Comparison of two MALDI-TOF MS systems for theidentification of clinically relevant anaerobic bacteriain Argentina

Abstract The aim of this study was to compare the performance of two MALDI-TOF MS systems in the identification of clinically relevant strict anaerobic bacteria. The 16S rRNA gene sequencing was the gold standard method when discrepancies or inconsistencies were observed between platforms. A total of 333 isolates were recovered from clinical samples of different centers in Buenos Aires City between 2016 and 2021. The isolates were identified in duplicate using two MALDI-TOF MS systems, BD Bruker Biotyper (Bruker Daltonics, Bremen, Germany) and Vitek MS (bioMèrieux, Marcy-l'Etoile, France). Using the Vitek MS system, the identification of anaerobic isolates yielded the following percentages: 65.5% (n: 218) at the species or species-complex level, 71.2% (n: 237) at the genus level, 29.4% (n: 98) with no identification and 5.1% (n: 17) with misidentification. Using the Bruker Biotyper system, the identification rates were as follows: 85.3% (n: 284) at the species or species-complex level, 89.7% (n: 299) at the genus level, 14.1% (n: 47) with no identification and 0.6% (n: 2) with misidentification. Differences in the performance of both methods were statistically significant (p-values <0.0001). In conclusion, MALDI-TOF MS systems speed up microbial identification and are particularly effective for slow-growing microorganisms, such as anaerobic bacteria, which are difficult to identify by traditional methods. In this study, the Bruker system showed greater accuracy than the Vitek system. In order to be truly effective, it is essential to update the databases of both systems by increasing the number of each main spectrum profile within the platforms.

Resumen El objetivo de este estudio fue comparar el desempeño de dos sistemas MALDI-TOF MS en la identificación de bacterias anaerobias estrictas de interés clínico. La secuenciación del gen 16S ARNr fue el método de referencia utilizado cuando se observaron discrepancias o inconsistencias entre plataformas. Se recuperaron 333 aislados de muestras clínicas de diferentes centros de la Ciudad Autónoma de Buenos Aires entre 2016 y 2021. Los aislados se identificaron por duplicado mediante dos sistemas MALDI-TOF MS: el BD Bruker Biotyper (Bruker Daltonics, Bremen, Alemania) y el Vitek MS (bioMèrieux, Marcy-l'Etoile, Francia). A través del sistema Vitek MS, los mismos fueron identificados a nivel de especie o complejo de especies en un 65,5% (n: 218) y de género en un 71,2% (n: 237), mientras que no se identificaron en un 29,4% (n: 98) y fue incorrecta en el 5,1% (n: 17). Mediante el sistema Bruker Biotyper, dichos valores fueron del 85,3% (n: 284), del 89,7% (n: 299), del 14,1% (n: 47) y del 0,6% (n: 2), respectivamente. La diferencia entre ambos métodos fue estadísticamente significativa (p<0,0001). En conclusión, los sistemas MALDI-TOF MS aceleran la identificación microbiana. Son especialmente útiles para los microorganismos de crecimiento lento, como las bacterias anaerobias, que son difíciles de identificar con los métodos tradicionales. El sistema Bruker demostró ser más preciso que el Vitek MS. Para que estos métodos sean realmente efectivos es fundamental actualizar las bases de datos de ambos sistemas e incrementar el número de espectros de referencia dentro de las plataformas.

Diferencia en la expresión del ligando de receptor activador para el factor nuclear K B, en tejido tumoral frente a la infección por diferentes patógenos periodontales / Difference in receptor activator of nuclear factor K B expression in tumoral tissue by different periodontal pathogens

La enfermedad periodontal es una de las principales causas de pérdida dentaria. Clínicamente, esta patología, mediada por la desregulación del sistema inmune producto de una disbiosis ocurrida en el surco gingival, inicia con la inflamación de la encía y evoluciona con el daño irreversible de los tejidos que rodean el diente. El hueso alveolar es uno de los tejidos afectados esta patología, esto debido a la activación de osteoclastos por la sobreexpresión de la proteína RANKL en el huésped. El propósito de este trabajo es determinar el nivel de sobreexpresión de RANKL, en un modelo de células tumorales U2OS, frente a la infección con Porphyromonas gingivalis y Prevotella intermedia. Para identificar el nivel de RANKL, se definieron cuatro grupos: Un grupo control, no tratado; Grupo PG, tratado con P. gingivalis; Grupo PI, tratado con P. Intermedia; y un grupo PG+PI, tratado con ambas bacterias. El nivel relativo de la proteína RANKL fue determinado en el sobrenadante y en los extractos celulares de manera independiente, mediante la técnica Western blot. En sobrenadantes, el grupo PG mostró mayores niveles de RANKL comparados con PI (p < 0,05). En extractos celulares los niveles fueron mayores en el grupo PG+PI (p < 0,05). El grupo PI mostró los niveles más bajos de RANKL. La infección polimicrobiana resulta en una mayor expresión de RANKL en células tumorales U2OS, mientras que frente a la infección P. gingivalis, se observó mayor cantidad de RANKL soluble.

