Anti-Leishmania effect of icetexanes from Salvia procurrens.

BACKGROUND AND PURPOSE: Leishmaniasis is a globally prevalent vector-borne disease caused by parasites of the genus Leishmania. The available chemotherapeutic drugs present problems related to efficacy, emergence of parasite resistance, toxicity and high cost, justifying the search for new drugs. Several classes of compounds have demonstrated activity against Leishmania, including icetexane-type diterpenes, previously isolated from Salvia and other Lamiaceae genera. Thus, in this study, compounds of Salvia procurrens were investigated for their leishmanicidal and immunomodulatory activities. METHODS: The exudate of S. procurrens was obtained by rapidly dipping the aerial parts in dichloromethane. The compounds were isolated by column and centrifugal planar chromatography over silica gel. The effects on L. amazonensis growth, survival, membrane integrity, reactive oxygen species (ROS) generation, mitochondrial membrane potential and cytotoxicity of the compounds towards human erythrocytes, peripheral blood mononuclear cells and macrophages were evaluated. The effects on intracellular amastigote forms, nitric oxide (NO) and TNF-α production were also investigated. RESULTS: The exudate from the leaves afforded the novel icetexane 7-hydroxyfruticulin A (1) as well as the known demethylisofruticulin A (2), fruticulin A (3) and demethylfruticulin A (4). The compounds (1-4) were tested against promastigotes of L. amazonensis and showed an effective inhibition of the parasite survival (IC50 = 4.08-16.26 µM). In addition, they also induced mitochondrial ROS production, plasma membrane permeability and mitochondrial dysfunction in treated parasites, and presented low cytotoxicity against macrophages. Furthermore, all diterpenes tested reduced the number of parasites inside macrophages, by mechanisms involving TNF-α, NO and ROS. CONCLUSION: The results suggest the potential of 7-hydroxyfruticulin A (1) as well as the known demethylisofruticulin A (2),fruticulin A (3) and demethylfruticulin A (4) as candidates for use in further studies on the design of anti-leishmanial drugs.

Resistance profile to antimicrobials agents in methicillin-resistant Staphylococcus aureus isolated from hospitals in South Brazil between 2014-2019.

INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA) is a common pathogen causing healthcare-associated infections. Owing to the restricted use of beta-lactams in MRSA infections, non-beta-lactam antimicrobials are required for treatment. However, MRSA can develop resistance mechanisms to non-beta-lactam antimicrobials, which reduces viable treatment options. Here, we evaluated the antimicrobial susceptibility and resistance genes of MRSA isolated from hospitalized patients in South Brazil. METHODS: The antimicrobial susceptibilities of hospital MRSA (217) isolates were determined by disk diffusion or microdilution methods. Additionally, the presence of 14 resistance genes and SCCmec typing was performed by PCR. RESULTS: Among the antimicrobials tested, we observed high erythromycin (74.2%), ciprofloxacin (64.5%), and clindamycin (46.1%) resistance rates and complete susceptibility to linezolid and vancomycin. Seventeen different patterns of MRSA antimicrobial resistance were observed, of which 42.9% represented multidrug resistance. Among erythromycin-resistant MRSA, 53.4%, 45.3%, 37.9%, 13.0%, and 6.8% carried ermA, msrA, msrB, ermC, and ermB genes, respectively. Among clindamycin-resistant MRSA, 83%, 17%, 10%, 4%, and 2% carried ermA, ermC, ermB, linA, and linB genes, respectively. Among gentamicin-resistant MRSA, 96.8%, 83.9%, and 9.7% carried aac(6')/aph(2''), aph(3')-IIIa, and ant(4')-Ia genes, respectively. Among tetracycline-resistant MRSA, 6.5% and 93.5% carried tetK and tetM genes, respectively. Lastly, among trimethoprim/sulfamethoxazole-resistant MRSA, 13.3% and 100% carried dfrA and dfrG genes, respectively. The SCCmec type IV isolates were detected more frequently, whereas the SCCmec type III isolates exhibited higher multidrug resistance. CONCLUSIONS: The study data provides information regarding the MRSA resistance profile in South Brazil that is associated with the clinical conditions of patients and can contribute to clinical decision-making.

Systematic review of descriptions of novel bacterial species: evaluation of the twenty-first century taxonomy through text mining.

