Influence of carbonyl cyanide m-chlorophenyl hydrazone on biofilm dynamics, protease, and siderophore production by Burkholderia pseudomallei.

Efflux pump inhibitors are a potential therapeutic strategy for managing antimicrobial resistance and biofilm formation. This article evaluated the effect of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) on the biofilm growth dynamics and the production of virulence factors by Burkholderia pseudomallei. The effects of CCCP on planktonic, growing, and mature biofilm, interaction with antibacterial drugs, and protease and siderophore production were assessed. CCCP MICs ranged between 128 and 256 µM. The CCCP (128 µM) had a synergic effect with all the antibiotics tested against biofilms. Additionally, CCCP reduced (p < .05) the biomass of biofilm growth and mature biofilms at 128 and 512 µM, respectively. CCCP also decreased (p < .05) protease production by growing (128 µM) and induced (p < .05) siderophore release by planktonic cells (128 µM) growing biofilms (12.8 and 128 µM) and mature biofilms (512 µM). CCCP demonstrates potential as a therapeutic adjuvant for disassembling B. pseudomallei biofilms and enhancing drug penetration.

Genetic diversity of Gerbich alleles in Brazilians reveals an unexpected prevalence of the GE:-2,-3,4 phenotype.

BACKGROUND AND OBJECTIVES: Gerbich (GE) blood group system carries high-frequency antigens and the absence of them leads to rare phenotypes: GE:-2,3,4, GE:-2,-3,4 and GE:-2,-3,-4. Their serological differentiation is limited and misclassification of Gerbich phenotypes may occur, but this can be avoided by molecular characterization. This study aimed to characterize the molecular background responsible for rare Gerbich phenotypes in Brazilian population. MATERIALS AND METHODS: We selected eight samples from patients with anti-Ge, six from their relatives and nine samples with normal expression of Gerbich antigens. Serological tests were performed in gel and red blood cells (RBCs) were tested with anti-Ge2 and anti-Ge3. Monocyte monolayer assay (MMA) was performed. Molecular investigation was performed with allele-specific polymerase chain reaction and DNA sequencing. RESULTS: Patient plasma samples reacted with all commercial RBCs. Patient RBCs showed negative results with anti-Ge2 and anti-Ge3. Using MMA two of eight antibodies were clinically significant. Exon 3 was not amplified in any of the patient samples and in two samples from relatives, suggesting the presence of GE*01.-03/GE*01.-03. By sequencing, we identified the genetic variability that interferes with the definition of deletion breakpoints, thus two options of genetic structure were suggested to be responsible for the GE:-2,-3,4 phenotype. CONCLUSION: This study showed for the first time the genetic diversity of GYPC alleles for carriers of Gerbich-negative phenotypes in a Brazilian population and showed an unexpected prevalence of the GE:-2,-3,4 phenotype. It also demonstrated the importance of using molecular tools to correctly classify Gerbich phenotypes for selection of variants in antigen-matched transfusions.

Virulence factors of Gram-negative bacteria from free-ranging Amazon river dolphins (Inia geoffrensis).

Freshwater cetaceans play a significant role as sentinel animals, providing important data on animal species and aquatic ecosystem health. They also may serve as potential reservoirs of emerging pathogens and host virulence genes in their microbiota. In this study, we evaluated virulence factors produced by Gram-negative bacteria recovered from individuals belonging to two populations of free-ranging Amazon river dolphins (Inia geoffrensis). A total of 132 isolates recovered from the oral cavity, blowhole, genital opening and rectum of 21 river dolphins, 13 from Negro River and 8 from Tapajós River, Brazil, were evaluated for the production of virulence factors, such as biofilms and exoproducts (proteases, hemolysins and siderophores), in planktonic and biofilm forms. In planktonic form, 81.1% (107/132) of the tested bacteria of free-ranging Amazon river dolphins were able to produce virulence factors, with 44/132 (33.4%), 65/132 (49,2%) and 54/132 (40,9%) positive for protease, hemolysin and siderophore production, respectively. Overall, 57/132 (43.2%) of the isolates produced biofilms and, under this form of growth, 66/132 (50%), 88/132 (66.7%) and 80/132 (60.6%) of the isolates were positive for protease, hemolysin and siderophore production. In general, the isolates showed a higher release of exoproducts in biofilm than in planktonic form (P < 0.001). The present findings show that Amazon river dolphins harbor potentially pathogenic bacteria in their microbiota, highlighting the importance of monitoring the micro-organisms from wild animals, as they may emerge as pathogens for humans and other animals.

In vitro effect of the iron chelator deferiprone on the antimicrobial susceptibility and biofilms of Burkholderia pseudomallei.

