resistance in the study hospital, and that previous use of polymyxin was an independent risk factor for carbapenem-resistant S. marcescens. There was a predominant clone, including the environmental isolates, suggesting that cross-transmission was involved in the dissemination of this pathogen.

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.

Clinical outcome from hematopoietic cell transplant patients with bloodstream infection caused by carbapenem-resistant P. aeruginosa and the impact of antimicrobial combination in vitro.

Bloodstream infection (BSI) caused by carbapenem-resistant P. aeruginosa (CRPA) has high mortality in hematopoietic stem cell transplant (HSCT) recipients. We performed MIC, checkerboard, time-kill assay, PFGE, PCR, and whole genome sequence and described the clinical outcome through Epi Info comparing the antimicrobial combination in vitro. Mortality was higher in BSI caused by CRPA carrying the lasB virulence gene. The isolates were 97% resistant to meropenem displaying synergistic effect to 57% in combination with colistin. Seventy-three percent of the isolates harbored blaSPM-1 and Tn4371 and belonged to ST277. The synergistic effect in vitro with meropenem with colistin appeared to be a better therapeutic option.

Conjugative transfer of plasmid p_8N_qac(MN687830.1) carrying qacA gene from Staphylococcus aureus to Escherichia coli C600: potential mechanism for spreading chlorhexidine resistance.

The methicillin resistant Staphylococcus aureus (MRSA) is recognized by its ability to acquire and transferring resistance genes through interspecies conjugative plasmids. However, transference of plasmids from Gram-positive cocci to Gram-negative bacilli is not well characterized. In this report, we describe the transfer of a conjugative plasmid carrying qacA from MRSA to Escherichia coli C600. We performed a conjugation experiment using a chlorhexidine resistant MRSA isolate (ST-105/SCCmec type III) carrying the gene qacA and qacC as the donor and a chlorhexidine susceptible E. coli C600 isolate as the receptor. Transconjugants were selected using MacConkey agar plates containing chlorhexidine in concentrations ranging from 0.25 to 16 g.L-1. To genotypically confirm the transfer of the resistance gene, the transconjugants were screened by Polymerase Chain Reaction (PCR) and submitted to Sanger's sequencing. MRSA isolates successfully transferred the chlorhexidine resistance gene (qacA) to the recipient E. coli strain C600. The E. coli transconjugant exhibited an important reduction of chlorhexidine susceptibility, with MICs increasing from ≤ 0.25 to ≥ 16 g.L-1 after conjugation. The qacA gene was detected by PCR as well as in the Sanger's sequencing analysis of DNA from transconjugant plasmids. To the best of our knowledge, this is the first report of the plasmid p_8N_qac(MN687830.1) carrying qacA and its transfer by conjugation from a MRSA to an E. coli. These findings increase concerns on the emergence of resistance dissemination across the genus and emphasizes the importance of continuous antiseptic stewardship.

Carbapenem-resistant Klebsiella pneumoniae colonization and infection is associated with lower overall survival in a cohort of haematopoietic stem-cell transplantation patients: mechanism of resistance and virulence by whole-genome sequencing.

Carbapenem-resistant Klebsiella pneumoniae (CRK) infections are a growing concern in immunocompromised patients. The aim of the present study was to evaluate the impact of CRK colonization and infection in overall mortality for haematopoietic stem-cell transplant (HSCT) patients. We also aimed to investigate resistance and virulence profiles of CRK isolates and assess their epidemiological and genetic relatedness. Patients in the HSCT unit were screened for colonization with CRK with weekly rectal swab or stool cultures and placed under contact precautions. We defined CRK colonization as positive culture from a swab or stool sample grown in MacConkey agar with meropenem at 1 µg ml-1. Demographic and clinical data were retrieved from the patients' charts and electronic records. According to resistance mechanisms and pulsed field gel electrophoresis profile, isolates were selected based on whole-genome sequencing (WGS) using MiSeq Illumina. Outcomes were defined as overall mortality (death up to D+100), and infection-related death (within 14 days of infection). We report a retrospective cohort of 569 haematopoietic stem-cell transplant patients with 105 (18.4 %) CRK colonizations and 30 (5.3 %) infections. blaKPC was the most frequent carbapenemase in our cohort with three isolates co-harbouring blaKPC and blaNDM. We found no difference in virulence profiles from the CRK isolates. There were also no significant differences in virulence profiles among colonization and infection isolates regarding genes encoding for type 1 and 3 fimbriae, siderophores, lipopolysaccharide and colibactin. In clonality analysis by PFGE and WGS, isolates were polyclonal and ST340 was the most prevalent. Overall survival at D+100 was 75.4 % in in CRK-colonized (P=0.02) and 35.7 % in infected patients and significantly lower than non-colonized patients (85.8 %; P<0.001). We found a higher overall mortality associated with colonization and infection; KPC was the main resistance mechanism for carbapenems. The polyclonal distribution of isolates and findings of CRK infection in patients not previously colonized suggest the need to reinforce antibiotic stewardship.

