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.

Sensitivity profile of blood culture isolates in a clinical analysis laboratory, Fortaleza, CE / Perfil de sensibilidade de isolados de hemocultura em um laboratório de análises clínicas, Fortaleza, CE / Perfil de sensibilidad de hemocultivos aislados en un laboratorio de análisis clínicos, Fortaleza, CE

Background and objectives: bacteremia is defined from the presence of bacteria in the bloodstream. Its clinical importance is associated with the high morbidity and mortality rate in the world. In severe cases, it can culminate in sepsis, with a constant increase in cases in Brazil. Therefore, this study aims to assess the main bacterial isolates in blood cultures and a possible change in their sensitivity profiles in a clinical analysis laboratory in Fortaleza, Ceará. Methods: an epidemiological, descriptive, retrospective study was carried out, with a quantitative approach of positive blood cultures, seeking to assess the main isolated microorganisms and their sensitivity profiles. The data used were obtained from the laboratory system through the EpiCenter→ software, from January 2019 to December 2020. Statistical analysis was performed using the Graphpad 7.0 software. Results: 840 microorganisms were identified from blood cultures, and the main ones were E. coli, K. pneumoniae, P. aeruginosa, S. epidermidis, S. aureus and S. haemolyticus. Some isolates show a change in the sensitivity profile, such as K. pneumoniae and P. aeruginosa, showing an increase in sensitivity to carbapenems and cephalosporins, while S. epidermidis showed a decrease in sensitivity to minocycline in the comparison between years 2019 and 2020.Conclusion: clinical isolates from blood cultures showed a change in the sensitivity profile between 2019 and 2020, taking into account that, for K. pneumoniae, P. aeruginosa, this change resulted in an increase in sensitivity, with an increase in resistance in S. epidermidis isolates.(AU)

Justificativa e objetivos: bacteremia é definida a partir da presença de bactérias na corrente sanguínea. Sua importância clínica está associada à alta taxa de morbidade e mortalidade no mundo. Nos casos graves, pode culminar em sepse, com constante aumento dos casos no Brasil. Portanto, o presente estudo tem como objetivo avaliar os principais isolados bacterianos em hemoculturas e uma possível alteração nos seus perfis de sensibilidade em um laboratório de análises clínicas de Fortaleza, Ceará. Métodos: foi realizado um estudo epidemiológico, descritivo, retrospectivo, com abordagem quantitativa de hemoculturas positivas, buscando avaliar os principais microrganismos isolados e seus perfis de sensibilidades. Os dados utilizados foram obtidos a partir do sistema laboratorial através do software EpiCenter→, referente ao período de janeiro de 2019 a dezembro de 2020. A análise estatística foi realizada pelo software Graphpad 7.0. Resultados: foram identificados 840 microrganismos a partir das hemoculturas, sendo os principais E. coli, K. pneumoniae, P. aeruginosa, S. epidermidis, S. aureus e S. haemolyticus. Alguns isolados apresentam uma alteração no perfil de sensibilidade, como K. pneumoniae e P. aeruginosa, apresentando um aumento na sensibilidade frente aos carbapenêmicos e as cefalosporinas, enquanto o S. epidermidis apresentou uma diminuição na sensibilidade frente à minociclina na comparação entre os anos de 2019 e 2020. Conclusão: os isolados clínicos de hemocultura apresentaram uma alteração no perfil de sensibilidade entre 2019 e 2020, levando em consideração que, para K. pneumoniae e P. aeruginosa, essa alteração resultou no aumento na sensibilidade, com aumento na resistência nos isolados de S. epidermidis.(AU)