This study evaluated the effect of the iron chelator deferiprone (DFP) on antimicrobial susceptibility and biofilm formation and maintenance by Burkholderia pseudomallei. Planktonic susceptibility to DFP alone and in combination with antibiotics was evaluated by broth microdilution and biofilm metabolic activity was determined with resazurin. DFP minimum inhibitory concentration (MIC) range was 4-64 µg/mL and in combination reduced the MIC for amoxicillin/clavulanate and meropenem. DFP reduced the biomass of biofilms by 21 and 12% at MIC and MIC/2, respectively. As for mature biofilms, DFP reduced the biomass by 47%, 59%, 52% and 30% at 512, 256, 128 and 64 µg/mL, respectively, but did not affect B. pseudomallei biofilm viability nor increased biofilm susceptibility to amoxicillin/clavulanate, meropenem and doxycycline. DFP inhibits planktonic growth and potentiates the effect of ß-lactams against B. pseudomallei in the planktonic state and reduces biofilm formation and the biomass of B. pseudomallei biofilms.

Effect of promethazine on biofilms of gram-positive cocci associated with infectious endocarditis.

This study evaluated the antimicrobial activity of promethazine against Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus mutans and its effect on the antimicrobial susceptibility of biofilms grown in vitro and ex vivo on porcine heart valves. Promethazine was evaluated alone and in combination with vancomycin and oxacillin against Staphylococcus spp. and vancomycin and ceftriaxone against S. mutans in planktonic form and biofilms grown in vitro and ex vivo. Promethazine minimum inhibitory concentration range was 24.4-95.31 µg/mL and minimum biofilm eradication concentration range was 781.25-3.125 µg/mL. Promethazine interacted synergistically with vancomycin, oxacillin and ceftriaxone against biofilms in vitro. Promethazine alone reduced (p < 0.05) the CFU-counts of biofilms grown on heart valves for Staphylococcus spp., but not for S. mutans, and increased (p < 0.05) the activity of vancomycin, oxacillin and ceftriaxone against biofilms of Gram-positive cocci grown ex vivo. These findings bring perspectives for repurposing promethazine as adjuvant in the treatment of infective endocarditis.

Ex vivo wound model on porcine skin for the evaluation of the antibiofilm activity of polyhexamethylene biguanide and ciprofloxacin.

This study aimed to standardize the use of an ex vivo wound model for the evaluation of compounds with antibiofilm activity. The in vitro susceptibility of Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 27853 to ciprofloxacin and polyhexamethylene biguanide (PHMB) was evaluated in planktonic and biofilm growth. The effects of ciprofloxacin and PHMB on biofilms grown on porcine skin explants were evaluated by colony-forming unit (CFU) counting and confocal microscopy. Minimum inhibitory concentrations (MICs) against S. aureus and P. aeruginosa were, respectively, 0.5 and 0.25 µg mL-1 for ciprofloxacin, and 0.78 and 6.25 µg mL-1 for PHMB. Minimum biofilm eradication concentrations (MBECs) against S. aureus and P. aeruginosa were, respectively, 2 and 8 µg mL-1 for ciprofloxacin, and 12.5 and >25 µg mL-1 for PHMB. Ciprofloxacin reduced (P < 0.05) log CFU counts of the biofilms grown ex vivo by 3 and 0.96 for S. aureus and P. aeruginosa, respectively, at MBEC, and by 0.58 and 8.12 against S. aureus and P. aeruginosa, respectively, at 2xMBEC. PHMB (100 µg/mL) reduced (P < 0.05) log CFU counts by 0.52 for S. aureus and 0.68 log for P. aeruginosa, leading to an overall decrease (P < 0.05) in biofilm biomass. The proposed methodology to evaluate the susceptibility of biofilms grown ex vivo led to reproducible and reliable results.

Antibiofilm activity of promethazine, deferiprone, and Manuka honey in an ex vivo wound model.

This study evaluated the antibiofilm activity of promethazine, deferiprone, and Manuka honey against Staphylococcus aureus and Pseudomonas aeruginosa in vitro and ex vivo in a wound model on porcine skin. The minimum inhibitory concentrations (MICs) and the effects of the compounds on biofilms were evaluated. Then, counting colony-forming units (CFUs) and confocal microscopy were performed on biofilms cultivated on porcine skin for evaluation of the compounds. For promethazine, MICs ranging from 97.66 to 781.25 µg/ml and minimum biofilm eradication concentration (MBEC) values ranging from 195.31 to 1562.5 µg/ml were found. In addition to reducing the biomass of both species' biofilms. As for deferiprone, the MICs were 512 and >1024 µg/ml, the MBECs were ≥1024 µg/ml, and it reduced the biomass of biofilms. Manuka honey had MICs of 10%-40%, MBECs of 20 to >40% and reduced the biomass of S. aureus biofilms only. Concerning the analyses in the ex vivo model, the compounds reduced (P < .05) CFU counts for both bacterial species, altering the biofilm architecture. The action of the compounds on biofilms in in vitro and ex vivo tests raises the possibility of using them against biofilm-associated wounds. However, further studies are needed to characterize the mechanisms of action and their effectiveness on biofilms in vivo.