Although described bacterial species increased in the twenty-first century, they correspond to a tiny fraction of the actual number of species living on our planet. The volume of textual data of these descriptions constitutes valuable information for revealing trends that in turn could support strategies for improvement of bacterial taxonomy. In this study, a text mining approach was used to generate bibliometric data to verify the state-of-art of bacterial taxonomy. Around 9700 abstracts of bacterial classification containing the expression 'sp. nov.' and published between 2001 and 2018 were downloaded from PubMed and analysed. Most articles were from PR China and the Republic of Korea, and published in the International Journal of Systematic and Evolutionary Microbiology. From about 10 800 species names detected, 93.33 % were considered valid according to the rules of the Bacterial Code, and they corresponded to 82.98 % of the total number of species validated between 2001 and 2018. Streptomyces, Bacillus and Paenibacillus each had more than 200 species described in the period. However, almost 40 % of all species were from the phylum Proteobacteria. Most bacteria were Gram-stain-negative, bacilli and isolated from soil. Thirteen species and one genus homonyms were found. With respect to methodologies of bacterial characterization, the use of terms related to 16S rRNA and polar lipids increased along these years, and terms related to genome metrics only began to appear from 2009 onward, although at a relatively lower frequency. Bacterial taxonomy is known as a conservative discipline, but it gradually changed in terms of players and practices. With the advent of the mandatory use of genomic analyses for species description, we are probably witnessing a turning point in the evolution of bacterial taxonomy.

Resistance profile to antimicrobials agents in methicillin-resistant Staphylococcus aureus isolated from hospitals in South Brazil between 2014-2019

Abstract INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA) is a common pathogen causing healthcare-associated infections. Owing to the restricted use of beta-lactams in MRSA infections, non-beta-lactam antimicrobials are required for treatment. However, MRSA can develop resistance mechanisms to non-beta-lactam antimicrobials, which reduces viable treatment options. Here, we evaluated the antimicrobial susceptibility and resistance genes of MRSA isolated from hospitalized patients in South Brazil. METHODS: The antimicrobial susceptibilities of hospital MRSA (217) isolates were determined by disk diffusion or microdilution methods. Additionally, the presence of 14 resistance genes and SCCmec typing was performed by PCR. RESULTS: Among the antimicrobials tested, we observed high erythromycin (74.2%), ciprofloxacin (64.5%), and clindamycin (46.1%) resistance rates and complete susceptibility to linezolid and vancomycin. Seventeen different patterns of MRSA antimicrobial resistance were observed, of which 42.9% represented multidrug resistance. Among erythromycin-resistant MRSA, 53.4%, 45.3%, 37.9%, 13.0%, and 6.8% carried ermA, msrA, msrB, ermC, and ermB genes, respectively. Among clindamycin-resistant MRSA, 83%, 17%, 10%, 4%, and 2% carried ermA, ermC, ermB, linA, and linB genes, respectively. Among gentamicin-resistant MRSA, 96.8%, 83.9%, and 9.7% carried aac(6')/aph(2''), aph(3')-IIIa, and ant(4')-Ia genes, respectively. Among tetracycline-resistant MRSA, 6.5% and 93.5% carried tetK and tetM genes, respectively. Lastly, among trimethoprim/sulfamethoxazole-resistant MRSA, 13.3% and 100% carried dfrA and dfrG genes, respectively. The SCCmec type IV isolates were detected more frequently, whereas the SCCmec type III isolates exhibited higher multidrug resistance. CONCLUSIONS: The study data provides information regarding the MRSA resistance profile in South Brazil that is associated with the clinical conditions of patients and can contribute to clinical decision-making.

Evaluation of Kindlin-1 and Ki-67 immunohistochemical expression in primary cutaneous malignant melanoma: a clinical series

Background: The capacity for prognostic prediction of cutaneous melanoma, one of the most aggressive cancers, is still difficult due to the tumor heterogeneity and lack of reliable tumor markers. The objective of this study is to correlate, through immunohistochemistry, a Ki-67 and Kindlin-1 staining in malignant melanomas with the prognosis of the disease. Methods: A historical cohort study. Immunohistochemistry, using mouse anti-human Kindlin-1 and Ki-67 monoclonal antibodies, was performed using tissue blocks from primary cutaneous melanoma patients treated between 2006 and 2014 at our institution. Information regarding pathological data and outcomes were retrieved from medical records. Statistical analyses were conducted in SPSS version 18.0. Results: Thirty patients were included. The median age was from 50.93 ± 15.31 years old. The expression of Ki-67 was detected in all patients with primary cutaneous melanoma, while Kindlin-1 was negative in two. Kindlin expression was not significantly correlated with Ki-67 expression by Spearman's rank correlation analysis (P = 0.46), as well as the expression of both markers and the clinical stage (P = 0.34 and 0.18, respectively). Breslow, Clark and mitotic rate were significantly correlated with AJCC stage (P = 0.001). Conclusion: Other studies investigating clinical evolution are needed to further test the potential of these markers as possible prognostic markers (AU)

