Geotricosis: fungemia en paciente con leucemia linfoblástica aguda / Geotrichosis: fungemia in a patient with acute lymphoblastic leukemia

La fungemia por Geotrichum spp. es poco frecuente y altamente letal. En el Instituto Nacional de Cancerología de Bogotá solo se han reportado dos casos: uno entre el 2001 y el 2007, y el otro entre el 2012 y el 2018. Este tipo de infección es más común en pacientes con algún grado de compromiso del sistema inmunitario, por lo que puede presentarse en pacientes con neoplasias hematológicas malignas. Se presenta el caso de un hombre de 27 años con recaída de leucemia linfoblástica aguda, que ingresó con poliartralgias de cinco días de duración. También cursaba con neutropenia febril, celulitis sin abscesos y bacteriemia por Staphylococcus aureus resistente a la meticilina para lo cual recibió terapia con oxacilina y cefepime. Sin embargo, persistía la neutropenia febril por lo que se sospechó una infección fúngica invasora. Se tomó un nuevo set de hemocultivos y se inició tratamiento antifúngico. En los hemocultivos se identificaron artroconidias y mediante espectrometría de masas por láser de matriz asistida de ionización-desorción se confirmó la presencia de Geotrichum spp. Se ajustó el tratamiento antifúngico con deoxicolato de anfotericina B por 14 días y voriconazol por cuatro semanas. Luego de una estancia prolongada se le dio de alta. Aunque la incidencia de la fungemia por Geotrichum spp. es baja, en pacientes con neoplasias hematológicas malignas debe considerarse en el contexto de una neutropenia febril que es persistente a pesar del tratamiento antimicrobiano de amplio espectro. La identificación de los agentes causantes de fungemias con herramientas de proteómica, como la espectrometría de masas mencionada, permite ajustar el tratamiento dirigido y reducir las complicaciones, la estancia hospitalaria y la mortalidad.

Fungemia caused by Geotrichum spp. is rare and highly lethal. The Instituto Nacional de Cancerología in Bogotá reported just two cases: one in the period 2001-2007 and the other in 2012-2018. This type of infection is more common in any kind of immunocompromised patients, so it can occur in those with hematological malignancies. Here we present the case of a 27-year-old man, diagnosed with acute lymphoblastic leukemia in relapse and admitted with polyarthralgia for five days, febrile neutropenia, non- abscessed cellulitis, and bacteremia due to methicillin-sensitive Staphylococcus aureus. The patient received therapy with oxacillin and cefepime, but the febrile neutropenia persisted. A new set of blood cultures was taken, and antifungal treatment was started because of the suspicion of invasive fungal infection. Arthroconidia were identified in blood cultures and Geotrichum spp. was confirmed using matrix-assisted laser desorption-ionization mass spectrometry. The antifungal treatment was adjusted with amphotericin B deoxycholate for 14 days and voriconazole for four weeks, and after a prolonged stay, the patient was discharged. Although the incidence of fungemia caused by Geotrichum spp. is low, it must be considered in patients with hematological malignancies and persistent febrile neutropenia despite the broadspectrum antimicrobial treatment. The confirmation of fungemia causing agents, with proteomic tools such as the mentioned mass spectrometry, allows treatment adjustment and decreases complications, hospital stay, and mortality.

Fusariosis en pacientes con cáncer: serie de 13 casos y revisión de la literatura / Fusariosis in cancer patients: 13 case series report and literature review

La fusariosis es una micosis oportunista producida por Fusarium spp. Su presentación clínica depende del estado inmunológico del huésped, especialmente, el de aquellos con enfermedades hematooncológicas, cuyas manifestaciones varían desde formas localizadas hasta infección fúngica invasora. El cultivo de piel o de sangre permite orientar el tratamiento antifúngico combinado con anfotericina B y voriconazol. Se presentan 13 casos de pacientes con cáncer en un periodo de once años que desarrollaron fusariosis diseminada; asimismo, se hizo con una revisión extensa de la literatura. En esta serie de casos, la mortalidad fue del 61,5 % (8/13), a pesar del uso del antifúngico. De los 13 pacientes, 11 tenían neoplasia hematológica y 2 neoplasia sólida. El factor de riesgo más importante fue la neutropenia profunda. El compromiso de la piel y los hemocultivos positivos facilitaron la prescripción del tratamiento combinado en la mayoría de los casos. La neutropenia febril persistente asociada a lesiones cutáneas, la onicomicosis, los nódulos o las masas pulmonares permitieron sospechar una infección fúngica invasora por Fusarium spp. El objetivo de la presentación de esta serie de casos es recordar el diagnóstico de fusariosis a la comunidad médica en contacto con pacientes oncológicos, con neutropenia febril profunda y persistentes.

The fusariosis is an opportunistic mycosis caused by Fusarium spp. Its clinical presentation depends on the immunological status of the host, especially in patients with hemato-oncological diseases, whose manifestations vary from localized to invasive fungal infections. Skin or blood culture helps to guide combined antifungal treatment with amphotericin B and voriconazole. Here, we present 13 cases in a period of eleven years of patients with cancer who developed disseminated fusariosis and their outcomes, together with a review of the related literature. In this series of cases, mortality was 61.5 % (8/13), despite the use of the antifungal. Out of the 13 cases, 11 had hematological neoplasia and 2 solid neoplasia. The most determinant risk factor was profound neutropenia. Skin involvement and positive blood cultures in most cases allowed combined treatment prescription. Persistent febrile neutropenia associated with skin lesions, onychomycosis, nodules, or lung masses lead to suspicion of Fusarium spp. fungal invasive infection. The aim of this series of cases is to remind healthcare professionals that oncological patients with deep and persistent febrile neutropenia can develop fusariosis.

Detection of Microorganisms in Clinical Sonicated Orthopedic Devices Using Conventional Culture and qPCR / Detecção de microrganismos em dispositivos ortopédicos sonicados clínicos usando cultura convencional e qPCR

Abstract Objective To evaluate the sensitivity and specificity of the quantitative real-time polymerase chain reaction (qPCR) for 16S rDNA gene screening using sonicated fluid from orthopedic implants. Methods A retrospective study was conducted on 73 sonicated fluids obtained from patients with infection associated with orthopedic implants. The samples were subjected to conventional culture and molecular testing using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and qPCR for 16S rDNA. The cycle threshold values were used to define a cut-off of the qPCR of the 16S rDNA for negative and positive cultures. Results No statistical differences were observed between the positive and negative culture groups based on the time from the first surgery to infection (p= 0.958), age (p =0.269), or general comorbidities. Nevertheless, a statistical difference was found between the mean duration of antibiotic use before device removal (3.41 versus 0.94; p =0.016). Bacterial DNA was identified in every sample from the sonicated fluids. The median cycle thresholds of the positive and negative cultures were of 25.6 and 27.3 respectively (p< 0.001). As a diagnostic tool, a cycle threshold cut-off of 26.89 demonstrated an area under the curve of the receiver operating characteristic of 0.877 (p≤ 0.001). Conclusion The presence of antimicrobial agents for more than 72 hours decreased culture positivity, but did not influence the qPCR results. Despite this, amplification of the 16S rDNA may overestimate infection diagnosis.