Justificación y objetivos: la bacteriemia se define por la presencia de bacterias en el torrente sanguíneo. Su importancia clínica está asociada con la alta tasa de morbimortalidad en el mundo. En casos severos, puede culminar en sepsis, con un aumento constante de casos en Brasil. Por tanto, este estudio tiene como objetivo evaluar los principales aislados bacterianos en hemocultivos y un posible cambio en sus perfiles de sensibilidad en un laboratorio de análisis clínicos en Fortaleza, Ceará. Métodos: se realizó un estudio epidemiológico, descriptivo, retrospectivo, con abordaje cuantitativo de hemocultivos positivos, buscando evaluar los principales microorganismos aislados y sus perfiles de sensibilidad. Los datos utilizados se obtuvieron del sistema de laboratorio a través del software EpiCenter→, para el período de enero de 2019 a diciembre de 2020. El análisis estadístico se realizó mediante el software Graphpad 7.0. Resultados: se identificaron 840 microorganismos a partir de hemocultivos, siendo los principales E. coli, K. pneumoniae, P. aeruginosa, S. epidermidis, S. aureus y S. haemolyticus. Algunos aislados muestran un cambio en el perfil de sensibilidad, como K.pneumoniae y P. aeruginosa, mostrando un aumento en la sensibilidad a los carbapenémicos y cefalosporinas, mientras que S. epidermidis mostró una disminución en la sensibilidad a la minociclina, en la comparación entre los años de 2019 y 2020. Conclusiones: los aislados clínicos de hemocultivos mostraron un cambio en el perfil de sensibilidad entre 2019 y 2020, teniendo en cuenta que para K. pneumoniae, P. aeruginosa, este cambio resultó en un aumento de la sensibilidad, con un aumento de la resistencia en los aislados de S. epidermidis

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.

Comparative biofilm-forming ability between Clostridioides difficile strains isolated in Latin America and the epidemic NAP1/027 strain.

Introduction: One of the challenges in treating Clostridioides difficile infection (CDI) is that the bacterium forms biofilms, a critical virulence mechanism known to promote antibiotic resistance and, as a result, consequently, a higher recurrence of the disease. The goal of this study was to compare the ability of three MLST Clade 2 strains to form a biofilm in vitro: ICC-45 (ribotype SLO231/UK[CE]821), a ST41 toxinotype IXb isolated in Brazil; and two epidemic NAP1/027/ST01 strains: NAP1/027/ST01 (LIBA5756), isolated during a 2010 outbreak in Costa Rica and the reference epidemic strain NAP1/027/ST01 (R20291); and ATCC700057, a non-toxigenic strain. Methods: The ability of strains to form biofilm was evaluated using crystal violet staining. In addition, samples were stained with the Film Tracer biofilm matrix (Invitrogen®) and the biofilm matrix thickness was measured using confocal microscopy. The matrix architecture was determined using Scanning electron microscop. Confocal microscopy was used to detect the presence of toxin A (tcdA) using an anti-Clostridioides difficile TcdA antibody. The expression of virulence genes (tcdA, tcdB, tcdC, cdtB, spo0A, slpA, cwp66 and cwp84) was examined, as well as the effect of antibiotics metronidazole (MTZ) and vancomycin (VAN) on biofilm growth. Results: All of the strains tested formed a moderate biofilm with 1.1 3.5. After 72h, biofilm biomass of the NAP1/027/ST01 epidemic strains (LIBA5756 and R20291) was significantly higher than ICC-45 and ATCC 700057 biofilms, as confirmed by electron and confocal microscopy. At 120h, the LIBA5756 biofilm biomass decreased compared to other strains. The toxigenic strains R20291 or LIBA 5756 had higher expression of genes tcdA, tcdB, tcdC, cdtA, slpA and spo0A than ICC-45, but there were no significant differences in the expression levels of cdtB, cwp66 and cwp84. In epidemic strains, VAN and MTZ inhibited biofilm formation; however, in the ICC-45 strain, MIC concentrations of VAN and MIC and 4MIC of MTZ did not inhibit biofilm formation. Conclusion: The three MLST Clade 2 isolated from different rybotipes, two of which were isolated from Latin America, are competent biofilm-forming bacteria, indicating their ability to induce C. difficile infection recurrence, making treatment difficult.

Exoproteomic analysis of two MLST clade 2 strains of Clostridioides difficile from Latin America reveal close similarities.