Características moleculares de Staphylococcus aureus susceptível à vancomicina poderia ajudar a prever falhas no tratamento devido a reduzida susceptibilidade à vancomicina / Molecular characteristics of vancomycin-susceptible Staphylococcus aureus could help to predict treatment failure due to reduced vancomycin susceptibility / Características moleculares de Staphylococcus aureus susceptible a la vancomicina podría ayudar a predecir fallas en el tratamiento debido a la reducida susceptibilidad a la vancomicina

Justificativa e Objetivos: Staphylococcus aureus resistente à meticilina (MRSA) é uma das causas mais frequentes de infecções relacionadas à assistência à saúde e comunitárias, e com seu avanço, a vancomicina tornou-se a principal opção terapêutica. Entretanto, o seu uso indiscriminado favoreceu o surgimento de MRSA com reduzida suscetibilidade à vancomicina, comumente associados com falhas no tratamento, bacteremia persistente, hospitalização prolongada e desfechos clínicos adversos. Este estudo avaliou a ocorrência de MRSA com reduzida suscetibilidade à vancomicina e determinou algumas características moleculares em comparação com MRSA suscetível à vancomicina (VS-MRSA). Métodos: Determinação do perfil de suscetibilidade aos antimicrobianos, a concentração inibitória mínima (CIM) e concentração bactericida mínima (CBM) para vancomicina, tolerância à vancomicina, tipagem do SCCmec e agr foram realizadas em um total de 177 MRSA. Posteriormente, foram triados para hVISA por BHIA-3V e BHIA-6V e confirmados com a Análise do Perfil Populacional - Área Abaixo da Curva (PAP-AUC). Resultados: Os fenótipos VT-MRSA e hVISA foram encontrados em 13,6% e 5,1% dos isolados clínicos de MRSA, respectivamente, e a presença de hVISA foi estatisticamente significativa entre os isolados de VT-MRSA (p<0,05). Em VT-MRSA, SCCmec tipo II foi significativamente mais frequente do que em não-VT-MRSA, assim como a presença do agr grupo II. Conclusão: Características moleculares encontradas em MRSA são importantes para a epidemiologia, bem como para demonstrar um padrão em isolados com reduzida suscetibilidade à vancomicina. Testes não-convencionais para detecção destas características podem ser realizados para evitar a identificação errada de VS-MRSA que, consequentemente, resulta em falhas no tratamento com vancomicina.(AU)

Background and Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most frequent causes of healthcare-associated and community-acquired infections and with its advancement, vancomycin became the main therapeutic option. However, its indiscriminate use favored the emergence of MRSA with reduced susceptibility to vancomycin, commonly associated with vancomycin treatment failure, persistent bacteremia, prolonged hospitalization and adverse clinical outcome. This study evaluated the occurrence of MRSA with reduced vancomycin susceptibility and determined some molecular characteristics in comparison with vancomycin-susceptible MRSA (VS-MRSA). Methods: Determination of antimicrobial susceptibility profile, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for vancomycin, vancomycin-tolerance, SCCmec and agr typing were performed in a total of 177 MRSA. Thereafter, they were screened for hVISA by BHIA-3V and BHIA-6V and confirmed with population analysis profile - area under the curve method (PAP-AUC). Results: VT-MRSA and hVISA phenotypes were found in 13.6% and 5.1% of clinical isolates of MRSA, respectively, and the presence of hVISA was statistically significant among VT-MRSA isolates (p<0.05). In T-MRSA, SCCmec type II was significantly more frequent than in non-VT-MRSA, as well as the presence of agr group II. Conclusion: Molecular characteristics found in MRSA are important for epidemiology, as well as demonstrate a pattern in reduced vancomycin susceptibility isolates. Non-conventional tests for detection of these characteristics might be performed to prevent misidentification of VS-MRSA that, consequently, results in vancomycin treatment failures.(AU)