Bloodstream Infections caused by Klebsiella pneumoniae and Serratia marcescens isolates co-harboring NDM-1 and KPC-2.

Carbapenem-resistant Enterobacteriaceae are a worldwide health problem and isolates carrying both blaKPC-2 and blaNDM-1 are unusual. Here we describe the microbiological and clinical characteristics of five cases of bloodstream infections (BSI) caused by carbapenem-resistant Klebsiella pneumoniae and Serratia marcescens having both blaKPC-2 and blaNDM-1. Of the five blood samples, three are from hematopoietic stem cell transplantation patients, one from a renal transplant patient, and one from a surgical patient. All patients lived in low-income neighbourhoods and had no travel history. Despite antibiotic treatment, four out of five patients died. The phenotypic susceptibility assays showed that meropenem with the addition of either EDTA, phenylboronic acid (PBA), or both, increased the zone of inhibition in comparison to meropenem alone. Molecular tests showed the presence of blaKPC-2 and blaNDM-1 genes. K. pneumoniae isolates were assigned to ST258 or ST340 by whole genome sequencing. This case-series showed a high mortality among patients with BSI caused by Enterobacteriae harbouring both carbapenemases. The detection of carbapenemase-producing isolates carrying both blaKPC-2 and blaNDM-1 remains a challenge when using only phenotypic assays. Microbiology laboratories must be alert for K. pneumoniae isolates producing both KPC-2 and NDM-1.

Genetic factors involved in fosfomycin resistance of multidrug-resistant Acinetobacter baumannii.

The treatment of infections caused by A. baumannii is a challenge and fosfomycin has been used as a combination therapy. Moreover, data regarding the fosfomycin resistance mechanism is scarce. The goals of this study were to evaluate fosfomycin susceptibility in polyclonal multi-resistant A. baumannii isolates and characterize the fosfomycin resistance. We analyzed 32 A. baumannii isolates from a Brazilian bacterial collection, followed by their minimum inhibitory concentration (MIC), and whole-genome sequence to detect fosfomycin resistance genes. The isolates showed a fosfomycin MIC ranging from 32 to ≥256 mg/L. All isolates were negative for fosA and fosB genes, and four isolates carried the fosX gene. Two different metabolic pathways that form peptidoglycan precursors were identified. Mutations were observed in the adenylate cyclase gene. All A. baumannii isolates studied showed Val132Ala substitutions in MurA. The analysis showed different ways that may lead to the intrinsic fosfomycin-resistance of A. baumannii, such as alterations on the glycerol-3-phosphate transporter system caused by adenylate cyclase mutations; and a possible connection of cell wall recycling by different metabolic pathways.

Colistin-resistant Escherichia coli belonging to different sequence types: genetic characterization of isolates responsible for colonization, community- and healthcare-acquired infections.

The plasmid-mediated colistin-resistance gene named mcr-1 has been recently described in different countries and it became a public health challenge. Of note, few studies have addressed the spread of Escherichia coli harboring the mcr-1 gene in both, community and hospital settings. A total of seven colistin-resistant E. coli carrying mcr-1, collected from 2016 to 2018, from community (n=4), healthcare-acquired infections (n=2) and colonization (n=1) were identified in three high complexity hospitals in Sao Paulo, Brazil. These colistin-resistant isolates were screened for mcr genes by PCR and all strains were submitted to Whole Genome Sequencing and the conjugation experiment. The seven strains belonged to seven distinct sequence types (ST744, ST131, ST69, ST48, ST354, ST57, ST10), and they differ regarding the resistance profiles. Transference of mcr-1 by conjugation to E. coli strain C600 was possible in five of the seven isolates. The mcr-1 gene was found in plasmid types IncX4 or IncI2. Three of the isolates have ESBL-encoding genes (blaCTX-M-2, n=2; blaCTX-M-8, n=1). We hereby report genetically distinct E. coli isolates, belonging to seven STs, harboring the mcr-1 gene, associated to community and healthcare-acquired infections, and colonization in patients from three hospitals in Sao Paulo. These findings point out for the potential spread of plasmid-mediated colistin-resistance mechanism in E. coli strains in Brazil.