Antimicrobial susceptibility and production of virulence factors by bacteria recovered from bitches with pyometra.

Pyometra is one of the most common diseases in adult female dogs, characterized by a suppurative bacterial infection of the uterus with accumulation of inflammatory exudate and a variety of local and systemic clinical manifestations. This study aimed to identify the bacteria within the uterine content and vaginal canal of bitches with pyometra and evaluate their antimicrobial susceptibility and production of virulence factors. Uterine and vaginal content were collected with sterile swabs from 30 bitches diagnosed with pyometra. Bacteria were identified and assessed for their antimicrobial susceptibility and production of virulence factors, including biofilms, siderophores, proteases and hemolysins, both in planktonic and biofilm forms. A total of 82 bacterial isolates (35 uterus, 47 vagina), belonging to 21 species, were identified, with Escherichia coli as the most prevalent species (32/82, 39%). As for susceptibility, 39/79 (49.4%) isolates were resistant to one or more drugs, with resistance proportion among Gram-positive bacteria (87.5%) higher (p < .05) than that observed for Gram-negative bacteria (32.7%). Four coagulase-negative Staphylococcus species were resistant to methicillin. Regarding virulence, the isolates had low production of biofilms, siderophores, proteases and hemolysins, suggesting that the occurrence of pyometra might be more associated with host-related factors than bacterial virulence.

Transfusion profile, clinical characteristics, comorbidities and outcomes of 3014 hospitalized patients diagnosed with COVID-19 in Brazil.

BACKGROUND: The novel coronavirus disease-2019 (COVID-19) caused a sudden and unexpected increase in the number of hospital admissions and deaths worldwide. The impact of social distancing on blood stocks was significant. Data on the use of blood products by patients with COVID-19 are scarce. MATERIAL AND METHODS: A retrospective observational study was conducted by analysing the medical records of 3014 hospitalized COVID-19 patients in 16 Brazilian hospitals. Individual data related to clinical, laboratory and transfusion characteristics and outcomes of these patients were collected. Patients characteristics association with mortality and transfusion need were tested independently by logistic regression models. RESULTS: Patients mean age was 57·6 years. In 2298 (76·2%) patients, there was an underlying clinical comorbidity. A total of 1657 (55%) patients required admission to intensive care unit (ICU), and 943 (31%) patients required ventilatory support and orotracheal intubation (OTI). There was a total of 471 (15·6%) deaths among all patients. 325 patients (10·7%) required blood transfusion; 3187 blood products were transfused: 1364 red blood cells in 303 patients, 1092 platelet units in 78 patients, 303 fresh frozen plasma in 49 patients and 423 cryoprecipitates in 21 patients. The mortality among patients who received transfusion was substantially higher than that among the total study population. CONCLUSION: Need for transfusion was low in COVID-19 patients, but significantly higher in patients admitted to ICU and in those who needed OTI. Knowledge of the transfusion profile of these patients allows better strategies for maintaining the blood stocks of hospitals during the pandemic.

Repurposing approved drugs as potential efflux pump inhibitors in multidrug-resistant Pseudomonas aeruginosa.

Aim: To evaluate the action of promethazine, fluoxetine and carbonyl cyanide 3-chlorophenylhydrazone as efflux pump inhibitors (EPIs) against multidrug-resistant Pseudomonas aeruginosa. Methods: The effect of the compounds was evaluated in planktonic cells and bacterial biofilms. Accumulation tests were performed with ethidium bromide to prove their action as EPIs. Then, they were associated with antimicrobials. Results: Effect on planktonic cells and biofilms was found. Assays with ethidium bromide indicate their action as EPIs. Significant reductions in the metabolic activity of biofilms were observed after the association with the antimicrobials, especially for meropenem. Conclusion: It is possible to prove the action of these compounds as EPIs for P. aeruginosa and demonstrate the relevance of efflux pumps in antimicrobial resistance.

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Effect of fluoxetine on planktonic and biofilm growth and the antimicrobial susceptibility of Burkholderia pseudomallei.

Aim: This study evaluated the effect of fluoxetine (FLU) on planktonic and biofilm growth and the antimicrobial susceptibility of Burkholderia pseudomallei. Materials & methods: The minimum inhibitory concentrations (MICs) for FLU were determined by broth microdilution. Its effect on growing and mature biofilms and its interaction with antibacterial drugs were evaluated by assessing biofilm metabolic activity, biomass and structure through confocal microscopy. Results: The FLU MIC range was 19.53-312.5 µg/ml. FLU eradicated growing and mature biofilms of B. pseudomallei at 19.53-312.5 µg/ml and 1250-2500 µg/ml, respectively, with no structural alterations and enhanced the antibiofilm activity of antimicrobial drugs. Conclusion: These results bring perspectives for the use of FLU in the treatment of melioidosis, requiring further studies to evaluate its applicability.