Justificación y objetivos: Staphylococcus aureus resistente a la meticilina (MRSA) es una de las causas más frecuentes de infecciones relacionadas con la asistencia sanitaria y comunitarias, y con su avance, a la vancomicina se ha convertido en la principal opción terapéutica. Sin embargo, su uso indiscriminado favoreció el surgimiento de MRSA con reducida susceptibilidad a la vancomicina, comúnmente asociados con fallas en el tratamiento, bacteriemia persistente, hospitalización prolongada y resultados clínicos adversos. Este estudio evaluó la ocurrencia de MRSA con reducida susceptibilidad a la vancomicina y determinó algunas características moleculares en comparación con MRSA susceptible a la vancomicina (VS-MRSA). Métodos: Determinación del perfil de susceptibilidad a los antimicrobianos, la concentración inhibitoria mínima (CIM) y la concentración bactericida mínima (CBM) para vancomicina, tolerancia a la vancomicina, tipificación del SCCmec y agr se realizaron en un total de 177 MRSA. Resultados: Los fenotipos VT-MRSA y hVISA se encontraron en el 13,6% y el 5,1% de los aislados clínicos de MRSA, respectivamente, y la presencia de hVISA fue estadísticamente significativa entre los aislados de VT-MRSA (p<0.05). En VT-MRSA, SCCmec tipo II fue significativamente más frecuente que en no-VT-MRSA, así como la presencia del agr grupo II. Conclusión: Características moleculares encontradas en MRSA son importantes para la epidemiología, así como para demostrar un patrón en aislados con reducida susceptibilidad a la vancomicina. Pruebas no convencionales para la detección de estas características pueden realizarse para evitar la identificación errónea de VS-MRSA que, consecuentemente, resulta en fallas en el tratamiento con vancomicina.(AU)

Coexistence of virulence genes in methicillin-resistant Staphylococcus aureus clinical isolates.

INTRODUCTION: The pathogenic versatility of Staphylococcus aureus is attributed to various virulence genes, including enterotoxins and hemolysins. METHODS: Here, the virulence genes in 177 nosocomial MRSA strains in Porto Alegre, Brazil were detected by PCR. RESULTS: The overall prevalence rates were as follows: sea, 4.5%; pvl, 18.6%; tst, 27.7%; hla, 87.6%; and hld, 90.4%. No strain contained all tested genes. However, there was frequent coexistence of tst with pvl and hla with hld (40.7% and 26.6%, respectively). CONCLUSIONS: Horizontal transfer of virulence genes is very common in S. aureus, as suggested by the frequent coexistence of several virulence genes.

Coexistence of virulence genes in methicillin-resistant staphylococcus aureus clinical isolates

Abstract INTRODUCTION: The pathogenic versatility of Staphylococcus aureus is attributed to various virulence genes, including enterotoxins and hemolysins. METHODS: Here, the virulence genes in 177 nosocomial MRSA strains in Porto Alegre, Brazil were detected by PCR. RESULTS: The overall prevalence rates were as follows: sea, 4.5%; pvl, 18.6%; tst, 27.7%; hla, 87.6%; and hld, 90.4%. No strain contained all tested genes. However, there was frequent coexistence of tst with pvl and hla with hld (40.7% and 26.6%, respectively). CONCLUSIONS: Horizontal transfer of virulence genes is very common in S. aureus, as suggested by the frequent coexistence of several virulence genes.

Coagulase-negative staphylococci in Southern Brazil: looking toward its high diversity.

INTRODUCTION: Coagulase-negative staphylococci (CoNS) are the most prevalent pathogens in nosocomial infections and may serve as a reservoir of mobile genetic elements such as the staphylococcal cassette chromosome mec (SCCmec) encoding methicillin resistance. Molecular characterization of SCCmec types combined with advanced molecular typing techniques may provide essential information for understanding the evolution and epidemiology of CoNS infections. We therefore aimed to investigate the SCCmec distribution, multidrug-resistance (MDR), and biofilm formation in CoNS blood culture isolates from a hospital in Southern Brazil. METHODS: We analyzed 136 CoNS blood culture isolates obtained during 2002-2004 from patients admitted to a tertiary care hospital in Brazil. SCCmec types I to V were determined using multiplex PCR. The clonal relationship of Staphylococcus epidermidis was determined using pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Molecular epidemiological data were interpreted along with data on biofilm formation, presence of the icaD gene, and MDR. RESULTS: The most prevalent species were S. epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis harboring mainly SCCmec types II, III, and V. Overall, the presence of multiple SCCmec was associated with non-MDR, except for S. epidermidis. S. epidermidis isolates showed a high prevalence of icaD, but had low phenotypic biofilm formation. PFGE and MLST revealed high genetic diversity in the S. epidermidis population. CONCLUSIONS: Our results suggest a major shift in SCCmec types within a short period and reveal a different behavior of S. epidermidis with regard to the association between the presence of multiple SCCmec types and MDR profile.