Resumo Objetivo Avaliar a sensibilidade e a especificidade da reação em cadeia de polimerase em tempo real quantitativa (quantitative real-time polymerase chain reaction, qPCR, em inglês) para a triagem do gene rDNA 16S, com a utilização do fluido sonicado de implantes ortopédicos. Métodos Um estudo retrospectivo foi realizado em 73 fluidos sonicados obtidos de pacientes com infecção associada aos implantes ortopédicos. As amostras foram submetidas a cultura convencional e a teste molecular utilizando ionização e dessorção a laser assistida por matriz com espectrometria de massa por tempo de voo (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF MS, em inglês) e qPCR para o gene rDNA 16S. Os valores limiares do ciclo foram usados para definir um ponto de corte para a qPCR do gene rDNA 16S para culturas negativas e positivas. Resultados Não foram observadas diferenças estatísticas entre os grupos de cultura positiva e negativa com base no tempo desde a primeira cirurgia até a infecção (p= 0,958), na idade (p= 0,269), ou nas comorbidades em geral. No entanto, uma diferença estatística foi encontrada entre a duração média do uso de antibióticos antes da remoção do dispositivo (3,41 versus 0,94; p= 0,016). O DNA bacteriano foi identificado em todas as amostras dos fluidos sonicados. Os limiares do ciclo médio de culturas positivas e negativas foram de 25,6 e 27,3, respectivamente (p< 0,001). Como uma ferramenta de diagnóstico, um corte do limite do ciclo de 26,89 demonstrou uma área sob a curva da característica de operação do receptor de 0,877 (p ≤ 0,001). Conclusão A presença de agentes antimicrobianos por mais de 72 horas diminuiu a positividade da cultura, mas não influenciou os resultados da qPCR. Apesar disso, a amplificação do rDNA 16S pode sobrestimar o diagnóstico de infecção.

Mannheimia varigena as the etiologic agent of lameness and coronary band lesion in cattle / Mannheimia varigena como agente etiológico de claudicação e lesão na banda coronária em bovinos

Mannheimia varigena was identified as the etiologic agent of lameness and coronary band lesion in 30% of cattle in a farm located in Rio Grande do Sul, Brazil. Swab samples from the lesions were cultured in McConkey Agar and Blood Agar for microbiological identification. Culture growth was submitted to Gram staining and Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) identification. Antimicrobial susceptibility test based on disc diffusion was performed for three antibiotics: ceftiofur, gentamicin and florfenicol. Furthermore, molecular characterization of 16S rDNA gene sequencing was performed and the data was used in a phylogenetic analysis. For that purpose, total DNA was extracted by thermo extraction directly from the bacterial colonies and the polymerase chain reaction (PCR) was performed. Gram-negative Mannheimia varigena strain LBV010/22 was identified as the causative of the lesions. The strain was susceptible to all antibiotics tested. The phylogenetic analysis demonstrated that the analyzed strain is closely related to M. varigena strains from pyelonephritis and respiratory tract. Overall, this is the first report of M. varigena as the causative agent of coronary band injury in bovine. Therefore, our findings show the importance of an accurate microbiological identification of infectious agent in lameness cases in order to prevent the occurrence and perform an appropriate treatment in the future.

Mannheimia varigena foi identificada como agente etiológico de claudicação e lesão de banda coronária em 30% dos bovinos de uma fazenda localizada no Rio Grande do Sul, Brasil. Amostras de swab das lesões foram cultivadas em Ágar McConkey e Ágar Sangue para identificação microbiológica. O crescimento da cultura foi submetido à coloração de Gram e identificação por Espectrometria de Massa de Ionização por Dessorção a Laser Assistida por Matriz (MALDI-TOF MS). O teste de suscetibilidade antimicrobiana baseado na difusão em disco foi realizado para três antibióticos: ceftiofur, gentamicina e florfenicol. Além disso, foi realizada a caracterização molecular do sequenciamento do gene 16S rDNA e o resultado utilizado para análise filogenética. Para tanto, o DNA total foi extraído por termoextração diretamente das colônias bacterianas e uma reação em cadeia da polimerase (PCR) foi realizada. Foi identificada como causadora das lesões a cepa gram-negativa de Mannheimia varigenaLBV010/22. Ela foi suscetível a todos os antibióticos testados. A análise filogenética demonstrou que a cepa analisada está intimamente relacionada às M. varigena presentes em pielonefrite e no trato respiratório. No geral, este é o primeiro relato de M. varigenacomo agente causador de lesão de banda coronária em bovinos. Portanto, nossos achados mostram a importância de uma identificação microbiológica precisa do agente infeccioso nos casos de claudicação, a fim de prevenir a ocorrência e realizar um tratamento adequado no futuro.

Identificación directa de microorganismos a partir de hemocultivos positivos utilizando espectrometría de masas (MALDI-TOF MS) / Direct identification of microorganisms in positive blood culture bottles by mass spectrometry (MALDI-TOF MS)

Resumen La espectrometría de masas (MALDI-TOF MS) permite la identificación de microorganismos directamente de las colonias en pocos minutos. En este estudio se ha desarrollado y evaluado un protocolo reducido para identificar microorganismos directamente de las botellas de hemocultivos positivos en 30 minutos con una alta sensibilidad y especificidad, utilizando MALDITOF. Un total de 2535 hemocultivos positivos fueron estudiados por el método directo de MALDI-TOF MS, a partir de una alícuota de sangre de las botellas y el método de colonia, utilizando los cultivos desarrollados en medios sólidos. Del total de hemocultivos positivos incluidos en este estudio, 2381 (93,9%) fueron monomicrobianos y 146 (5,8%) polimicrobianos. Mil trescientos treinta (55,9%) de los aislamientos correspondieron a cocos gram positivos, 922 (38,7%) a bacilos gram negativos, 60 (2,5%) a anaerobios, 36 (1,5%) a bacilos gram positivos y 13 a levaduras. La concordancia global entre ambos métodos fue del 81,7% a nivel de especie (90,0% para bacilos gram negativos, 76,7% para cocos gram positivos y 33,3% para bacilos gram positivos). Se identificó al menos un germen en el 88% de las botellas positivas con desarrollo polimicrobiano. Los resultados del presente estudio demostraron que el protocolo basado en MALDI-TOF MS permite la identificación microbiana directamente de hemocultivos positivos en un tiempo corto, con una alta precisión, con excepción de los bacilos gram positivos.

Abstract Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) enables the identification of microorganisms directly from colonies within minutes. In this study this technology was adapted and tested for use with blood culture bottles, thus allowing identification in 30 minutes once the blood culture is detected as positive by the automate. A total of 2535 blood culture bottles reported as positive were tested by MALDI-TOF MS directly from positive blood culture bottles and colonies. A total of 2381 (93.9%) and 146 (5.8%) of the positive blood cultures were monomicrobial and polymicrobial, respectively. And 1330 (55.9%), 922 (38.7%), 60 (2.5%), 36 (1.5%) and 13 of the isolates were gram-positive cocci (GPC), gram-negative bacilli (GNB), anaerobic bacteria, gram-positive bacilli (GPB) and yeast respectively. Concordance between both methods was 81.7% (76.7% of GPC, 90% of GNB, 74.2% of anaerobic bacteria and 33.3% of GPB) in monomicrobial cultures. Eighty eight per cent of the polymicrobial cultures were identified correctly in at least one of the two bacteria. The results of the present study show that this fast, MALDI-TOF MS based method allows microbial identification directly from positive blood culture in a short time, with a high accuracy, with the exception of gram-positive bacilli.