One Health Implications of Antimicrobial Resistance in Bacteria from Amazon River Dolphins.

Studies on the microbiota of freshwater cetaceans are scarce and may provide important data on animal and environmental health. This study aimed to evaluate the antimicrobial susceptibility of Gram-negative bacteria recovered from two populations of free-ranging Amazon river dolphins (Inia geoffrensis). Twenty-one animals were captured and released, 13 from Negro River and 8 from Tapajós River, Brazil. Swab samples were obtained from the oral cavity, blowhole, genital opening and rectum and were cultured on MacConkey agar. Isolates were biochemically identified, and antimicrobial susceptibility was assessed by disk diffusion method. Overall, 132 isolates were recovered, of which 71 were recovered from animals from Negro River and 61 from Tapajós River. The most commonly recovered bacterial species were Enterobacter cloacae, Morganella morganii, Klebsiella pneumoniae and Pseudomonas aeruginosa. Overall, 51.6% (63/122) of the isolates were not-susceptible (intermediate resistance and resistance), of which 28/122 (22.9%) were resistant to at least one antimicrobial. Cephalothin, cefuroxime and cefepime were the drugs to which more resistant and intermediate results were observed (P < 0.001). The results indicate that free-ranging Amazon river dolphins host resistant bacteria, contributing for their maintenance in the environment. This study highlights the importance of the One Health approach to monitor the emergence of antimicrobial resistance. Summary Gram-negative bacteria recovered from 21 free-ranging Amazon river dolphins (Inia geoffrensis) from the Negro River and the Tapajós River populations were evaluated for their antimicrobial susceptibility. Overall, 51.6% (63/122) of the isolates were not-susceptible (intermediate resistance and resistance), of which 28/122 (22.9%) were resistant to at least one antimicrobial. Cephalothin, cefuroxime and cefepime were the drugs to which more resistant and intermediate results were observed. Thus, free-ranging Amazon river dolphins, never treated with antimicrobials, host resistant bacteria, contributing for their maintenance in the environment and highlighting the importance of the One Health approach to monitor the emergence of antimicrobial resistance.

Avaliação do impacto familiar em pais de crianças diagnosticadas com microcefalia pelo Zika Vírus / Evaluation of family impact in parents of children diagnosed with microcephaly by Zika Virus

Objetiva-se avaliar o impacto familiar em pais de crianças diagnosticadas com microcefalia pelo Zika vírus. Trata-se de um estudo transversal analítico com abordagem quantitativa, utilizando-se de um questionário sociodemográfico e a escala de impacto familiar (EIF). Foram pesquisados 76 pais com filhos em tratamento de reabilitação e readaptação em um centro de referência de Goiânia/GO. Foram realizadas análises descritivas utilizando-se frequência relativa e absoluta, média e desvio padrão. O teste utilizado para avaliar a existência ou não de diferença estatisticamente significativa (p≤0,05) foi a análise de variância (ANOVA) Scheffé. O pré-natal foi o período predominante do recebimento do diagnóstico de microcefalia. Foi evidenciado uma dificuldade por parte dos pais em encontrar pessoas de confiança para cuidar do filho, bem como, falta de compreensão de outras pessoas pelo fardo que é cuidar do filho deficiente. Os participantes que apresentaram maior tendência em empreender atividades com amigos, festas e bares, também se mostraram mais propensos a atividades físicas e de lazer. Após o choque inicial do recebimento do diagnóstico, os pais passam reorganizar e adaptar aos desafios, alterando a rotina familiar. É fundamental o envolvimento dos profissionais de saúde, fornecendo suporte e orientação a essas famílias

Aimed to evaluate the family impact in parents of children diagnosed with microcephaly by the Zika virus. This is a crosssectional analytical study with a quantitative approach, using a sociodemographic questionnaire and the Family Impact Scale (FIA). A total of 76 parents with children in rehabilitation and rehabilitation treatment were surveyed at a reference center in Goiânia / GO. Descriptive analyzes were performed using relative and absolute frequency, mean and standard deviation. The test used to evaluate the existence or not of a statistically significant difference (p≤0.05) was the analysis of variance (ANOVA) Scheffé. Prenatal care was the predominant period for the diagnosis of microcephaly. It was evidenced a difficulty for the parents to find reliable people to take care of the child, as well as lack of understanding of others for the burden of caring for the disabled child. Participants who showed a greater tendency to engage in activities with friends, parties and bars were also more prone to physical and leisure activities. After the initial shock of receiving the diagnosis, the parents reorganize and adapt to the challenges, changing the family routine. The involvement of health professionals is essential, providing support and guidance to these families