Coagulase-negative staphylococci in Southern Brazil: looking toward its high diversity

Abstract: INTRODUCTION: Coagulase-negative staphylococci (CoNS) are the most prevalent pathogens in nosocomial infections and may serve as a reservoir of mobile genetic elements such as the staphylococcal cassette chromosome mec (SCCmec) encoding methicillin resistance. Molecular characterization of SCCmec types combined with advanced molecular typing techniques may provide essential information for understanding the evolution and epidemiology of CoNS infections. We therefore aimed to investigate the SCCmec distribution, multidrug-resistance (MDR), and biofilm formation in CoNS blood culture isolates from a hospital in Southern Brazil. METHODS: We analyzed 136 CoNS blood culture isolates obtained during 2002-2004 from patients admitted to a tertiary care hospital in Brazil. SCCmec types I to V were determined using multiplex PCR. The clonal relationship of Staphylococcus epidermidis was determined using pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Molecular epidemiological data were interpreted along with data on biofilm formation, presence of the icaD gene, and MDR. RESULTS: The most prevalent species were S. epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis harboring mainly SCCmec types II, III, and V. Overall, the presence of multiple SCCmec was associated with non-MDR, except for S. epidermidis. S. epidermidis isolates showed a high prevalence of icaD, but had low phenotypic biofilm formation. PFGE and MLST revealed high genetic diversity in the S. epidermidis population. CONCLUSIONS: Our results suggest a major shift in SCCmec types within a short period and reveal a different behavior of S. epidermidis with regard to the association between the presence of multiple SCCmec types and MDR profile.

Evaluation of four different DNA extraction methods in coagulase-negative staphylococci clinical isolates.

Currently there are several methods to extract bacterial DNA based on different principles. However, the amount and the quality of the DNA obtained by each one of those methods is highly variable and microorganism dependent, as illustrated by coagulase-negative staphylococci (CoNS) which have a thick cell wall that is difficult to lyse. This study was designed to compare the quality and the amount of CoNS DNA, extracted by four different techniques: two in-house protocols and two commercial kits. DNA amount and quality determination was performed through spectrophotometry. The extracted DNA was also analyzed using agarose gel electrophoresis and by PCR. 267 isolates of CoNS were used in this study. The column method and thermal lyses showed better results with regard to DNA quality (mean ratio of A260/280 = 1.95) and average concentration of DNA (), respectively. All four methods tested provided appropriate DNA for PCR amplification, but with different yields. DNA quality is important since it allows the application of a large number of molecular biology techniques, and also it's storage for a longer period of time. In this sense the extraction method based on an extraction column presented the best results for CoNS.

Upregulation of icaA, atlE and aap genes by linezolid but not vancomycin in Staphylococcus epidermidis RP62A biofilms.

Biofilms are complex bacterial structures protected by a self-produced polymer matrix that enables survival in hostile environments. Biofilms are more resistant to antibiotics than their planktonic counterparts and are therefore more difficult to eradicate. The aim of this study was to investigate the influence of vancomycin and linezolid on the maintenance of staphylococcal biofilms and their effect on the expression of biofilm-associated genes in Staphylococcus epidermidis. Pre-formed biofilms of S. epidermidis RP62A were challenged with linezolid and vancomycin at different concentrations as well as at their clinically relevant target concentration (15 mg/L) over time. Expression of icaA, atlE, aap, rnaIII, luxS, sarA, rsbU and icaR genes following 2h of exposure to these antibiotics was determined by quantitative PCR. Vancomycin did not significantly affect the biofilm under the tested conditions. However, linezolid affected the biofilm structure at concentrations of ≥ 2 mg/L (P<0.05); moreover, the exposure time to this antibiotic was a determinant for biofilm eradication. The level of transcription of icaA, aap and atlE increased by 5.18-, 2.58- and 3.06-fold, respectively, in biofilms exposed to linezolid, but no changes were observed for vancomycin. The other genes were not affected by these antibiotics. This study demonstrated that linezolid was effective in eradicating biofilms formed by S. epidermidis RP62A. Under the conditions tested, linezolid upregulated biofilm-associated genes probably due to the stress caused by low-dose antibiotic stimulation. In this study, linezolid showed better performance than vancomycin against staphylococcal biofilms.

Evaluation of fuur different dna extraction metods in coagulase-negative staphhylococci clinical isolates / Avaliacao de quatro metodos diferentes de extracao de DNA em isolados clinicos de estafilococos coagulase negativos (SCoN)

Currently there are several methods to extract bacterial DNA based on different principles. However, the amount and the quality of the DNA obtained by each one of those methods is highly variable and microorganism dependent, as illustrated by coagulase-negative staphylococci (CoNS) which have a thick cell wall that is difficult to lyse. This study was designed to compare the quality and the amount of CoNS DNA, extracted by four different techniques: two in-house protocols and two commercial kits. DNA amount and quality determination was performed through spectrophotometry. The extracted DNA was also analyzed using agarose gel electrophoresis and by PCR. 267 isolates of CoNS were used in this study. The column method and thermal lyses showed better results with regard to DNA quality (mean ratio of A260/280 = 1.95) and average concentration of DNA (), respectively. All four methods tested provided appropriate DNA for PCR amplification, but with different yields. DNA quality is important since it allows the application of a large number of molecular biology techniques, and also it's storage for a longer period of time. In this sense the extraction method based on an extraction column presented the best results for CoNS.