Resumo A espectrometria de massa (MALDI-TOF MS) permite a identificação de microorganismos diretamente das colônias em minutos. Nesse estudo, foi desenvolvido um protocolo reduzido para identificar microrganismos diretamente das garrafas de hemoculturas positivas em 30 minutos com alta sensibilidade e especificidade, utilizando MALDI-TOF. Um total de 2535 hemoculturas positivas foram relatadas -o método direto de MALDI-TOF MS, a partir de uma alíquota de sangue dos vidros e o método de colônia, a partir das culturas desenvolvidas em meios sólidos. Do total de hemoculturas positivas incluídas neste estudo, 2.381 (93,9%) eram monomicrobianas e 146 (5,8%) eram polimicrobianas. Mil trezentos e trinta (55,9%) dos isolados corresponderam a cocos gram-positivos, 922 (38,7%) bacilos gram-negativos, 60 (2,5%) anaeróbios, 36 (1,5%) bacilos gram-positivos e 13 leveduras. A concordância geral entre os dois métodos foi de 81,7% em nivel de especie (90,0% para bacilos gram-negativos, 76,7% para cocos gram-positivos e 33,3% para bacilos gram-positivos). Pelo menos um germe foi identificado em 88% dos vidros positivos com desenvolvimento polimicrobiano. Os resultados do presente estudo demonstraram que o protocolo baseado em MALDI-TOF MS permite a identificação microbiana diretamente de hemoculturas positivas em um curto espaço de tempo, com alta precisão, com exceção de bacilos gram-positivos.

Use of MALDI-TOF for identification and surveillance of gramnegative bacteria in captive wild psittacines / Uso do MALDI-TOF para identificação e monitoramento de bactérias Gram negativas em psitacídeos cativos

Microbiological studies of the sanitary and health status of psittacine birds that will be reintroduced is important in evaluating whether these animals act as carriers of pathogenic agents to other animals and humans. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a faster and more accurate method to identify bacteria than conventional microbiology methods. The aim of this study was to evaluate the health status of psittacines housed in captivity, by assessment of Gram-negative bacteria from fecal microbiota through MALDI- TOF MS identification. The results indicate high frequency of Gram-negative bacteria in feces (96.5%), especially from the Enterobacteriaceae family (88.7%). The most prevalent bacteria were Escherichia coli (39.0%), Proteus vulgaris (12.2%), Klebsiella spp. (12.1%) and Raoultella ornithinolytica (8.7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. and Escherichia hermannii were isolated with lower frequency. . All these agents are potentially pathogenic for parrots and can cause systemic infections in other animals and humans. These findings reinforce that MALDI- TOF MS proved to be a rapid and accurate method of identification of the microorganism and evaluation of the health status of psittacines, providing relevant data to assist decision-making regarding the sanitary protocols in wildlife centers, and possible future reintroduction of wild birds.

Estudos microbiológicos da sanidade de psitacídeos que serão reintroduzidos são importantes para avaliar se esses animais atuam como portadores de agentes patogênicos para outros animais e humanos. A espectrometria de massa por ionização/dessorção de matriz assistida por laser/tempo de vôo (MALDI-TOF MS) é um método mais rápido e preciso para identificar bactérias na comparação com métodos convencionais de microbiologia. O objetivo deste estudo foi avaliar o estado de saúde de psitacídeos cativos, identificando bactérias Gram-negativas da microbiota fecal por MALDI -TOF MS. Os resultados indicaram alta frequência de bactérias Gram-negativas nas fezes (96,5%), principalmente da família Enterobacteriaceae (88,7%). As mais prevalentes foram Escherichia coli (39,0%), Proteus vulgaris (12,2%), Klebsiella spp. (12,1%) e Raoultella ornithinolytica (8,7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. e Escherichia hermannii foram isolados com menor frequência. Todos esses agentes são potencialmente patogênicos para os papagaios e podem causar infecções sistêmicas em outros animais e seres humanos. Esses achados reforçam que o MALDI- TOF MS é um método rápido e preciso de identificação do microrganismo e avaliação do estado de saúde dos psitacídeos, fornecendo dados relevantes para auxiliar na tomada de decisões sobre os protocolos sanitários em centros de triagem de animais selvagens e sobre a possibilidade de reintrodução futura.

Species identification and antimicrobial susceptibility profile of bacteria associated with cow mastitis in southern Brazil / Identificação de espécies e perfil de suscetibilidade aos antimicrobianos de bactérias associadas à mastite bovina no sul do Brasil

Bovine mastitis is the most common disease in dairy cattle and responsible for economic losses in the milk industry. The present study aimed to identify the main species and to evaluate the antimicrobial susceptibility of bacterial isolates from cow herds with mastitis in dairy farms from southern Brazil. A total of 107 milk samples were collected from different cow herds in one important dairy producing region in southern Brazil, including farms located in ten cities from the Northeast region in the Rio Grande do Sul state. Bacterial strains were isolated and submitted to presumptive identification by classical bacteriological methods. Bacterial species were also identified by MALDI-TOF MS and antimicrobial susceptibility testing was performed with 12 antimicrobials commonly used in dairy farms. Fifty-one bacterial strains were isolated and the presumptive identification demonstrated the occurrence of Staphylococcus spp. (82.3%), Bacillus spp. (3.9%), Klebsiella spp. (3.9%), Streptococcus spp. (3.9%), Corynebacterium sp. (2%), Enterococcus sp. (2%) and Serratia sp. (2%). Forty-one isolates were successfully identified in the MALDI-TOF analysis, including 35 isolates from eleven different bacterial species. Importantly, there were eight different Staphylococcus species, with a high frequency of Staphylococcus chromogenes (48.6%) and Staphylococcus aureus (20%). Overall, bacterial isolates demonstrated resistance to penicillin (46.3%), tetracycline (39%), amoxicillin (36.6%), ampicillin (34.1%) and sulfamethoxazole/trimethoprim (31.7%). Enrofloxacin was the unique antimicrobial that all isolates were susceptible. In addition, there were six multidrug resistant isolates (five S. chromogenes and one S. aureus). This study highlights that bacterial pathogens with resistance to several antimicrobials were identified in cows from dairy farms in a very important milk producing region located in southern Brazil. Microbial identification of the bovine mastitis pathogens and determination of the antimicrobial profile is necessary for the rational use of the medicines.

A mastite bovina é a doença mais comum em gado leiteiro e responsável por perdas econômicas na indústria de laticínios. O presente estudo teve como objetivo identificar as principais espécies e avaliar a suscetibilidade antimicrobiana de isolados bacterianos de rebanhos bovinos com mastite em fazendas leiteiras no sul do Brasil. Um total de 107 amostras de leite foram coletadas em diferentes rebanhos bovinos em uma importante região produtora de leite do sul do Brasil, incluindo fazendas localizadas em 10 cidades da região Nordeste do estado do Rio Grande do Sul. As cepas bacterianas foram isoladas e submetidas à identificação presuntiva por métodos bacteriológicos clássicos. A identificação bacteriana foi confirmada por MALDI-TOF MS e o teste de sensibilidade antimicrobiana foi realizado com antimicrobianos comumente usados em fazendas leiteiras. Cinquenta e uma cepas bacterianas foram isoladas e a identificação presuntiva demonstrou a ocorrência de Staphylococcus spp. (82,3%), Bacillus spp. (3,9%), Klebsiella spp. (3,9%), Streptococcus spp. (3,9%), Corynebacterium sp. (2%), Enterococcus sp. (2%) e Serratia sp. (2%). Os 41 isolados foram identificados com sucesso na análise MALDI-TOF, incluindo 35 isolados de onze espécies bacterianas diferentes. É importante ressaltar que houve a ocorrência de oito espécies diferentes de Staphylococcus, com alta frequência de Staphylococcus chromogenes (48,6%) e Staphylococcus aureus (20%). No geral, os isolados bacterianos tiveram alta resistência à penicilina (46,3%), tetraciclina (39%), amoxicilina (36,6%), ampicilina (34,1%) e sulfametoxazol/trimetoprima (31,7%). A enrofloxacina foi o único antimicrobiano que todos os isolados foram suscetíveis. Além disso, havia seis isolados multirresistentes (cinco S. chromogenes e um S. aureus). Este estudo destaca que os patógenos bacterianos com resistência aos antimicrobianos estão presentes em fazendas leiteiras de subsistência em uma importante região produtora no sul do Brasil. É necessário o monitoramento constante dos patógenos da mastite bovina e a determinação de seu perfil antimicrobiano para o uso racional dos medicamentos.