Clostridioides difficile BI/NAP1/ribotype 027 is an epidemic hypervirulent strain found worldwide, including in Latin America. We examined the genomes and exoproteomes of two multilocus sequence type (MLST) clade 2 C. difficile strains considered hypervirulent: ICC-45 (ribotype SLO231/UK[CE]821), isolated in Brazil, and NAP1/027/ST01 (LIBA5756), isolated during a 2010 outbreak in Costa Rica. C. difficile isolates were cultured and extracellular proteins were analyzed using high-performance liquid chromatography-tandem mass spectrometry. Genomic analysis revealed that these isolates shared most of the gene composition. Only 83 and 290 NAP1/027 genes were considered singletons in ICC-45 and NAP1/027, respectively. Exoproteome analysis revealed 197 proteins, of which 192 were similar in both strains. Only five proteins were exclusive to the ICC-45 strain. These proteins were involved with catalytic and binding functions and indirectly interacted with proteins related to pathogenicity. Most proteins, including TcdA, TcdB, flagellin subunit, and cell surface protein, were overrepresented in the ICC-45 strain; 14 proteins, including mature S-layer protein, were present in higher proportions in LIBA5756. Data are available via ProteomeXchange with identifier PXD026218. These data show close similarity between the genome and proteins in the supernatant of two strains with hypervirulent features isolated in Latin America and underscore the importance of epidemiological surveillance of the transmission and emergence of new strains.

Predictive factors, outcomes, and molecular epidemiology of Clostridioides difficile diarrhea in Brazilian hospitals.

Little is known about the role of lineage of strains of Clostridioides difficile (CD) on the clinical presentation of CD infection (CDI) in Latin America, especially regarding the treatment response. We conducted a multicenter, prospective study to investigate the predictive factors and treatment outcomes of CDI in hospitalized patients and to performed phenotypical and molecular characterization of CD strains. A total of 361 diarrheic patients at 5 hospitals from different regions of the country were enrolled. All stool samples were tested for glutamate dehydrogenase (GDH), toxins A and B, and toxin genes using a nucleic acid amplification test (NAAT). Specimens were cultured and susceptibility profile and whole-genome sequencing (WGS) were performed. CDI positivity was 15% (56/377). Predictive factors for CDI were prior use of meropenem (OR 4.09, 95% CI 2.097-7.095; p<0.001), mucus in stools (OR 3.29; 95% CI 1.406-7.722; p=0.006) and neutrophil left-shift with >20% of bands (OR 3.77; 95% IC 1.280-11.120; p=0.016). Overall mortality was 19%, with no deaths attributed to CDI. Oral metronidazole was used in 74% of cases, with 85% of cure and 14% of recurrence. A total of 35 CD isolates were recovered, all of them susceptible to metronidazole and vancomycin. The WGS revealed 17 different STs, six of which were novel. ST42 was the most common ST and hypervirulent strains were not found. Severe CDI were caused by ST42, ST5, ST8, ST48, ST33 and a novel ST667. The ermB gene was more frequently found in isolates of ST42 (p=0.004).

Inhibitory effect of Brazilian red propolis on planktonic and biofilm forms of Clostridioides difficile.

Clostridioides difficile is a Gram-positive, spore-forming, anaerobic bacillus which is the leading cause of health-care-associated infective diarrhea. The rising incidence of antibiotic resistance in pathogens such as C. difficile makes researches on alternative antibacterial products very important, especially those exploring natural products like propolis. Brazilian Red Propolis, found in the Northeast region of Brazil, is composed by products from regional plants that have the antimicrobial properties. This study aimed to evaluate the in vitro activity of Brazilian Red Propolis (BRP) against C. difficile strains in planktonic and biofilm forms. The susceptibility of four strains of C. difficile to BRP was analyzed by broth microdilution method and vancomycin was included as control drug. BRP-exposed C. difficile cells were evaluated by scanning electron microscopy (SEM). Then, the effects of BRP on growing and mature C. difficile biofilms were also evaluated. BRP minimum inhibitory concentration was 625 µg/mL against all tested strains, while vancomycin MIC range was 0.5-2 µg/mL. SEM showed the loss of homogeneity in bacterial cell wall and cell fragmentation, after BRP-exposure. BRP, at MIC, reduced (P < 0.05) the biomass, matrix proteins and matrix carbohydrates of growing biofilms, and, at 8xMIC, reduced (P < 0.05) the biomass and matrix proteins of mature biofilms. The present study demonstrated that BRP inhibits planktonic growth, damages cell wall, decreases biofilm growth and harms mature biofilms of C. difficile.