SUMMARY: Periodontal disease is one of the main causes of tooth loss. Clinically, this pathology, mediated by the deregulation of the immune system due to a dysbiosis occurred in the gingival sulcus, begins with the inflammation of the gum and evolves with the irreversible damage of the tissues that surround the tooth. Alveolar bone is one of the most affected tissues by this disease, due to the activation of osteoclasts by the upregulation of RANKL in the host. The aim of this study is to determine the increase of RANKL, in a U2OS tumor cells model, inoculated with Porphyromonas gingivalis and Prevotella intermedia. To identify the level of RANKL, four groups were defined: A control group, not treated; PG group, treated with P.gingivalis; PI group, treated with P. intermedia; and a PG+PI group, treated with both bacteria. The relative level of RANKL was determined in the supernatant and cell extracts independently, using the Western blot technique. In supernatants, the PG group showed higher RANKL levels compared to PI (p < 0.05). In cell extracts the levels were higher in the PG+PI group (p < 0.05.). The PI group showed the lowest levels of RANKL.Polymicrobial infection results in a greater expression of of soluble RANKL was observed.

Empiema pleural por Parvimonas micra en un paciente inmunocompetente: reporte de un caso / Pleural empyema by Parvimonas micra in an immunocompetent patient: a case report

Se presenta el caso de un paciente joven inmunocompetente, con antecedente de tuberculosis pulmonar, que acude al hospital por un cuadro clínico subagudo de fiebre persistente, baja de peso, disnea y abolición del murmullo vesicular. La tomografía de tórax mostró un extenso empiema en hemitórax izquierdo. Se le toman muestras para detección de gérmenes comunes y se le colocan un tubo de drenaje torácico y se inicia antibioticoterapia. La prueba de MALDI-TOF MS identificó a Parvimonas micra, una bacteria anaerobia, comensal de la flora oral, asociado a periodontitis severa, escasamente reportado en empiema pleural, especialmente, en personas inmunocompetentes. En la evaluación odontológica se realizó el diagnóstico de gingivitis y pericoronaritis de la tercera molar. El paciente evolucionó favorablemente. Se sugiere que, en casos de empiemas pleurales subagudos o crónicos, se debe considerar, además de las micobacterias, como agente etiológico al Parvimonas micra, y optar por exámenes como MALDI-TOF MS o secuenciamiento del 16S rRNA, colocación de tubo de tórax, cobertura antibiótica empírica y evaluación odontológica.

We present the case of a young immunocompetent patient, with a history of pulmonary tuberculosis, who attended the hospital with a subacute clinical picture of persistent fever, weight loss, dyspnea and abolition of vesicular murmur. Chest CT scan showed an extensive empyema in the left hemithorax. Samples were taken for detection of common germs. Then, a chest drainage tube was placed and antibiotic therapy started. The MALDI-TOF MS test identified Parvimonas micra, an anaerobic bacterium, commensal to the oral flora, associated with severe periodontitis, but rarely reported in cases of pleural empyema, especially in immunocompetent patients. Gingivitis and pericoronaritis of the third molar were diagnosed during oral evaluation. The patient progressed favorably. Parvimonas micra should be considered as a possible etiological agent in cases of subacute or chronic pleural empyema, in addition to mycobacteria. Tests such as MALDI-TOF MS or 16S rRNA sequencing, chest tube placement, empirical antibiotic coverage and an adequate oral evaluation should be considered in these cases.

Bacterial and C linical P rofile of D iabetic F oot U lcer using O ptimal C ulture T echniques: A Prospective Hospital Based Study

Background: Diabetic foot ulcers (DFU) are the complications of diabetes mellitus. The infection of diabetic foot ulcer is microbes in nature. If they are not recognized and controlled it may lead to many devastating consequences like limb amputation, sepsis, and even mortality. So, the present study was aimed that to determine the bacterial and clinical profile of diabetic foot ulcer using optimal culture techniques. Methods: This prospective study was conducted on Type 2 Diabetic patients with foot ulcer. Around 206 total cases were enrolled on the basis of inclusion and exclusion criteria. The duration of study was over a period of two years. Results: The result of this study revealed that causative agent of DFU were found due to aerobic in n = 170 cases whereas anae robic bacteria in n = 36 cases. Conclusion: This study concludes that that proper care should be offered, causative organisms should be timely identified and antibiotic should be sensibly chosen to cut short the morbidity and mortality of DFU.

Diagnostic and therapeutic approaches in deep neck infections: an analysis of 74 consecutive patients / Abordagens diagnósticas e terapêuticas em infecções cervicais profundas: uma análise de 74 pacientes consecutivos

Abstract Introductıon: Deep neck infections are a group of diseases with serious complications and mortality, which can occur as a result of common diseases in the community and which have surgical and medical treatment options. Objectives: Patients ages, genders, complaints, physical examination findings, hospitalization complaints, history of antibiotic use before the application, additional diseases, radiological tests and analysis of examinations, type of treatment method, antibiotic agents selected in treatment, bacterial culture results, duration of hospitalization, complications, mortality rates were systematically recorded. In the study, anaerobic bacterial factors, which are difficult to produce in routine, were produced by considering special transport conditions and culture media. Methods: A total of 74 patients who were hospitalized in the Department of Otorhinolaryngology, University of Mersin, between 01.07.2016 and 01.07.2017 for deep neck infection were evaluated prospectively. The study included 37 female and 37 male patients. The ages of the patients ranged from 1 to 69 and the mean age was 31 years. Results: According to the analysis of the obtained data, there was a statistically significant relationship between the patients with additional diseases and the treatment modalities of the patients (p = 0.017). The surgical treatment rate was increased in this group of patients. In patients with a history of antibiotic use, it was found that patients in the pediatric group were in hospital longer in terms of length of stay compared to adults (p = 0.036). In adult patients who underwent surgery, the absorptive long axis was found to be longer in mm than in patients receiving isolated medical treatment (p = 0.008). Conclusions: Deep neck infections is a disease group that seriously concerns public health, with significant mortality and morbidity. Ensuring airway safety of patients should be the first intervention. Abscesses located lateral to the tonsil capsule may not drain adequately without concomitant tonsillectomy.