Use of MALDI-TOF for identification and surveillance of gram-negative bacteria in captive wild psittacines / Uso do MALDI-TOF para identificação e monitoramento de bactérias Gram-negativas em psitacídeos cativos

Microbiological studies of the sanitary and health status of psittacine birds that will be reintroduced is important in evaluating whether these animals act as carriers of pathogenic agents to other animals and humans. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a faster and more accurate method to identify bacteria than conventional microbiology methods. The aim of this study was to evaluate the health status of psittacines housed in captivity, by assessment of Gram-negative bacteria from fecal microbiota through MALDI- TOF MS identification. The results indicate high frequency of Gram-negative bacteria in feces (96.5%), especially from the Enterobacteriaceae family (88.7%). The most prevalent bacteria were Escherichia coli (39.0%), Proteus vulgaris (12.2%), Klebsiella spp. (12.1%) and Raoultella ornithinolytica (8.7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. and Escherichia hermannii were isolated with lower frequency. . All these agents are potentially pathogenic for parrots and can cause systemic infections in other animals and humans. These findings reinforce that MALDI- TOF MS proved to be a rapid and accurate method of identification of the microorganism and evaluation of the health status of psittacines, providing relevant data to assist decision-making regarding the sanitary protocols in wildlife centers, and possible future reintroduction of wild birds.

Estudos microbiológicos da sanidade de psitacídeos que serão reintroduzidos são importantes para avaliar se esses animais atuam como portadores de agentes patogênicos para outros animais e humanos. A espectrometria de massa por ionização/dessorção de matriz assistida por laser/tempo de vôo (MALDI-TOF MS) é um método mais rápido e preciso para identificar bactérias na comparação com métodos convencionais de microbiologia. O objetivo deste estudo foi avaliar o estado de saúde de psitacídeos cativos, identificando bactérias Gram-negativas da microbiota fecal por MALDI -TOF MS. Os resultados indicaram alta frequência de bactérias Gram-negativas nas fezes (96,5%), principalmente da família Enterobacteriaceae (88,7%). As mais prevalentes foram Escherichia coli (39,0%), Proteus vulgaris (12,2%), Klebsiella spp. (12,1%) e Raoultella ornithinolytica (8,7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. e Escherichia hermannii foram isolados com menor frequência. Todos esses agentes são potencialmente patogênicos para os papagaios e podem causar infecções sistêmicas em outros animais e seres humanos. Esses achados reforçam que o MALDI- TOF MS é um método rápido e preciso de identificação do microrganismo e avaliação do estado de saúde dos psitacídeos, fornecendo dados relevantes para auxiliar na tomada de decisões sobre os protocolos sanitários em centros de triagem de animais selvagens e sobre a possibilidade de reintrodução futura.

Species identification and antimicrobial susceptibility profile of bacteria associated with cow mastitis in southern Brazil / Identificação de espécies e perfil de suscetibilidade aos antimicrobianos de bactérias associadas à mastite bovina no sul do Brasil

Bovine mastitis is the most common disease in dairy cattle and responsible for economic losses in the milk industry. The present study aimed to identify the main species and to evaluate the antimicrobial susceptibility of bacterial isolates from cow herds with mastitis in dairy farms from southern Brazil. A total of 107 milk samples were collected from different cow herds in one important dairy producing region in southern Brazil, including farms located in ten cities from the Northeast region in the Rio Grande do Sul state. Bacterial strains were isolated and submitted to presumptive identification by classical bacteriological methods. Bacterial species were also identified by MALDI-TOF MS and antimicrobial susceptibility testing was performed with 12 antimicrobials commonly used in dairy farms. Fifty-one bacterial strains were isolated and the presumptive identification demonstrated the occurrence of Staphylococcus spp. (82.3%), Bacillus spp. (3.9%), Klebsiella spp. (3.9%), Streptococcus spp. (3.9%), Corynebacterium sp. (2%), Enterococcus sp. (2%) and Serratia sp. (2%). Forty-one isolates were successfully identified in the MALDI-TOF analysis, including 35 isolates from eleven different bacterial species. Importantly, there were eight different Staphylococcus species, with a high frequency of Staphylococcus chromogenes (48.6%) and Staphylococcus aureus (20%). Overall, bacterial isolates demonstrated resistance to penicillin (46.3%), tetracycline (39%), amoxicillin (36.6%), ampicillin (34.1%) and sulfamethoxazole/trimethoprim (31.7%). Enrofloxacin was the unique antimicrobial that all isolates were susceptible. In addition, there were six multidrug resistant isolates (five S. chromogenes and one S. aureus). This study highlights that bacterial pathogens with resistance to several antimicrobials were identified in cows from dairy farms in a very important milk producing region located in southern Brazil. Microbial identification of the bovine mastitis pathogens and determination of the antimicrobial profile is necessary for the rational use of the medicines.(AU)

A mastite bovina é a doença mais comum em gado leiteiro e responsável por perdas econômicas na indústria de laticínios. O presente estudo teve como objetivo identificar as principais espécies e avaliar a suscetibilidade antimicrobiana de isolados bacterianos de rebanhos bovinos com mastite em fazendas leiteiras no sul do Brasil. Um total de 107 amostras de leite foram coletadas em diferentes rebanhos bovinos em uma importante região produtora de leite do sul do Brasil, incluindo fazendas localizadas em 10 cidades da região Nordeste do estado do Rio Grande do Sul. As cepas bacterianas foram isoladas e submetidas à identificação presuntiva por métodos bacteriológicos clássicos. A identificação bacteriana foi confirmada por MALDI-TOF MS e o teste de sensibilidade antimicrobiana foi realizado com antimicrobianos comumente usados em fazendas leiteiras. Cinquenta e uma cepas bacterianas foram isoladas e a identificação presuntiva demonstrou a ocorrência de Staphylococcus spp. (82,3%), Bacillus spp. (3,9%), Klebsiella spp. (3,9%), Streptococcus spp. (3,9%), Corynebacterium sp. (2%), Enterococcus sp. (2%) e Serratia sp. (2%). Os 41 isolados foram identificados com sucesso na análise MALDI-TOF, incluindo 35 isolados de onze espécies bacterianas diferentes. É importante ressaltar que houve a ocorrência de oito espécies diferentes de Staphylococcus, com alta frequência de Staphylococcus chromogenes (48,6%) e Staphylococcus aureus (20%). No geral, os isolados bacterianos tiveram alta resistência à penicilina (46,3%), tetraciclina (39%), amoxicilina (36,6%), ampicilina (34,1%) e sulfametoxazol/trimetoprima (31,7%). A enrofloxacina foi o único antimicrobiano que todos os isolados foram suscetíveis. Além disso, havia seis isolados multirresistentes (cinco S. chromogenes e um S. aureus). Este estudo destaca que os patógenos bacterianos com resistência aos antimicrobianos estão presentes em fazendas leiteiras de subsistência em uma importante região produtora no sul do Brasil. É necessário o monitoramento constante dos patógenos da mastite bovina e a determinação de seu perfil antimicrobiano para o uso racional dos medicamentos.(AU)

Identification of BRAF V600E mutation in odontogenic tumors by high-performance MALDI-TOF analysis / 国际口腔科学杂志·英文版