Atualmente, para extrair o DNA bacteriano, existem diversos métodos baseados em diferentes princípios. Entretanto, a quantidade e qualidade do DNA obtido por cada um destes métodos é variável e depende do tipo de micro-organismo em questão; os estafilococos coagulase-negativos (CoNS), por exemplo, possuem parede celular espessa difícil de lisar. O objetivo deste estudo foi comparar a quantidade e a qualidade do DNA extraído de isolados clínicos de CoNS utilizando quatro metodologias diferentes: dois protocolos caseiros e dois kits comerciais. A determinação da quantidade e da qualidade do DNA foi realizada por espectrofotometria. O DNA extraído também foi analisado em eletroforese em gel de agarose e por PCR. A concentração média de DNA foi mais alta no método de lise térmica (). Entretanto, com relação à qualidade do DNA, o kit comercial que utiliza um método de extração baseado em uma coluna de separação apresentou melhor resultado (média da relação A260/280 = 1,95). As quatro técnicas testadas forneceram DNA passível de amplificação por PCR, porém com diferentes rendimentos. A qualidade do DNA extraído de bactérias é importante, pois possibilita a realização de maior número de técnicas de biologia molecular e também armazenamento do material por maior período de tempo. Neste sentido, a técnica de extração por coluna de separação apresentou melhor desempenho frente aos CoNS.

Inhibition of biofilm maturation by linezolid in meticillin-resistant Staphylococcus epidermidis clinical isolates: comparison with other drugs.

Biofilm resistance mechanisms are multifactorial and vary from one organism to another. The purpose of this study was to investigate the efficacy of linezolid against indwelling device-related meticillin-resistant Staphylococcus epidermidis (MRSE) biofilm, and compare this with other antimicrobials. MICs, minimum biofilm inhibitory concentrations (MBICs) and minimum biofilm eradication concentrations (MBECs) were determined by the microtitre plate method. Fourteen and thirteen isolates from patients with indwelling device-related bacteraemia (IDB) and indwelling device colonization not associated with bacteraemia, respectively, were assessed. High MBIC was associated with a high intensity of biofilm formation (gentamicin r=0.796; linezolid r=0.477; rifampicin r=0.634; tigecycline r=0.410; and vancomycin r=0.771), but this correlation was not observed with MBEC. Linezolid demonstrated better in vitro antimicrobial activity than other antimicrobials (MBIC - gentamicin P<0.001, rifampicin P=0.019, vancomycin P=0.008; MBEC - gentamicin P<0.001, rifampicin P=0.002, vancomycin P<0.001). Biofilm growth inhibition was strongly associated with biofilm formation intensity; however, biofilm eradication was not cell number dependent. MRSE biofilm eradication would represent a huge advance for IDB, although high concentrations of gentamicin, linezolid, rifampicin, tigecycline and vancomycin were required for that. In general, linezolid reached better in vitro concentrations and was demonstrated to be highly active against MRSE biofilms by inhibiting their growth during biofilm formation.

Rifampicin fails to eradicate mature biofilm formed by methicillin-resistant Staphylococcus aureus.

INTRODUCTION: Antimicrobial activity on biofilms depends on their molecular size, positive charges, permeability coefficient, and bactericidal activity. Vancomycin is the primary choice for methicillin-resistant Staphylococcus aureus (MRSA) infection treatment; rifampicin has interesting antibiofilm properties, but its effectivity remains poorly defined. METHODS: Rifampicin activity alone and in combination with vancomycin against biofilm-forming MRSA was investigated, using a twofold serial broth microtiter method, biofilm challenge, and bacterial count recovery. RESULTS: Minimal inhibitory concentration (MIC) and minimal bactericidal concentration for vancomycin and rifampicin ranged from 0.5 to 1mg/l and 0.008 to 4mg/l, and from 1 to 4mg/l and 0.06 to 32mg/l, respectively. Mature biofilms were submitted to rifampicin and vancomycin exposure, and minimum biofilm eradication concentration ranged from 64 to 32,000 folds and from 32 to 512 folds higher than those for planktonic cells, respectively. Vancomycin (15mg/l) in combination with rifampicin at 6 dilutions higher each isolate MIC did not reach in vitro biofilm eradication but showed biofilm inhibitory capacity (1.43 and 0.56log10 CFU/ml reduction for weak and strong biofilm producers, respectively; p<0.05). CONCLUSIONS: In our setting, rifampicin alone failed to effectively kill biofilm-forming MRSA, demonstrating stronger inability to eradicate mature biofilm compared with vancomycin.