Resumo Introdução: As infecções cervicais profundas constituem um grupo de doenças com graves complicações e mortalidade, que podem ocorrer como resultado de doenças comuns na comunidade e que têm opções de tratamento médico e cirúrgico. Objetivo: Detectar bactérias anaeróbias e comparar a eficácia do tratamento médico-cirúrgico em diferentes faixas etárias. Método: Foram sistematicamente registrados idade dos pacientes, sexo, queixas, achados do exame físico, queixas na hospitalização, histórico de uso de antibióticos, doenças adicionais, exames radiológicos e análise dos exames, tipo de tratamento, agentes antibióticos selecionados no tratamento, resultados de cultura bacteriana, duração da hospitalização, complicações e taxas de mortalidade. No estudo, culturas bacterianas anaeróbias, difíceis de obter rotineiramente, mesmo considerando-se condições especiais de transporte e meios, foram obtidas. Foram avaliados prospectivamente 74 pacientes internados no Departamento de Otorrinolaringologia da Universidade de Mersin, entre 01.07.2016 e 07.07.2017, devido a infecção cervical profunda. O estudo incluiu 37 pacientes do sexo feminino e 37 do masculino. A idade dos pacientes variou de 1 a 69 anos e a média foi de 31. Resultados: De acordo com a análise dos dados obtidos, houve uma relação estatisticamente significante entre os pacientes com doenças adicionais e as modalidades de tratamento dos pacientes (p = 0,017). A taxa de tratamento cirúrgico foi maior nesse grupo de pacientes. Em pacientes com histórico de uso de antibióticos, verificou-se que aqueles do grupo pediátrico permaneceram mais tempo hospitalizados em comparação aos adultos (p = 0,036). Nos pacientes adultos submetidos à cirurgia, verificou-se que o eixo longo absortivo era mais longo em mm do que nos pacientes que receberam tratamento médico isolado (p = 0,008). Conclusão: As infecções cervicais profundas constituem um grupo de doenças que são preocupantes na saúde pública, com sua mortalidade e morbidade. Garantir a segurança das vias aéreas dos pacientes deve ser a primeira intervenção. Os abscessos localizados lateralmente à cápsula tonsilar podem não apresentar drenagem sem a tonsilectomia.

The effectiveness and safety of Morinidazole for acute complicated appendicitis undergoing laparoscopic appendectomy / 中华普通外科杂志

Objective:To analyze the effectiveness and safety of Morinidazole combined laparoscopic appendectomy for acute purulent appendicitis and acute gangrene appendicitis.Methods:We retrospectively retrieved 387 acute appendicitis patients who have received surgery or conservative treatment from Jan 2017 to Jun 2019. Firty four patients with Morinidazole + Etimicin bigeminy combined laparoscopic surgery were set to experimental group. The other 54 cases using ornidazole + levofloxacin bigeminy combined laparoscopic appendectomy were enrolled into control group after Propensity Score Matching.Results:After 1∶1 propensity score matching, there was no statistically significant difference between the two groups and control group in clinical features. The white blood cell count, C-reactive protein and calcitonin original inflammation index change were higher in experimental group than in the control group,both preoperatived and on post-op day1 and day 3 (all P<0.05). In the experimental group the length of hospital stay and analgesic drug usetime were shorter than in the control group (all P<0.05). Conclusion:Morinidazole combined laparoscopic appendectomy can effectively control the intra-abdominal infection caused by acute purulent appendicitis and acute gangrene appendicitis with less adverse reactions.

Use of wide-field optical fluorescence for visualization of oral biofilm in a patient with peri-implant mucositis: a new approach / Uso da fluorescência óptica de campo amplo para visualização de biofilme oral em paciente com mucosite peri-implantar: uma nova abordagem

ABSTRACT Peri-implant diseases, caused by bacteria from biofilm related to dental implants, are one of the main causes of late loss of implants. In this sense, peri-implant diseases are divided into peri-implant mucositis, when it affects only the soft tissues, and peri-implantitis, when there is a bone involvement, which can lead to the failure of dental implant therapy. Thus, biofilm removal is essential for peri-implant health, allowing long-term success in implant therapy. To improve the visualization of oral biofilm, which is usually transparent or colorless, disclosing agents have been routinely used. However, disclosing agents have allergenic potential and can cause staining extrinsically in restorative and prosthetic materials, leading to aesthetic impairment. Thus, the use of fluorescence has been studied as an alternative for visualization of oral biofilm. Therefore, this report describes the use of wide-field optical fluorescence for visualization of oral biofilm associated with implants and teeth, in a routine appointment and follow-up of a partially edentulous patient with peri-implant mucositis. In addition, this report showed wide-field optical fluorescence can be used in a clinical routine of care of patients with dental implants. In this sense, wide-field optical fluorescence allowed easy and immediate visualization of the mature oral biofilm for its adequate removal, evaluation of the quality of restoration to sealing of screw access-hole of implant and identification of cariogenic lesions, without risk of allergic reactions or staining of prostheses and restorations.