Odontogenic tumors are rare lesions with unknown etiopathogenesis. Most of them are benign, but local aggressiveness, infiltrative potential, and high recurrence rate characterize some entities. The MAP-kinase pathway activation can represent a primary critical event in odontogenic tumorigenesis. Especially, the BRAF V600E mutation has been involved in 80-90% of ameloblastic lesions, offering a biological rationale for developing new targeted therapies. The study aims to evaluate the BRAF V600E mutation in odontogenic lesions, comparing three different detection methods and focusing on the Sequenom MassARRAY System. 81 surgical samples of odontogenic lesions were subjected to immunohistochemical analysis, Sanger Sequencing, and Matrix-Assisted Laser Desorption/Ionization-Time of Flight mass spectrometry (Sequenom). The BRAF V600E mutation was revealed only in ameloblastoma samples. Moreover, the presence of BRAF V600E was significantly associated with the mandibular site (ρ = 0.627; P value <0.001) and the unicystic histotype (ρ = 0.299, P value <0.001). However, any significant difference of 10-years disease-free survival time was not revealed. Finally, Sequenom showed to be a 100% sensitive and 98.1% specific, suggesting its high-performance diagnostic accuracy. These results suggest the MAP-kinase pathway could contribute to ameloblastic tumorigenesis. Moreover, they could indicate the anatomical specificity of the driving mutations of mandibular ameloblastomas, providing a biological rational for developing new targeted therapies. Finally, the high diagnostic accuracy of Sequenom was confirmed.

Spatial distribution characteristics of metabolities in rhizome of Paris polyphylla var. yunnanensis: based on MALDI-MSI / 中国中药杂志

In this study, a method was established for in-situ visualization of metabolite distribution in the rhizome of Paris polyphylla var. yunnanensis. To be specific, through matrix-assisted laser desorption/ionization-mass spectrometry imaging(MALDI-MSI), the spatial locations of steroidal saponins, amino acids, organic acids, phytosterols, phytoecdysones, nucleosides, and esters in rhizome of the medicinal plant were directly analyzed, and six unknown compounds with differential distribution in rhizome tissues were identified. The specific procedure is as follows: preparation of rhizome tissue section, matrix screening and optimization, and MALDI-MSI analysis. The results showed that the steroidal saponins were mainly distributed in the central, amino acids in epidermis and cortex, low-molecular-weight organic acids in central epidermis, phytosterols in the epidermis and lateral cortex, the phytoecdysones in epidermis and cortex, nucleosides(uneven distribution) in epidermis and cortex, growth hormones around the epidermis and cortex, particularly outside the cortex, and esters in cortex with unobvious difference among different tissues. In this study, the spatial distribution of meta-bolites in the rhizome of P. polyphylla var. yunnanensis was characterized for the first time. The result can serve as a reference for identifying and extracting endogenous metabolites of P. polyphylla var. yunnanensis, exploring the synthesis and metabolism mechanisms of the metabolites, and evaluating the quality of medicinal materials.

Evaluation of the MALDI-TOF MS method for routine identification of Candida species in a tertiary care hospital in Thailand

Aims@#Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used to identify Candida spp. in diagnostic laboratories due to its strength in providing accurate information results, speed and cost-effectiveness. However, its accuracy varies on instrument platform, reference database, sample preparation techniques and interlaboratory comparisons. Therefore, the use of MALDI-TOF MS for species identification was evaluated against traditional biochemical identification, namely BrillianceTM Candida and Remel RapIDTM Yeast Plus System.@*Methodology and results@#To evaluate and compare identification efficiency, turnaround time and consumable cost, 194 clinical isolates of Candida were collected. The results showed overall 85.6% concordant identification between two methods with 94.9-99.5% and 100% accuracy in traditional and MALDI-TOF MS, respectively, in the identification of four common Candida species; C. albicans, C. tropicalis, C. parapsilosis sensu stricto and C. glabrata sensu stricto. Other Candida species were also identified with 85.6% and 97.5% accuracy rates by traditional and MALDI-TOF MS, respectively. Additionally, identification using MALDI-TOF MS reduced overall turnaround time and cost by approximately 99.8% and 86.5%, respectively.@*Conclusion, significance and impact of study@#This study highlights the performance of MALDI-TOF MS, which is more accurate in identifying Candida spp. with a less hands-on approach, cheaper cost and shorter turnaround time.

Interações populacionais mediadas por quorum sensing na microbiota do soro-fermento utilizado na produção do queijo Canastra / Population interactions mediated by quorum sensing in the microbiota of the endogenous culture used in the production of canasta cheese

Bactérias regulam a expressão de diversos fenótipos de acordo com a sua densidade populacional, em um comportamento conhecido como quorum sensing. Em micro-organismos de origem alimentar, o quorum sensing pode influenciar na formação de biofilmes, produção de toxinas e de enzimas hidrolíticas. Em bactérias Gram-negativas a sinalização é normalmente mediada por moléculas de N-acilhomoserina lactona (AHLs), conhecidas por autoindutor 1 (AI-1). Estudos revelam a inibição do quorum sensing nestas bactérias por enzimas que degradam as AHLS, em um processo denominado quorum quenching. Tipicamente brasileiro, o queijo Canastra é um produto artesanal maturado, produzido a partir de leite cru e do pingo, um tipo de soro-fermento coletado e utilizado diariamente na produção. A composição microbiana do pingo é diversificada e característica da região produtora. Essa combinação de bactérias, única em cada queijaria, resulta em aroma e textura típicos. Enquanto a microbiota Gram-positiva contribui para o desenvolvimento de sabor, textura e aroma no produto, bactérias Gram-negativas nesses queijos são geralmente associadas à formação de olhaduras, aromas desagradáveis, má coagulação da massa e até à patogenicidade. Este trabalho visou analisar a interação entre a microbiota Gram-positiva e Gram-negativa presente no pingo pela detecção dos sistemas de quorum sensing e quorum quenching nas amostras. A presença de AHLs foi avaliada em 45 amostras de pingo, a partir da extração em acetato de etila acidificado e da avaliação dos extratos por meio de bioensaios com Agrobacterium tumefaciens WCF47(pCF218)(pCF372) e KYC55(pJZ410)(pJZ372)(pJZ384), resultando em apenas uma amostra positiva. Em seguida, 350 isolados foram obtidos a partir de 11 amostras de pingo, sendo 200 isolados classificados como Gram-positivos e 150 Gram-negativos. Os Gramnegativos foram avaliados quanto à produção de AHLs in vitro através de ensaio em placa utilizando as estirpes biossensoras A. tumefaciens WCF47(pCF218)(pCF372), Chromobacterium violaceum CV026 e Escherichia coli pSB403, resultando em 39 isolados produtores de AHLs, provenientes de 10 pingos diferentes. Já os isolados Gram-positivos foram analisados quanto à capacidade de inibição do QS utilizando as estirpes biossensoras C. violaceum CV026 e A. tumefaciens WCF47(pCF218)(pCF372), em meio suplementado com C6-HSL ou 3-oxo-C12-HSL. Foi detectada a inibição total da resposta ao quórum por 78 isolados testados, enquanto a inibição parcial foi provocada por outros 63. A inibição do crescimento das estirpes biossensoras também foi observada para 24 isolados. Os isolados promotores de inibição parcial foram recultivados em meio mínimo com C6-HSL ou 3-oxo-C12-HSL como únicas fontes de carbono. Foram recuperados 28 isolados, e a ação desses sobre diferentes substratos foi avaliada, resultando em 22 isolados produtores de lactonases e 6 produtores de acilase. Os 39 isolados Gram-negativos e os 28 isolados Gram-positivos finais foram identificados por MALDI-TOF MS, resultando, segundo o conhecimento do autor, no primeiro relato de produção de AHLs por Pseudomonas fulva, Enterobacter xiangfangensis e Lelliottia amnigena, bem como a produção de lactonases por Staphylococcus xylosus e a produção de acilase por S. aureus, Microbacterium maritypicum e Rothia kristinae. Este trabalho mostrou que interações populacionais mediadas por quorum sensing dependente de AHLs na microbiota do soro-fermento são possíveis. Porém, essas interações estão propensas a serem inibidas por meio de lactonases e acilases produzidas por parte das bactérias Gram-positivas