Mini-review: from in vitro to ex vivo studies: an overview of alternative methods for the study of medical biofilms.

Microbial biofilms are a natural adaptation of microorganisms, typically composed of multiple microbial species, exhibiting complex community organization and cooperation. Biofilm dynamics and their complex architecture are challenging for basic analyses, including the number of viable cells, biomass accumulation, biofilm morphology, among others. The methods used to study biofilms range from in vitro techniques to complex in vivo models. However, animal welfare has become a major concern, not only in society, but also in the academic and scientific field. Thus, the pursuit for alternatives to in vivo biofilm analyses presenting characteristics that mimic in vivo conditions has become essential. In this context, the present review proposes to provide an overview of strategies to study biofilms of medical interest, with emphasis on alternatives that approximate experimental conditions to host-associated environments, such as the use of medical devices as substrata for biofilm formation, microcosm and ex vivo models.

Prevalence of Clostridioides difficile associated diarrhea in hospitalized patients in five Brazilian centers: A multicenter, prospective study.

Epidemiological data on CD infection (CDI) in Latin American are scarce. CDI prevalence and strains characterization were prospectively evaluated in 5 Brazilian hospitals from different regions. Prevalence rates of CDI were 15%, ranging from 0 to 37%. ST42 was the most common Sequence Type and hypervirulent strains were not identified.

Molecular epidemiology of Clostridium difficile infection in a Brazilian cancer hospital.

Clostridium difficile is a Gram-positive spore forming anaerobic bacterium and the main cause of healthcare-associated diarrhea. This study aimed to perform the phenotypic characterization and molecular typing of Clostridium difficile isolates among patients at a cancer hospital in Brazil. During 18 months, 48 diarrheic fecal samples were collected, of these 48% were positive in either one or both of the performed tests: detection of toxins A/B and culture. Clostridium difficile was recovered from four samples (17%). All strains carried toxin A and B genes, and the isolates belonged to PCR-ribotype 014/020, PGFE-type NAP4 and toxinotype XVIII. On the other hand, one isolate belonged to a novel PCR-ribotype, and PFGE-type, likewise to toxinotype IXb. The isolates showed susceptibility to metronidazole, vancomycin and moxifloxacin, and were resistant to ciprofloxacin. Finally, the findings indicate high positivity between the samples tested, suggesting an expressive importance of this infection, including detection of a novel ribotype/PFGE-type of Clostridium difficile, and show for the first time the detection of community-associated Clostridium difficile infection (CA-CDI) in these patients in Northeast Brazil. These data emphasize the importance to a better understanding of the epidemiological situation of this infection in Brazilian hospitals.

A MLST Clade 2 Clostridium difficile strain with a variant TcdB induces severe inflammatory and oxidative response associated with mucosal disruption.

The epidemiology of Clostridium difficile infections is highly dynamic as new strains continue to emerge worldwide. Here we present a detailed analysis of a new C. difficile strain (ICC-45) recovered from a cancer patient in Brazil that died from severe diarrhea. A polyphasic approach assigned a new PCR-ribotype and PFGE macrorestriction pattern to strain ICC-45, which is toxigenic (tcdA(+), tcdB(+) and ctdB(+)) and classified as ST41 from MLST Clade 2 and toxinotype IXb. Strain ICC-45 encodes for a variant TcdB that induces a distinct CPE in agreement with its toxinotype. Unlike epidemic NAP1/027 strains, which are also classified to MLST Clade 2, strain ICC-45 is susceptible to fluoroquinolones and does not overproduce toxins TcdA and TcdB. However, supernatants from strain ICC-45 and a NAP1/027 strain produced similar expression of pro-inflammatory cytokines, epithelial damage, and oxidative stress response in the mouse ileal loop model. These results highlight inflammation and oxidative stress as common features in the pathogenesis of C. difficile Clade 2 strains. Finally, this work contributes to the description of differences in virulence among various C. difficile strains.