RESUMO Doenças peri-implantares, causadas por bactérias de biofilme relacionadas a implantes dentários, são uma das principais causas de perda tardia de implantes. Nesse sentido, as doenças peri-implantares são divididas em mucosite peri-implantar, quando afeta apenas tecidos moles, e peri-implantite, quando há comprometimento ósseo, o que pode levar ao fracasso da terapia com implantes dentários. Assim, a remoção do biofilme é essencial para a saúde peri-implantar, permitindo sucesso a longo prazo na terapia com implantes. A fim de melhorar a visualização do biofilme oral, que geralmente é transparente ou incolor, agentes reveladores têm sido rotineiramente utilizados. No entanto, esses agentes têm potencial alergênico e podem causar manchas extrinsecamente em materiais restauradores e protéticos, levando a prejuízo estético. Assim, o uso da fluorescência tem sido estudado como alternativa para visualização do biofilme oral. Este relato descreve o uso da fluorescência óptica de campo amplo para visualização do biofilme oral associado a implantes e dentes em uma consulta de acompanhamento de rotina de uma paciente parcialmente edêntula com mucosite peri-implantar. Além disso, este relato evidenciou que a fluorescência óptica de campo amplo pode ser utilizada dentro da rotina clínica de atendimento de pacientes com implantes dentários. Nesse sentido, a fluorescência óptica de campo amplo permitiu a visualização fácil e imediata do biofilme oral maduro para sua remoção adequada, a avaliação da qualidade da restauração do selamento do orifício de acesso do parafuso do implante e a identificação de lesões cariogênicas, sem risco de reações alérgicas ou manchamento de próteses e restaurações.

Anal abscess microbiology as an anal fistula predictor / Microbiologia de abscesso anal como preditivo de fístula anal

ABSTRACT In order to determine the microbiology of anal abscess as a predictor of anal fistulas in patients who attended the external consultation of the Coloproctology unit of the Dr. Antonio María Pineda University Central Hospital, a prospective, descriptive, longitudinal study was conducted from September 2018 to July 2019. In this study, the population consisted of patients with a diagnosis of anal abscess, without associated comorbidities or contraindications for surgery, who agreed to be included in the study. A non-probabilistic, intentional sample consisting of 42 patients was determined. An appointment-based study protocol was applied by outpatient for patients who met the inclusion criteria applied, to perform due medical history through anamnesis, physical examination and culture taking of suppuration from the anal abscess to subsequently establish medical and surgical behavior thereof. The results were expressed in absolute numbers and percentages, a prevalence of ischiorectal abscesses was observed, followed by deep post-anal space abscesses. Anaerobic bacteria were isolated in 100% of the sample. In all fistulized patients, E. Coli was isolated as a predominant germ.

RESUMO Um estudo prospectivo, descritivo e longitudinal foi realizado de setembro de 2018 a Julho 2019 para determinar a microbiologia do abscesso anal como preditivo de fístulas anais em pacientes que compareceram à consulta externa da unidade de Coloproctologia do Hospital Central da Universidade Dr. Antonio María Pineda. Neste estudo, a população foi composta por pacientes com diagnóstico de abscesso anal, sem comorbidades ou contraindicações associadas à cirurgia, que concordaram em participar do estudo. Uma amostra intencional não probabilística, composta por 42 pacientes foi determinada. Um protocolo de estudo com base na consulta em regime ambulatorial foi aplicado aos pacientes que atenderam os critérios de inclusão estabelecidos, para realizar a anamnese, o exame físico e a devida cultura da supuração do abscesso anal para posteriormente estabelecer o comportamento médico e cirúrgico. Os resultados foram expressos em números absolutos e porcentagens, observando-se a prevalência de abscessos isquiorretais, seguidos por abscessos profundos no espaço pós-anal. Bactérias anaeróbias foram isoladas em 100% das amostras. Em todos os pacientes com fístulas, E. Coli foi isolada como um germe predominante.

Bacteriology of peritonsillar abscess: the changing trend and predisposing factors / Bacteriologia do abscesso peritonsilar: tendência de mudança e fatores predisponentes

Abstract Introduction: Peritonsillar abscess is the most common deep neck infection. The infectious microorganism may be different according to clinical factors. Objective: To identify the major causative pathogen of peritonsillar abscess and investigate the relationship between the causative pathogen, host clinical factors, and hospitalization duration. Methods: This retrospective study included 415 hospitalized patients diagnosed with peritonsillar abscess who were admitted to a tertiary medical center from June 1990 to June 2013. We collected data by chart review and analyzed variables such as demographic characteristics, underlying systemic disease, smoking, alcoholism, betel nut chewing, bacteriology, and hospitalization duration. Results: A total of 168 patients had positive results for pathogen isolation. Streptococcus viridans (28.57%) and Klebsiella pneumoniae (23.21%) were the most common microorganisms identified through pus culturing. The isolation rate of anaerobes increased to 49.35% in the recent 6 years (p = 0.048). Common anaerobes were Prevotella and Fusobacterium spp. The identification of K. pneumoniae increased among elderly patients (age > 65 years) with an odds ratio (OR) of 2.76 (p = 0.03), and decreased in the hot season (mean temperature > 26 °C) (OR = 0.49, p = 0.04). No specific microorganism was associated with prolonged hospital stay. Conclusion: The most common pathogen identified through pus culturing was S. viridans, followed by K. pneumoniae. The identification of anaerobes was shown to increase in recent years. The antibiotics initially selected should be effective against both aerobes and anaerobes. Bacterial identification may be associated with host clinical factors and environmental factors.