Bacteria regulate the expression of different phenotypes according to their population density, in a behavior known as quorum sensing. In food-borne microorganisms, quorum sensing can influence the formation of biofilms, production of toxins and hydrolytic enzymes. In Gram-negative bacteria, signaling is normally mediated by Nacyl homoserine lactone molecules (AHLs), known as autoinducer 1 (AI-1). Studies reveal the inhibition of quorum sensing in these bacteria by enzymes that degrade AHLS, in a process called quorum quenching. Typically Brazilian, Canastra cheese is a matured artisanal product, produced from raw milk and pingo, a type of endogenous culture collected and used daily in production. The microbial composition of pingo is diverse and characteristic of the producing region. This combination of bacteria, unique in each cheese factory, results in a typical aroma and texture. While the Gram-positive microbiota contributes to the development of flavor, texture and aroma in the product, Gram-negative bacteria in these cheeses are generally associated with the formation of eyes, off-flavors, poor curd coagulation and even pathogenicity. Thus, this work aimed to analyze the interaction between the Gram-positive and Gram-negative microbiota present in this culture by detecting quorum sensing and quorum quenching systems in the samples. The presence of AHLs was evaluated in 45 samples of pingo, with extraction with acidified ethyl acetate and the evaluation of the extracts through bioassays with Agrobacterium tumefaciens WCF47(pCF218)(pCF372) and KYC55(pJZ410)(pJZ372)(pJZ384 ), resulting in only one positive sample. Then, 350 isolates were obtained from 11 endogenous culture samples, with 200 being classified as Gram-positive and 150 Gram-negative. Gram-negatives were evaluated for the production of AHLs in vitro by plaque assay using the biosensor strains A. tumefaciens WCF47(pCF218)(pCF372), Chromobacterium violaceum CV026 and Escherichia coli pSB403, resulting in 39 AHL-producing isolates from 10 different samples. Gram-positive isolates were analyzed for their ability to inhibit quorum sensing using biosensor strains C. violaceum CV026 and A. tumefaciens WCF47(pCF218)(pCF372), in medium supplemented with N-hexanoyl-L-homoserine lactone or 3-oxo-dodecanoyl-Lhomoserine lactone. Total inhibition of the quorum response was detected by 78 tested isolates, while partial inhibition was caused by 63. Growth inhibition of biosensor strains was also observed for 24 isolates. Partial inhibition promoter isolates were recultured on minimal medium with C6-HSL or 3-oxo-C12-HSL as sole carbon sources. Twenty-eight isolates were recovered, and the action of these isolates on different substrates was evaluated, resulting in 22 lactonase producers and 6 acylase producers. The 39 Gram-negative isolates and the final 28 Grampositive isolates were identified by MALDI-TOF MS, resulting, to the best of the author's knowledge, in the first report of AHL production by Pseudomonas fulva, Enterobacter xiangfangensis and Lelliottia amnigena, as well as the lactonase production by Staphylococcus xylosus and acylase production by S. aureus, Microbacterium maritypicum and Rothia kristinae. This work demonstrated that population interactions mediated by AHLs-dependent quorum sensing in Canastra cheese endogenous culture microbiota are possible. However, these interactions are prone to inhibition by lactonases and acylases produced by Gram-positive bacteria

Antimicrobial activity and partial chemical structure of acylpolyamines isolated from the venom of the spider Acanthoscurria natalensis

Background: Acylpolyamines are one of the main non-peptide compounds present in spider venom and represent a promising alternative in the search for new molecules with antimicrobial action. Methods: The venom of Acanthoscurria natalensis spider was fractionated by reverse-phase liquid chromatography (RP-HPLC) and the antimicrobial activity of the fractions was tested using a liquid growth inhibition assay. The main antimicrobial fraction containing acylpolyamines (ApAn) was submitted to two additional chromatographic steps and analyzed by MALDI-TOF. Fractions of interest were accumulated for ultraviolet (UV) spectroscopy and ESI-MS/MS analysis and for minimum inhibitory concentration (MIC) and hemolytic activity determination. Results: Five acylpolyamines were isolated from the venom with molecular masses between 614 Da and 756 Da, being named ApAn728, ApAn614a, ApAn614b, ApAn742 and ApAn756. The analysis of UV absorption profile of each ApAn and the fragmentation pattern obtained by ESI-MS/MS suggested the presence of a tyrosyl unit as chromophore and a terminal polyamine chain consistent with structural units PA43 or PA53. ApAn presented MIC between 128 µM and 256 µM against Escherichia coli and Staphylococcus aureus, without causing hemolysis against mouse erythrocytes. Conclusion: The antimicrobial and non-hemolytic properties of the analyzed ApAn may be relevant for their application as possible therapeutic agents and the identification of an unconventional chromophore for spider acylpolyamines suggests an even greater chemical diversity.(AU)

Novel neuroprotective peptides in the venom of the solitary scoliid wasp Scolia decorata ventralis

Background Solitary wasp venoms may be a rich source of neuroactive substances, since their venoms are used for paralyzing preys. We have been exploring bioactive constituents of solitary wasp venoms and, in this study, the component profile of the venom from a solitary scoliid wasp, Scolia decorata ventralis, was investigated through a comprehensive analysis using LC-MS. Two peptides were synthesized, and their neuroprotective properties were evaluated. Methods A reverse-phase HPLC connected to ESI-MS was used for LC-MS analyses. Online mass fingerprinting was performed from TIC, and data-dependent tandem mass spectrometry gave the MS/MS spectra. The sequences of two major peptide components were determined by MALDI-TOF/TOF MS analysis, confirmed by solid phase synthesis. Using the synthetic peptides, biological activities were assessed. Cell integrity tests and neuroprotection analyzes using H2O2 as an oxidative stress inducer were performed for both peptides. Results Online mass fingerprinting revealed that the venom contains 123 components, and the MS/MS analysis resulted in 33 full sequences of peptide components. The two main peptides, α-scoliidine (DYVTVKGFSPLR) and β-scoliidine (DYVTVKGFSPLRKA), present homology with the bradykinin C-terminal. Despite this, both peptides did not behave as substrates or inhibitors of ACE, indicating that they do not interact with this metallopeptidase. In further studies, β-scoliidine, but not α -scoliidine, showed protective effects against oxidative stress-induced neurotoxicity in PC12 cells through integrity and metabolism cell assays. Interestingly, β-scoliidine has the extension of the KA dipeptide at the C-terminal in comparison with α-scoliidine. Conclusion Comprehensive LC-MS and MS/MS analyses from the Scolia decorata ventralis venom displayed the component profile of this venom. β-scoliidine showed an effective cytoprotective effect, probably due to the observed increase in the number of cells. This is the first report of solitary wasp venom peptides showing neuroprotective activity.(AU)

Mass Spectrometry Imaging and Its Application in Forensic Toxicology / 法医学杂志

Mass spectrometry imaging （MSI） is a new imaging technology that can simultaneously detect and record the spatial distribution information of multiple molecules on the sample surface without labeling. The main principle of MSI is to combine mass spectrometry with imaging technology and irradiate the sample slice with ion beam or laser to ionize the molecules on its surface, obtain the mass spectrometry signal through the detector, convert the obtained data into pixel points by the imaging software, and then construct the spatial distribution image of the target compound on the tissue surface. The sample preparation for MSI include： sample collection and storage, tissue section, tissue pretreatment, selection and application of matrix. At present, this technology has been widely used in the fields of biomedicine, new drug development and proteomics, and its application in the field of forensic toxicology has also gradually attracted attention. This article reviews the principles and sample preparation process of MSI, describes the application of MSI in abused substances and metabolites of various material matrices, herbal mixtures, latent fingerprints, hair and animal and plant tissues, and discusses the prospects of the application of this technology in forensic toxicology, in order to provide ideas and references for the application of MSI technology in forensic toxicology.