Rifampicin fails to eradicate mature biofilm formed by methicillin-resistant Staphylococcus aureus / Rifampicina falha na erradicação de biofilmes maduros formados por Staphylococcus aureus resistentes à meticilina

INTRODUCTION: Antimicrobial activity on biofilms depends on their molecular size, positive charges, permeability coefficient, and bactericidal activity. Vancomycin is the primary choice for methicillin-resistant Staphylococcus aureus (MRSA) infection treatment; rifampicin has interesting antibiofilm properties, but its effectivity remains poorly defined. METHODS: Rifampicin activity alone and in combination with vancomycin against biofilm-forming MRSA was investigated, using a twofold serial broth microtiter method, biofilm challenge, and bacterial count recovery. RESULTS: Minimal inhibitory concentration (MIC) and minimal bactericidal concentration for vancomycin and rifampicin ranged from 0.5 to 1mg/l and 0.008 to 4mg/l, and from 1 to 4mg/l and 0.06 to 32mg/l, respectively. Mature biofilms were submitted to rifampicin and vancomycin exposure, and minimum biofilm eradication concentration ranged from 64 to 32,000 folds and from 32 to 512 folds higher than those for planktonic cells, respectively. Vancomycin (15mg/l) in combination with rifampicin at 6 dilutions higher each isolate MIC did not reach in vitro biofilm eradication but showed biofilm inhibitory capacity (1.43 and 0.56log10 CFU/ml reduction for weak and strong biofilm producers, respectively; p<0.05). CONCLUSIONS: In our setting, rifampicin alone failed to effectively kill biofilm-forming MRSA, demonstrating stronger inability to eradicate mature biofilm compared with vancomycin.

INTRODUÇÃO: A atividade dos antimicrobianos em biofilmes depende do seu peso molecular, de cargas positivas, coeficiente de permeabilidade e atividade bactericida. Vancomicina é a escolha primária para o tratamento de infecções causadas por Staphylococcus aureus resistentes à meticilina (MRSA) e rifampicina possui interessante propriedade antibiofilme, apesar da sua efetividade ainda ser fracamente definida. MÉTODOS: Foi investigada a atividade da rifampicina sozinha e em combinação com vancomicina frente à MRSA formadores de biofilme, utilizando o método das microplacas com diluição seriada e recuperação bacteriana em biofilme após exposição antimicrobiana. RESULTADOS: Concentração inibitória minima (MIC) e concentração bactericida mínima (MBC) para vancomicina e rifampicina foi de 0,5-1mg/l e 0,008-4mg/l; 1-4mg/l e 0,06-32mg/l, respectivamente. Biofilmes maduros foram expostos à vancomicina e rifampicina, e a concentração mínima para erradicar o biofilme (MBEC) foi 64-32.000 e 32-512 vezes maior do que para células planctônicas, respectivamente. A combinação de vancomicina (15mg/l) com rifampicina (6-diluições maior do que o MIC de cada isolado) não atingiu erradicação do biofilme in vitro, porém apresentou capacidade inibitória do biofilme formado (redução de 1,43 e 0,56log10 UFC/ml para produtores fracos e fortes, respectivamente; p<0,05). CONCLUSÕES: Rifampicina sozinha falhou em efetivamente matar MRSA formadores de biofilme, demonstrando fraca habilidade para erradicação de biofilmes maduros comparado com vancomicina.

Enhancement of antistaphylococcal activities of six antimicrobials against sasG-negative methicillin-susceptible Staphylococcus aureus: an in vitro biofilm model.

This study was designed to evaluate antimicrobial activities against methicillin-susceptible Staphylococcus aureus in both sessile and planktonic forms and to detect genes associated with this biofilm phenotype. Minimal biofilm inhibition and eradication concentrations (MBIC and MBEC, respectively) were determined by an in vitro biofilm model, and icaA, atlA, and sasG genes were detected by polymerase chain reaction. Vancomycin and tigecycline presented better biofilm inhibitory activity (MBIC range: 4-8 µg/mL) (P ≤ 0.05) and lower MBEC/MIC ratios (P ≤ 0.001) than other antimicrobials. All isolates harbored icaA and atlA, whereas sasG was present only in strong biofilm formers (P ≤ 0.05). Interestingly, antimicrobial activities against sasG- weak biofilm formers were significantly higher than those against sasG+ strong biofilm formers (P ≤ 0.05), demonstrating that number of cells in a biofilm matrix affected the antimicrobial activity, which was also variable, and might be associated with specific genetic determinants. To our knowledge, this was the first study reporting the presence of sasG in clinical isolates of S. aureus in South America.