Resumo Introdução: O Abscesso Peritonsilar é a infecção cervical profunda mais comum. O microrganismo infeccioso pode ser diferente de acordo com os fatores clínicos. Objetivo: Identificar o principal agente causador do abscesso peritonsilar e investigar a relação entre o patógeno causador, os fatores clínicos do hospedeiro e a duração da hospitalização. Método: Este estudo retrospectivo incluiu 415 pacientes hospitalizados diagnosticados com abscesso peritonsilar que foram internados em um centro médico terciário de junho de 1990 a junho de 2013. Coletamos dados através da análise dos arquivos médicos dos pacientes e analisamos variáveis como características demográficas, doença sistêmica subjacente, tabagismo, alcoolismo, hábito de mascar noz de betel, bacteriologia e duração da hospitalização. Resultados: Um total de 168 pacientes apresentaram resultados positivos para isolamento de patógenos. Streptococcus viridans (28,57%) e Klebsiella pneumoniae (23,21%) foram os microrganismos mais comuns identificados pela cultura da secreção. A taxa de isolamento de anaeróbios aumentou para 49,35% nos últimos 6 anos (p = 0,048). Os anaeróbios comuns foram Prevotella e Fusobacterium spp. A identificação de K. pneumoniae aumentou em pacientes idosos (idade > 65 anos) com razão de chances (Odds Ratio - OR) de 2,76 (p = 0,03) e diminuiu na estação do calor (temperatura média > 26 °C) (OR = 0,49, p = 0,04). Nenhum microrganismo específico foi associado à hospitalização prolongada. Conclusão: O patógeno mais comumente identificado através da cultura de secreção foi S. viridans, seguido por K. pneumoniae. A identificação de anaeróbios mostrou ter aumentado nos últimos anos. Os antibióticos selecionados inicialmente devem ser efetivos contra aeróbios e anaeróbios. A identificação bacteriana pode estar associada a fatores clínicos e fatores ambientais do hospedeiro.

Evaluation of MALDI-TOF mass spectrometry (VITEK-MS) compared to the ANC card (VITEK 2) for the identification of clinically significant anaerobes / Avaliação da espectrometria de massas MALDI-TOF (VITEK-MS) diante do cartão ANC (VITEK 2) na identificação de anaeróbios clinicamente significantes

ABSTRACT Introduction: The identification of anaerobic bacteria by conventional methods employed in clinical laboratories requires a lot of work and a long response time [turnaround time (TAT)]. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is an accurate, rapid and inexpensive technique with promising results for bacterial identification. Objective: To evaluate the MALDI-TOF mass spectrometry (VITEK-MS, bioMérieux, France) compared to the ANC card (VITEK 2, bioMérieux, France) for the identification of anaerobes, and also veriying the cost variation between both methodologies. Methods: 421 anaerobes were concomitantly identified by ANC (VITEK 2) and MALDI-TOF (VITEK MS). The conflicting results or those presenting low differentiation of the species were subjected to the 16S ribosomal ribonucleic acid (rRNA) sequencing. Results: Thirty-five strains were not identified by ANC (VITEK 2), and only one isolate was not identified by MALDI-TOF (VITEK MS). From the 386 anaerobes identified by the two methodologies, 97% agreement was observed on the identification of genus and species between the methodologies. Thirteen (3%) isolates were submitted to 16S sequencing. The agreement observed was 70% using ANC (VITEK 2) using 92% by MALDI-TOF (VITEK MS). Conclusion: Both methodologies showed an excellent performance for the identification of the strains tested with great differences in relation to cost-benefit. MALDI-TOF MS allowed 35 additional identifications and a saving of BRL$ 7,786 with the release of culture positive result five days ahead of the ANC (VITEK 2). TAT reduction may contribute to a successful clinical resolution.

RESUMO Introdução: A identificação das bactérias anaeróbias por métodos convencionais empregados nos laboratórios clínicos demanda muito trabalho e um longo tempo de resposta (TAT). A espectrometria de massa por ionização e dessorção a laser assistida por matriz (MALDI-TOF MS) é uma técnica precisa, rápida e barata, com resultados promissores para a identificação bacteriana. Objetivo: Avaliar a espectrometria de massas MALDI-TOF (VITEK MS, bioMérieux, France) diante do cartão ANC (VITEK 2, bioMérieux, France) para a identificação de anaeróbios, bem como verificar a variação de custos entre as metodologias. Métodos: Foram identificados 421 anaeróbios concomitantemente pelo ANC (VITEK 2) e pelo MALDI-TOF (VITEK MS). Os resultados discordantes ou que apresentaram baixa discriminação das espécies foram submetidos ao sequenciamento do 16S do ácido ribonucleico ribossonal (rRNA). Resultados: Trinta e cinco cepas não foram identificadas pelo ANC (VITEK 2), e somente um isolado ficou sem identificação pelo MALDI-TOF (VITEK MS). Dos 386 anaeróbios identificados pelas duas metodologias, a concordância na identificação de gênero e espécie foi observada em 97%. Treze (3%) isolados foram submetidos ao sequenciamento do 16S; a concordância observada foi de 70% com o ANC (VITEK 2) e 92% com MALDI-TOF (VITEK MS). Conclusão: Ambas as metodologias demonstraram ótimo desempenho para identificação das cepas testadas com grandes diferenças em relação ao custo-benefício. O MALDI-TOF MS permitiu 35 identificações adicionais e uma economia de R$ 7.786,00 com a liberação do resultado positivo da cultura cinco dias à frente do ANC (VITEK 2). A redução do TAT pode contribuir para uma resolução clínica bem-sucedida.