PEGuilação do peptídeo Lunatina-1 visando novo candidato à fármaco antimicrobiano / PEGylation of the peptide Lunatin-1 aiming at novel candidate for antimicrobial drug

As infecções relacionadas à assistência à saúde (IRAS) podem ser causadas por bactérias, vírus e fungos, sendo de extrema importância para o sistema de tratamento e pacientes. Com o alarmante avanço no surgimento de bactérias resistentes, tem havido uma preocupação crescente com as IRAS de origem bacteriana. Nesse sentido, várias pesquisas buscam alternativas para os fármacos antimicrobianos convencionais, sendo que os peptídeos antimicrobianos (AMPs), como a lunatina-1, aparecem como moléculas promissoras. No entanto, os AMPs geralmente apresentam rápida degradação proteolítica no trato gastrointestinal e meia-vida curta na corrente sanguínea, principais fatores limitantes para sua aplicação no tratamento de IRAS. Entre as estratégias empregadas para superar esses inconvenientes, a PEGuilação apresenta-se como alternativa eficaz que aumenta o tempo de circulação in vivo dos AMPs, resultando na melhora farmacocinética e, em alguns casos, também farmacodinâmica. A PEGuilação consiste na ligação covalente de cadeias de polietileno glicol (PEG) ao peptídeo, que pode ser efetuada por meio de uma reação aleatória ou sítio-específica. Neste trabalho, desenvolveu-se uma PEGuilação sítio-específica no N-terminal da lunatina-1 empregando-se mPEG-NHS de 2 kDa em tampão fosfato 100 mM, visando o aumento da solibilidade deste peptídeo, bem como para avaliar sua ação antimicrobiana. Com relação à reação de PEGuilação, avaliou-se a influência da razão molar PEG:peptídeo (10:1 ou 15:1) a pH 8,5. Foi obtido um rendimento de PEGuilação de 92%, através da análise por RP-HPLC quantitativo. Quanto à purificação da lunatina-1 PEGuilada, foi empregada a técnica semi-preparativa de RP-HPLC utilizando a coluna C18. A caracterização da lunatina-1 PEGuilada, incluindo determinação do grau de PEGuilação, foi realizada por MALDI-TOF Autoflex Speed (Bruker), mostrando que a molécula foi monoPEGuilada na região N-terminal. A atividade antimicrobiana de lunatina-1 livre e bioconjugada frente a diferentes cepas bacterianas, sendo duas Gram-negativas (ATCC 25922 de Escherichia coli e ATCC 9027 de Pseudomonas aeruginosa) e uma Gram-positiva (CECT 239 de Staphylococcus aureus), foi estudada por determinação da concentração inibitória mínima (CIM) em microplaca, sendo que foram obtidos valores de CIM de 86 e 140 µM para o peptídeo liver e PEGuilado, respetivamente. O potencial hemolítico também foi estudado, sendo que a forma PEGuilada mostrou significativa redução da atividade hemolítica em comparação à forma livre. Em suma, a PEGuilação da lunatina-1, aumenta a sua solubilidade e reduz a atividade hemolítica. Porém, para viabilizar esta estratégia a PEGuilação deve ser reversível, pois a conjugação ao polímero reduz atividade antimicrobiana

Health care-related infections (HAIs) caused by bacteria, viruses and fungi are extremely important for patients and health systems. With the alarming advance in the emergence of resistant bacteria, a growing concern with HAIs of bacterial origin is observed. In this sense, several studies investigate alternatives to conventional antimicrobial drugs and antimicrobial peptides (AMPs), such as lunatin-1, appear as promising molecules. However, AMPs generally show rapid proteolytic degradation in the gastrointestinal tract and short half-life in the bloodstream, the main limiting factors for their therapeutic application to treat HAIs. Among the strategies used to overcome these drawbacks, PEGylation presents itself as an effective alternative that increases the in vivo circulation time of AMPs, resulting in improved pharmacokinetics and, in some cases, also pharmacodynamics. PEGylation consists on the covalent attachment of polyethylene glycol (PEG) chains to the peptide, which can be carried out by means of a random or site-specific reaction. In this work, a site-specific PEGylation was developed at the N-terminus of lunatin-1 using 2 kDa mPEG-NHS to increase the solubility of this peptide, as well as to evaluate its antimicrobial activity. Regarding the PEGylation reaction, the influence of the molar ratio PEG: peptide (10: 1 or 15: 1) at pH 8.5 was evaluated and a PEGylation yield of 92% was obtained, based on quantitative RP-HPLC analysis. As for the purification of PEGylated lunatin-1, semi-preparative RP-HPLC was used. The characterization of PEGylated lunatin-1, including determination of the degree of PEGylation, was performed by MALDI-TOF Autoflex Speed (Bruker), showing that the peptide was monoPEGylated in the N-terminal region. The antimicrobial activity of free and bioconjugated lunatin-1 against different bacterial strains, two Gram-negative (ATCC 25922 from Escherichia coli and ATCC 9027 from Pseudomonas aeruginosa), and one Gram positive (CECT 239 from Staphylococcus aureus), was studied by determining the minimum inhibitory concentration (MIC) in a microplate, resulting in MIC values of 86 and 140 µM for the free and PEGylated peptide, respectively. The hemolytic potential was also studied and the PEGylated form showed a significant reduction in hemolytic activity compared to the free form. In short, the PEGylation of lunatin-1 increases its solubility and reduces hemolytic activity. However, to make this strategy feasible, PEGylation must be reversible, since the conjugation to the polymer reduces antimicrobial activity

Diseño, estandarización e implementación de una nueva técnica en Guatemala, para el diagnóstico rápido de hemocultivos positivos, utilizando la tecnología Maldi-tof / Design, standardization and implementation of a new technique in Guatemala, for the rapid diagnosis of positive blood cultures, using Maldi-tof technology

Las enfermedades infecciosas son un problema de salud que a pesar de los avances médicos siguen cobrando vi-das en todo el mundo; como las septicemias. La presente investigación tuvo por objetivo diseñar, estandarizar e implementar un protocolo inexistente en Guatemala, para el diagnóstico rutinario de hemocultivos positivos dentro de las instalaciones del Laboratorio Clínico del Hospital General San Juan de Dios, lugar en donde se encuentra el único espectrómetro de masas de tipo Maldi-tof (Matrix Assisted Laser Desorption Ionization-Time of flight-mass spectrometry).Se utilizaron 240 muestras de pacientes de los diferentes servicios. El diagnóstico se realizó compa-rando las identificaciones obtenidas a partir de cultivos microbiológicos puros con muestras directas de botellas con caldo BHI(Brain Heart Infusion).Los resultados de las dos metodologías fueron evaluados con el diseño estadístico "apareado o emparejado en grupo". La comparación no evidenció discordancia en las identificaciones; pero sí en los tiempos de respuesta. La reducción de tiempo fue de 42.9 h para bacterias Gram positivo, 45.0 h para bacterias Gram negativo y 126.2 h para levaduras, todos a favor de identificaciones a partir de muestras directas. Con esta investigación se pretende ofrecer una nueva alternativa que permitirá brindar un diagnóstico rápido, confiable y certero a la población guatemalteca. También permitirá reducir la morbimortalidad de los pacientes con septicemias, promover el ahorro de insumos hospitalarios, disminuir el tiempo de estancia hospitalaria, ahorrar el consumo de antibióticos innecesarios y contribuir indirectamente a combatir la resistencia antimicrobiana; un problema actual de gran importancia a nivel mundial.