High biofilm production by invasive multiresistant staphylococci.

Biofilm-forming staphylococci are known for being opportunistic and invasive pathogens that cause severe disease, mostly catheter-related infections. Early detection and pathogenic strains carrying highly transferable resistance cassettes epidemiology are essential for infection spread control. Hence, this study was designed to evaluate staphylococci biofilm formation and SCCmec typing. Biofilm production and SCCmec typing were evaluated using a semi-quantitative method based on microtiter plates and a multiplex PCR for types, I-V, respectively. Blood cultures and peripheral intravenous device (IVD) staphylococci were consecutively enrolled and allocated into two different groups (invasive and colonizing) based on clinical and microbiological criteria. Seventy-four invasive and 30 colonizing isolates from distinct patients were studied. Vancomycin was the most administrated antimicrobial agent among these patient's treatments. Biofilm formation was observed in 89% of invasive and 64% of colonizing isolates (p < 0.05). There was significant difference regarding SCCmec typing between colonizing and invasive isolates when harboring SCCmec types IV or V (p < 0.05), but no correlation between biofilm intensity and SCCmec types was verified. The SCCmec elements spread are still ongoing and for that reason, antimicrobial resistance evolution in invasive and colonizing biofilm-forming staphylococci is highly relevant.

Distribution of erm genes and low prevalence of inducible resistance to clindamycin among staphylococci isolates

INTRODUCTION: Resistance to macrolides, lincosamides and streptogramins B (MLS B antibiotics) in staphylococci may be due to modification in ribosomal target methylase encoded by erm genes. The expression of MLS B resistance lead to three phenotypes, namely constitutive resistance (cMLS B), inducible resistance (iMLS B), and resistance only to macrolides and streptogramins B (MS B). The iMLS B resistance is the most difficult to detect in the clinical laboratory. OBJECTIVE: This study investigated the expression of MLS B resistance and the prevalence of the erm genes among 152 clinical isolates of Staphylococcus aureus and coagulase-negative Staphylococcus (CNS) from Hospital de Clínicas de Porto Alegre. METHODS: Primary MLS B resistance was detected by the disk diffusion method. Isolates with iMLS B phenotype were tested by double-disk induction method. All isolates were tested by a genotypic assay, PCR with specific primers. RESULTS: A total of 46.7 percent of staphylococci were positive for cMLS B; 3.3 percent for iMLS B and 3.3 percent for MS B. One or more erm genes were present in 50.1 percent of isolates. The gene ermA was detected in 49 isolates, ermC in 29 and ermB in 3. CONCLUSION: The prevalence of the ermA, ermB and ermC genes were 29.6 percent, 17.1 percent and 0.66 percent respectively, and constitutive resistance was the most frequent as compared to the other two phenotypes.

High prevalence of methicillin-resistant Staphylococcus aureus with SCCmec type III in cystic fibrosis patients in southern, Brazil.

INTRODUCTION: Bacterial colonization of the lungs is the main cause of morbidity in cystic fibrosis (CF). Pathogens such as Staphylococcus aureus are very well adapted to the pulmonary environment and may persist for years in the same patient. Genetic determinants of these bacteria, such as the presence of SCCmec have recently emerged as a problem in this population of patients. METHODS: Staphylococcus aureus isolates obtained from different clinical materials coming from CF and non-CF patients attended at a cystic fibrosis reference hospital were compared according to SCCmec type and antibiotic susceptibility profile. RESULTS: Three hundred and sixty-four single-patient Staphylococcus aureus isolates were collected, of which 164 (45%) were from CF patients. Among the latter, 57/164 (44.5%) were MRSA, and among the non-CF patients, 89/200 (35%) were MRSA. Associated pathogens were found in 38 CF patients. All 57 MRSA from CF patients harbored the multiresistant cassette type III. In contrast, 31/89 MRSA from non-CF patients harbored SCCmec type I (35%) and 44/89 harbored type III (49%). The antibiotic susceptibility pattern was similar between CF and non-CF patients. CONCLUSIONS: The high prevalence of multiresistant SCCmec type III among CF patients compared with non-CF patients in our institution may make it difficult to control disease progression through antibiotic therapy for promoting the survival of this kind of patient.