Inactivation of MarR gene homologs increases susceptibility to antimicrobials in Bacteroides fragilis

ABSTRACT Bacteroides fragilis is the strict anaerobic bacteria most commonly found in human infections, and has a high mortality rate. Among other virulence factors, the remarkable ability to acquire resistance to a variety of antimicrobial agents and to tolerate nanomolar concentrations of oxygen explains in part their success in causing infection and colonizing the mucosa. Much attention has been given to genes related to multiple drug resistance derived from plasmids, integrons or transposon, but such genes are also detected in chromosomal systems, like the mar (multiple antibiotic resistance) locus, that confer resistance to a range of drugs. Regulators like MarR, that control expression of the locus mar, also regulate resistance to organic solvents, disinfectants and oxygen reactive species are important players in these events. Strains derived from the parental strain 638R, with mutations in the genes hereby known as marRI (BF638R_3159) and marRII (BF638R_3706) were constructed by gene disruption using a suicide plasmid. Phenotypic response of the mutant strains to hydrogen peroxide, cell survival assay against exposure to oxygen, biofilm formation, resistance to bile salts and resistance to antibiotics was evaluated. The results showed that the mutant strains exhibit statistically significant differences in their response to oxygen stress, but no changes were observed in survival when exposed to bile salts. Biofilm formation was not affected by either gene disruption. Both mutant strains however, became more sensitive to multiple antimicrobial drugs tested. This indicates that as observed in other bacterial species, MarR are an important resistance mechanism in B. fragilis.

Anaerobic Bacteria in Oral Cavities and Dental Health

@#Oral cavity is a very suitable habitat for a wide range of bacteria of which a significant proportion is facultative or strict anaerobes. In healthy individuals, specific sites of the oral cavity are colonized by specific microbial communities, and a balance of the species within the community, known as “microbial homeostasis”, is maintained. When this balance is disrupted by ecological perturbations, the biofilm composition changes leading to the initiation of local infections that may ultimately lead to tooth loss. At the onset of the infections, Gram-positive bacteria dominate the biofilm composition, but if left undisturbed, a more complex biofilm builds up where Gram-negative anaerobic and proteolytic rods become dominant.

Anaerobic bacteria in the intestinal microbiota of Brazilian children

OBJECTIVE: Changes in the neonatal gut environment allow for the colonization of the mucin layer and lumen by anaerobic bacteria. The aim of the present study was to evaluate Bifidobacterium, Lactobacillus and Lactococcus colonization through the first year of life in a group of 12 Brazilian infants and to correlate these data with the levels of Escherichia coli. The presence of anaerobic members of the adult intestinal microbiota, including Eubacterium limosum and Faecalibacterium prausnitzii, was also evaluated. METHODS: Fecal samples were collected during the first year of life, and 16S rRNA from anaerobic and facultative bacteria was detected by real-time PCR. RESULTS: Bifidobacterium was present at the highest levels at all of the studied time points, followed by E. coli and Lactobacillus. E. limosum was rarely detected, and F. prausnitzii was detected only in the samples from the latest time points. CONCLUSION: These results are consistent with reports throughout the world on the community structure of the intestinal microbiota in infants fed a milk diet. Our findings also provide evidence for the influence of the environment on intestinal colonization due to the high abundance of E. coli. The presence of important anaerobic genera was observed in Brazilian infants living at a low socioeconomic level, a result that has already been well established for infants living in developed countries.

Presencia de bacilos negro pigmentantés en bolsas periodontales de pacientes diabéticos tipo 2 y su relación con el control glicémico / Presence of bacilli black pigments in periodontal pockets of patients with type 2 diabetes and its relationship with the glycemic level

Objetivos: El estudio tuvo como objetivo identificar la presencia de bacilos negro pigmentantés (BNP) en bolsas periodontales de pacientes diabéticos tipo 2 y determinar una relación con su control glicémico. Material y métodos: Se evaluó un número total de 46 pacientes que cumplían con los criterio de inclusión en el estudio, de los cuales quedaron 20 por haber concluido con todas las pruebas realizadas en el estudio, como el estudio microbiológico de sus muestras de placa subgingival y las pruebas de hemoglobina glicosilada que se realizó en forma periódica en los 3 meses que se evaluó a los pacientes. Resultados: De los 20 pacientes evaluados que completaron todas sus pruebas, 14 eran mujeres y 6 varones, con un rango de edad de 40 a 68 años, pudiéndose identificar estos bacilos negro pigmentantés (BNP) en el 20 % de casos con diagnóstico de periodontitis moderada a severa (periodontitis moderada: bolsa periodontal de 6 a 7 mm y Periodontitis severa: bolsa periodontal mayor de 7 mm), presentando estos pacientes un control glicémico de condición bueno a moderado (control bueno: = 6.9 % HbAc1y control moderado: 7 a 7.9 % HbAc1). La prueba estadística realizada para valorar la relación presencia BNP y el control glicémico fue de Chi cuadrado el cual no determino significancia de la relación (P > 5 %). Conclusiones: Se identificó la presencia de BNP en pacientes diabéticos tipo 2 pero más relacionado a pacientes con cuadros de periodontitis moderada a severa y con un control glicémico de bueno a moderado, no presentando significancia estadística.

Objectives: The study aimed to identify the presence of bacilli black pigments in periodontal pockets of type 2 diabetic patients and determine a relationship with glycemic control. Material and methods: A total of 46 patients who met the criteria for inclusion in the study, of which were 20 for having completed all tests in the study, such as microbiological analysis of samples of subgingival plaque and evaluated glycosylated hemoglobin tests held periodically in the 3 months patients were assessed. Results: Of the 20 patients evaluated, 14 were women and 6 men, with an age range of 40 to 68 years, being able to identify these black bacilli pigments (BNP) in 20% of cases diagnosed with moderate to severe periodontitis (periodontitis. moderate: periodontal pocket of 6 to 7 mm and severe periodontitis: greater periodontal pocket of 7 mm), presenting these patients glycemic control in good condition to moderate (good control: = 6,9 % HbAc1 and moderate control: 7 to 7,9 % HbAc1) . The statistical test performed to assess the presence relationship BNP and glycemic control was Chi square which does not determine significance of the relationship (P> 5%). Conclusions: It was possible to identify the presence of BNP in patients with type 2 diabetes but more related to patients with moderate to severe pictures periodontitis and with good glycemic control to moderate, showing no statistical significance.

