The influence of growth time on the identification of Bartonella henselae strains by MALDI-TOF mass spectrometry

ABSTRACT Bartonella spp. are bacteria responsible for neglected diseases worldwide. Bartonella henselae is the species most associated with human infections. It is associated with a large spectrum of clinical manifestations and is potentially fatal. The identification of Bartonella spp. is considered a challenge in clinical routine. These bacteria are fastidious, and the time required to isolate them varies from one to six weeks. MALDI-TOF mass spectrometry has emerged as an application for research on Bartonella spp. , and has still been little explored. We investigated whether three different B. henselae strains with different growth times—14 and 28 days—could be correctly identified by MALDI-TOF mass spectra fingerprint comparison and matching. We found that the spectra from strains with different growth times do not match each other, leading to misidentification. We suggest creating database entries with multiple spectra from strains with different growth times to increase the chances of accurate identification of Bartonella spp. by MALD-TOF MS.

Actinomyces europaeus (Gleimia europaea) asociado con absceso cerebral: comunicación de tres casos / Actinomyces europaeus (Gleimia europaea) associated with brain abscess: A reportof three cases

Resumen El absceso cerebral es una infección focal caracterizada por acumulación de pus enel parénquima cerebral; su diagnóstico es de urgencia debido a la alta mortalidad que acarrea.Presentamos tres casos de pacientes con abscesos cerebrales con foco otogénico de origen poli-microbiano, que presentaron en común el aislamiento de Actinomyces europaeus, agente nodescrito hasta el momento en esta localización. A. europaeus fue identificado por la metodo-logía convencional, por espectrometría de masas por desorción/ionización asistida por matriz(MALDI-TOF MS) y por secuenciación del gen ARNr 16S. La sensibilidad antibiótica se evaluó porel método epsilométrico. Todos los aislados presentaron sensibilidad a penicilina, vancomicinay linezolid, mientras que la sensibilidad a clindamicina y eritromicina fue variable. La iden-tificación por MALDI-TOF MS permitió arribar a nivel de especie de forma rápida y confiabley dar una respuesta oportuna y efectiva, evitando el retraso en el tratamiento, lo que sueleincrementar la morbimortalidad del cuadro clínico.

Orquiepididimitis por Haemophilus parainfluenzae: caso clínico y revisión de la literatura / Orchiepididymitis due to Haemophilus parainfluenzae: case report and review of the literature / Orquiepididimite por Haemophilus parainfluenzae: caso clínico e revisão da literatura

Resumen Haemophilus parainfluenzae forma parte de la microbiota normal de la cavidad oral y del tracto respiratorio superior. Es un reconocido agente causal de endocarditis y, con menor frecuencia, de enfermedades como neumonía, sepsis, osteomielitis, celulitis, meningitis y gastroenteritis aguda. Aquí se presenta un caso de orquiepididimitis en un joven adulto donde H. parainfluenzae, confirmado por espectrometría de masas (MALDI-TOF MS), fue el único patógeno detectado. Este caso contribuye a valorar el rol de H. parainfluenzae como patógeno humano, aislado a partir de sitios diferentes del torrente sanguíneo y las vías respiratorias.

Abstract Haemophilus parainfluenzae is part of the normal microbiota of the oral cavity and the upper respiratory tract. It is a recognised causal agent of endocarditis and, less frequently, of diseases such as pneumonia, sepsis, osteomyelitis, cellulitis, meningitis, and acute gastroenteritis. A case of orchiepididymitis in a young adult is reported, where H. parainfluenzae, confirmed by mass spectrometry (MALDI-TOF MS), was the only pathogen detected. This case contributes to assess the role of H. parainfluenzae as a human pathogen, isolated from sites other than the bloodstream and the respiratory tract.

Resumo Haemophilus parainfluenzae faz parte da microbiota normal da cavidade oral e do trato respiratório superior. É um reconhecido agente causal de endocardite e, menos frequentemente, de doenças como pneumonia, sepse, osteomielite, celulite, meningite e gastroenterite aguda. Aqui é relatado um caso de orquiepididimite em um adulto jovem onde H. parainfluenzae, confirmado por espectrometria de massa (MALDI-TOF MS), foi o único patógeno detectado. Este caso contribui para avaliar o papel do H. parainfluenzae como patógeno humano, isolado de outros locais que não sejam a corrente sanguínea e o trato respiratório.

Acid-fast bacteria as causative agents of skin and soft tissue infections: case presentations and literature review

ABSTRACT Acid-fast bacteria can be implicated in skin and soft tissue infections. Diagnostic identification can be challenging or not feasible by routine laboratory techniques, especially if there is no access to the Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) technology. Here, we present two cases of skin and soft tissue infections caused by two different acid-fast bacteria, Nocardia brasiliensis and Mycobacterium marinum. They both grew on Löwenstein-Jensen medium, Sabouraud agar medium and blood agar medium. Both bacteria appeared acid-fast by Ziehl-Neelsen stain and Gram-positive by Gram stain. The identification was performed by MALDI-TOF MS and gene analysis. N. brasiliensis and nontuberculous mycobacterium M. marinum represent rare pathogens that cause severe skin and soft tissue infections. Failure to identify the causative agent and subsequent inappropriate or inadequate treatment may lead to severe complications or even disseminated disease, especially in immunocompromised individuals.