Infectious diseases are a health problem that despite medical advances in terms of diagnosis continue to take lives worldwide, such is the case of sepsis. The purpose of this research was to design, standardize and implement a non-existent protocol in Guatemala, for the routine diagnosis of positive blood cultures, within the facilities of the clinical laboratory of the San Juan de Dios General Hospital; where the only Maldi-tof (Matrix Assisted Laser Desorp-tion Ionization-Time of flight-mass spectrometry) type mass spectrometer is located. For this, 240 samples of positive blood cultures were used, coming from patients of the different services. The microbiological diagnosis was made by comparing the identification data obtained from pure microbiological cultures and direct samples of BHI broth (Brain Heart Infusion) bottles. The results of the two methodologies were evaluated based on "paired or matched in groups" statistical design. The Maldi-tof technique did not show disagreement regarding identification between the two types of samples; but it did in the response time. The time reduction was 42.9 h for Gram positive bacteria, 45.0 h for Gram negative and 126.2 h for yeasts, supporting identification from direct samples. This research aims to provide a new diagnostic alternative that will allow access to fast, reliable, and accurate results for the Guatemalan population. It will also help to reduce e morbidity and mortality rates of patients with sepsis, to promote hospital supplies savings, decrease the patient length of stay, save unnecessary antibiotics and indirectly contribute to combating antimicrobial resistance; a critical problem faced by the world today.

Identificación de aislamientos de Candida auris recuperados a través de la vigilancia por laboratorio en Colombia: un reto para el diagnóstico / Identification of Candida auris isolates recovered through laboratory surveillance in Colombia: a challenge for diagnostic

Resumen Objetivo: Comparar los resultados obtenidos de diferentes sistemas de identificación de C. auris. Métodos: Análisis descriptivo con datos recopilados durante 2016-19 mediante la vigilancia nacional. Se evaluaron los resultados generados por los sistemas MicroScan, Phoenix BD, VITEK 2 y MALDI-TOF MS de instituciones hospitalarias de 843 aislamientos clínicos sospechosos de C. auris remitidos al INS y se compararon con los resultados generados de confirmación a través de MALDI- TOF MS (Bruker Daltonics) o PCR. Resultados: De los 843 aislamientos clínicos remitidos al INS, el 81,7% fueron confirmados como C. auris mediante MALDI- TOF MS o PCR en el INS y el resto, 18,3%, fueron identificados como otras especies de Candida spp. Las identificaciones correctas enviadas por los laboratorios representaron el 42,4%. MicroScan identificó C. auris principalmente como C. haemulonii, C. guilliermondii, C. albicans y C. famata; Phoenix BD, VITEK 2 y MALDI-TOF MS identificó C. auris como C. haemulonii. Discusión: Estudios señalan que C. auris exhibe una estrecha relación filogenética con C. haemulonii. Las identificaciones discrepantes pueden darse debido a que las bases de datos de los sistemas de diagnóstico son limitadas para este patógeno. Las deficiencias de los sistemas comerciales para la identificación de C. auris deben ser complementados con otros sistemas como MALDI-TOF MS o pruebas moleculares.

Abstract Objective: To compare the identification results obtained by different identification systems of C. auris isolates. Methods: A descriptive study with data collected during the years 2016-19 through surveillance. The results generated by the MicroScan, Phoenix BD, VITEK 2 and MALDI-TOF MS systems of 843 clinical isolates of C. auris submitted to the INS were evaluated and compared with the results generated from confirmation through MALDI-TOF MS (Bruker Daltonics) or PCR. Results: Out of 843 clinical isolates submitted to the INS, 81.7% were confirmed as C. auris by MALDITOF MS or PCR in the INS and the rest, 18.3%, were identified as other species of Candida spp. The correct identifications sent by the laboratories was 42.4%. MicroScan identified C. auris as C. haemulonii, C. guilliermondii, C. albicans and C. famata; Phoenix BD, VITEK 2 and MALDI-TOF MS identified C. auris as C. haemulonii. Discussion: Studies indicate that C. auris exhibits a close phylogenetic relationship with C. haemulonii. In addition, discrepant identifications may occur because the databases of diagnostic systems are limited with reference to this pathogen. The deficiencies of commercial systems for the identification of C. auris must be complemented with other systems such as MALDI-TOF MS or molecular tests.

Burkholderia cepacia complex: 11 years of surveillance in patients with Cystic Fibrosis in Posadas, Argentina

Abstract Cystic fibrosis patients with Burkholderia cepacia complex pulmonary infections have high morbidity and mortality. Worldwide, this disease is undergoing substantial epidemiological changes. Advances in the diagnosis and treatment have conditioned an increase in child sur-vival as well as in the proportion of affected adults. In order to know our reality, we refer to an epidemiological study in 64 CF patients during 11 years of surveillance, focusing on infections caused by Burkholderia species. Conventional and automated phenotypic tests, restriction fragment length polymorphism-recA, recA gene sequencing, and matrix-assisted laser desorp-tion ionization-time of flight (MALDI-TOF) mass spectrometry were applied. Bacterial isolates were also tested for antimicrobial susceptibility patterns. The prevalence of Burkholderia cepacia complex was 9.4%. Based on recA gene sequencing, the most common species identified were Burkholderia cenocepacia (67.3%) and Burkholderia vietnamiensis (20.3%). Ceftazidime and meropenem were the most active, inhibiting 53% and 46% of isolates, respectively. This report represents the first systematic study of Burkholderia infections in our CF population since beginning of monitoring and treatment and highlights the importance of continued longitudinal studies.

Resumen Los pacientes con fibrosis quística (FQ) con infecciones pulmonares causadas por especies del complejo Burkholderia cepacia tienen una alta morbimortalidad. En todo el mundo, esta enfermedad está experimentando cambios epidemiológicos sustanciales. Los avances en el diagnóstico y el tratamiento han condicionado un aumento en la supervivencia infantil, así como en la proporción de adultos afectados. Para conocer nuestra realidad, nos referimos a un estudio epidemiológico en 64 pacientes con FQ durante 11 años de vigilancia, focalizando las infecciones causadas por especies del género Burkholderia. Se aplicaron pruebas fenotípicas convencionales y automatizadas, polimorfismo de longitud de fragmentos de restricción-recA, secuenciación del gen recA y espectrometría de masa MALDI-TOF. Los aislados bacterianos también se analizaron para determinar los patrones de susceptibilidad antimicrobiana. La prevalencia de complejo B. cepacia fue del 9,4%. Con base en la secuenciación del gen recA, las especies más comunes identificadas fueron Burkholderia cenocepacia (67,3%) y Burkholderia vietnamiensis (20,3%). Ceftazidima y meropenem fueron los antibióticos más activos e inhibieron el 53 y el 46% de los aislamientos, respectivamente. Este informe representa el primer estudio sistemático de las infecciones por Burkholderia en nuestra población desde el comienzo de la monitorización y el tratamiento, y resalta la importancia de continuar los estudios de vigilancia longitudinales.