Molecular-level evaluation of selected periodontal pathogens from subgingival regions in canines and humans with periodontal disease

Dogs commonly serve as a model for various human conditions, including periodontal diseases. The aim of this study was to identify the anaerobic bacteria that colonize the subgingival areas in dogs and humans by using rapid real-time polymerase chain reaction (RT-PCR)-based tests and to compare the results obtained in each species. Bacterial microflora evaluations, both quantitative and qualitative, were performed by applying ready-made tests on twelve dogs and twelve humans. Five samples were collected from each subject's deepest gingival pockets and joined to form a collective sample. The results of the study revealed interspecies similarities in the prevalences of Porphyromonas (P.) gingivalis, Treponema denticola, Tannerella forsythia, and Fusobacterium nucleatum. Red complex bacteria comprised the largest portion of the studied bacterial complexes in all study groups, with P. gingivalis being the most commonly isolated bacterium. The results show similarities in the prevalence of bacterial microflora in dogs and humans. Microbiological analysis of gingival pockets by using rapid real-time PCR-based tests in clinical practice, both veterinary and human, can facilitate the choice of appropriate pharmacological treatment and can provide a basis for subsequent verification of the treatment's effectiveness.

Culture-dependent approaches to explore the prevalence of root canal pathogens from endodontic infections

Abstract: Endodontic infections are considered to be caused by the presence of various microorganisms within the root canal system. Recognition of this microbiota contributes to the successful treatment of infected root canals. This study investigated the microorganisms associated with primary and secondary endodontic infections via culture methods, biochemical tests, and molecular approaches in an Iranian population. Microbial specimens were collected from 36 patients with primary endodontic infection and 14 patients with a history of root canal therapy. Advanced microbiological culture techniques were used to isolate microbiota; subsequently, biochemical tests and 16S ribosomal RNA gene sequencing were performed to identify the microorganisms. Within the total 218 cultivable isolates, Veillonella parvula (20.6%) was found to occur with the highest frequency in primary endodontic infection, followed by Porphyromonas gingivalis (14.1%), and Aggregatibacter actinomycetemcomitans (9.2%). Enterococcus faecalis (36.6%) was the most predominant microorganism in secondary endodontic infections, followed by Candida albicans, Propionibacterium acnes, and V. parvula with frequencies of 20%, 2%, and 2%, respectively. It was concluded that V. parvula and E. faecalis was most frequently found in primary and secondary endodontic infections, respectively.

The Dynamics of Subcutaneous Tissue Response to Microorganisms Associated with the Extract of Araçá (Psidium cattleianum): An Edemogenic and Microscopic Analysis / A Dinâmica da Resposta do Tecido Subcutâneo para Associação de Microorganismos com o Extrato de Araçá (Psidium cattleianum): Uma Análise Microscópica e Edemogênica

Objective: To evaluate in vivo tissue reaction to the extract of araçá (Psidium cattleianum) associated with inactivated microorganisms. Material and Methods: A 0.1 mL suspension was used containing Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, Enterococcus faecalis, Peptostreptococcus micros, and Porphyromonas endodontalis, which were inactivated by heat and mixed into a 1.0 mL saline (control group), an aqueous solution, or a hydroalcoholic extract of araçá. Eighteen male rats (Rattus norvegiccus) under general anesthesia received 0.2 mL of 1% intravenous Evans blue. Thirty minutes later, 0.1 mL of one of the associations was injected into the animals' dorsal region. The animals were euthanized after 3 and 6 hours, and the materials obtained were placed in formamide for 72 hours then analyzed in a spectrophotometer (λ=630 ηm). For the morphological analysis, 30 rats received polyethylene tubes implants with the extracts or the saline with the associations in the dorsal region and euthanized after 7 and 30 days to be analyzed according to an inflammation cell score. Results: No significant difference (p>0.05) was observed in the edema among groups. The optical microscopy results showed a repair in the 30-day-period, which was higher when compared to the 7-day-period (p< 0.0001). Nevertheless, in the 7-day-period, the hydroalcoholic extract presented a significant response compared to the aqueous extract (p=0.05) and a trend for better results than the control group. Conclusion: The aqueous and hydroalcoholic araçá extracts associated with inactivated microorganisms showed similar responses to control, indicating no interference on the toxic effects of the bacterial components in tissue repair. (AU)

Objetivo: Avaliar in vivo a reação tecidual do extrato de araçá (Psidium cattleianum) associado com microorganismos inativados. Material e Métodos: Uma suspensão de 0.1mL foi usada contendo Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, Enterococcus faecalis, Peptostreptococcus micros e Porphyromonas endodontalis dos quais foram inativos por aquecimento e misturados a 1,0 mL de soro fisiológico (grupo controle), uma solução aquosa ou hidroalcoólica de araçá. Dezoito ratos machos (Rattus norvegiccus) sob anestesia geral receberam 0,2mL de Azul de Evans a 1% intravenoso. Após trinta minutos, 0,1mL de um dos extratos (associado com microorganismos inativos) foi injetado nos animais na região dorsal. Os animais foram eutanasiados após 3 e 6 horas, e os materiais obtidos colocados em formamida por 72 horas para análise em espectrofotômetro (λ=630 ηm). Para análise morfológica, 30 ratos receberam implante subcutâneo de tubo de polietileno com as associações na região dorsal, eutanasiados após 7 e 30 dias para serem analisados de acordo com um escore de células inflamatórias. Resultados: Não houve diferença significativa (p>0,05) no edema entre os grupos. Os resultados obtidos em microscópio óptico apontaram reparo em 30 dias superior ao de 7 dias (p< 0,0001). No período de 7 dias a solução hidroalcoólica apresentou resposta superior a solução aquosa (p=0,05) e uma tendência de melhor resultado que o controle. Conclusão: A solução aquosa e hidroalcoólica de extrato de araçá associadas a microrganismos inativados apresentaram respostas biológicas semelhantes ao controle, indicando que não há interferência sobre os efeitos tóxicos advindos dos componentes bacterianos, no sentido de favorecer o reparo(AU)

Detection of cfxA2, cfxA3, and cfxA6 genes in beta-lactamase producing oral anaerobes

ABSTRACT Purpose The aim of this study was to identify β-lactamase-producing oral anaerobic bacteria and screen them for the presence of cfxA and BlaTEM genes that are responsible for β-lactamase production and resistance to β-lactam antibiotics. Material and Methods Periodontal pocket debris samples were collected from 48 patients with chronic periodontitis and anaerobically cultured on blood agar plates with and without β-lactam antibiotics. Presumptive β-lactamase-producing isolates were evaluated for definite β-lactamase production using the nitrocefin slide method and identified using the API Rapid 32A system. Antimicrobial susceptibility was performed using disc diffusion and microbroth dilution tests as described by CLSI Methods. Isolates were screened for the presence of the β-lactamase-TEM (BlaTEM) and β-lactamase-cfxA genes using Polymerase Chain Reaction (PCR). Amplified PCR products were sequenced and the cfxA gene was characterized using Genbank databases. Results Seventy five percent of patients carried two species of β-lactamase-producing anaerobic bacteria that comprised 9.4% of the total number of cultivable bacteria. Fifty one percent of β-lactamase-producing strains mainly Prevotella, Porphyromonas, and Bacteroides carried the cfxA gene, whereas none of them carried blaTEM. Further characterization of the cfxA gene showed that 76.7% of these strains carried the cfxA2 gene, 14% carried cfxA3, and 9.3% carried cfxA6. The cfxA6 gene was present in three Prevotella spp. and in one Porphyromonas spp. Strains containing cfxA genes (56%) were resistant to the β-lactam antibiotics. Conclusion This study indicates that there is a high prevalence of the cfxA gene in β-lactamase-producing anaerobic oral bacteria, which may lead to drug resistance and treatment failure.

The value of isolation and identification of anaerobic bacteria causing diabetic foot deep ulcers / 国际检验医学杂志

Objective To investigate the deep ulcer infection of anaerobic bacteria in patients with diabetic foot ,while make clini-cal value analysis .Methods 87 specimens collected from the patients with deep ulcers of diabetic foot were performed by bacterio-logical smear and bacterial culture respectively .Results The bacteriological smear positive cases were 81 ,the aerobic culture posi-tive cases were 67 and the anaerobic culture positive cases were 17 .Conclusion The deep ulcers of diabetic foot often are infected by anaerobic bacteria .Aerobic culture and anaerobic culture are both necessary to define the pathogen .It must be noted that the an-aerobic culture positive rate would be interfered by incorrect sampling ,no timely examination ,using antibiotics and incorrect culture conditions .

Distribution and Antimicrobial Susceptibility of Anaerobic Bacteria in Periodontal Pocket of 84 Patients with Chronic Periodontitis / 昆明医科大学学报

Objective To study the distribution and antimicrobial susceptibilities of anaerobic bacteria from periodontal pockets of patients with chronic periodontitis, so as to guide the anti-infection therapy. Methods Eighty-four patients with chronic periodontitis were enrolled in this study. The anaerobes were isolated from periodontal pockets of patients with chronic periodontitis. Antimicrobial susceptibility of the anaerobes to seven kinds of antibiotics (like metronidazole, penicillin, cefmetazole, etc.) was tested with the agar dilution technique. Results A total of 305 cultivable anaerobes were isolated from periodontal pockets of 84 patients with chronic periodontitis. All patients were infected by anaerobes. 59.34%of cultivable anaerobes were gram-positive anaerobes, and 40.66%were gram-negative anaerobes. The P.melanin ogenica and P.gingivalis were the most common among the cultivable anaerobes, accounting for 51.19% and 55.95%, followed by P.intermedia (36.90%), P.micros (35.71%), F.mucleatum (33.33%), Capnocytophaga ssp (27.38%), Actinomyces ssp (10.71%), and so on. Most isolated anaerobes were sensitive to seven antibiotics. All isolated anaerobes were susceptible to Chloromycetin and Imipenem. 98.03%of isolated anaerobes were susceptible to Amoxicillin. Approximately 76%to 88%of isolated anaerobes were susceptible to Cefmetazole and Metronidazole. Susceptibility to Clindamycinum and Penicillin were 65.91%and 55.73%. Conclusions There is a close relationship between chronic periodontitis and anaerobes. P.melanin ogenica and P.gingivalis are the most frequently isolated anaerobes from periodontal pockets of patients with chronic periodontitis. Chloromycetin and Imipenem can be used for the treatment of anaerobes infected chronic periodontitis.