Identification and cluster analysis of Streptococcus suis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry / 公共卫生与预防医学

Objective To evaluate the feasibility of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for identification and cluster analysis of Streptococcus suis. Methods Eighteen clinical isolates biochemically identified as Streptococcus suis were pre-treated by smearing formic acid method and formic acid-acetonitrile extraction method. The identification results and protein profiles of MALDI-TOF-MS method were analyzed and compared. A self-constructed database of profiling with better pretreatment method was established, and cluster analysis was performed on the 18 strains of Streptococcus suis. At the same time, PFGE homology analysis was performed to compare the results of the two genotyping. Results Both pretreatment methods could accurately identify Streptococcus suis with scores above 2.1. The protein fingerprint of formic acid and acetonitrile extraction method had a smoother baseline, fewer miscellaneous peaks and more identifiable ion peaks. Comparison of the results of homology typing showed that the homology results of MALDI-TOF-MS were significantly different from those of PFGE. Conclusion MALDI-TOF-MS can accurately identify Streptococcus suis for the strains pre-treated with formic acid method or formic acid-acetonitrile extraction method, and the formic acid-acetonitrile extraction method can obtain a better protein mapping. MALDI-TOF-MS can play a certain role in typing, but it still has some limitations and cannot completely replace the PFGE.

Progress in protein extraction methods for fungal identification with MALDI-TOF MS / 中华微生物学和免疫学杂志

With the development of techniques for rapid microbial identification, MALDI-TOF MS has become an important tool for clinical identification of fungi. Problems such as the applicability and standardization of protein extraction methods have hindered the development of MALDI-TOF MS technology in the fungal field. This paper analyzed the complex structure of fungal cell walls, introduced the protein extraction methods recommended by MALDI-TOF MS commercial mass spectrometry systems, discussed the protein extraction methods for the identification of various genera of yeast-like fungi and filamentous fungi by MALDI-TOF MS, such as direct smear method, formic acid acetonitrile extraction method and magnetic bead grinding method, and summarized the current status and drawbacks of protein extraction methods in fungal identification by MALDI-TOF MS with a view to providing theoretical reference for subsequent research.

Etiologic identification and drug susceptibility analysis of a Citrobacter freundii food poisoning event / 中国热带医学

@#Abstract: Objective　In order to provide reference for emergency treatment of a sudden food poisoning incident, pathogen detection and drug resistance analysis were carried out. Methods　Diarrheal stool and surplus food samples were detected by GB 4789 and the isolates were identified by VITEK2 and matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), at the same time, the bacterial drug sensitivity test was carried out by using the method of microbroth dilution, and the isolates from different sources were molecularly classified by pulsed field gel electrophoresis (PFGE), and the correlation between the strains was analyzed by BioNumerics software. Results　Totaly 13 leftovers and 3 diarrhea patients were isolated and identified, The total number of colonies and coliforms in 7 leftovers samples all exceeded the standard, and Citrobacter freundii was detected in 5 leftovers and 2 stools. The results of drug sensitivity test showed that seven strains of Citrobacter freundii were sensitive to ciprofloxacin, tetracycline, chloramphenicol, gentamicin, amikacin, cefotaxime and meropenem, but completely resistant to ampicillin, and there was no multiple drug resistance. The results of pulsed field gel electrophoresis (PFGE) showed that 7 strains of Citrobacter freundii had the same PFGE bands and 100% homology, showing the same clone. Conclusions　This food poisoning incident was caused by Citrobacter freundii. The pathogen of food poisoning can be quickly and accurately determined by MALDI-TOF MS, which is beneficial to the early diagnosis and treatment of infectious diseases. It is suggested to strengthen the corresponding management, improve food safety awareness and prevent similar incidents.

Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry azole susceptibility assessment in Candida and Aspergillus species

BACKGROUND Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS) allows rapid pathogen identification and potentially can be used for antifungal susceptibility testing (AFST). OBJECTIVES We evaluated the performance of the MALDI-TOF MS in assessing azole susceptibility, with reduced incubation time, by comparing the results with the reference method Broth Microdilution. METHODS Resistant and susceptible strains of Candida (n = 15) were evaluated against fluconazole and Aspergillus (n = 15) against itraconazole and voriconazole. Strains were exposed to serial dilutions of the antifungals for 15 h. Microorganisms' protein spectra against all drug concentrations were acquired and used to generate a composite correlation index (CCI) matrix. The comparison of autocorrelations and cross-correlations between spectra facilitated by CCI was used as a similarity parameter between them, enabling the inference of a minimum profile change concentration breakpoint. Results obtained with the different AFST methods were then compared. FINDINGS The overall agreement between methods was 91.11%. Full agreement (100%) was reached for Aspergillus against voriconazole and Candida against fluconazole, and 73.33% of agreement was obtained for Aspergillus against itraconazole. MAIN CONCLUSIONS This study demonstrates MALDI-TOF MS' potential as a reliable and faster alternative for AFST. More studies are necessary for method optimisation and standardisation for clinical routine application.

Descripción de un caso de sepsis abdominal por Desulfovibrio desulfuricans / Description of a case of abdominal sepsis due to Desulfovibrio desulfuricans

Resumen Desulfovibrio spp. son bacterias anaerobias estrictas, ubicuas en la naturaleza, quepueden formar parte del tracto gastrointestinal humano o animal, pero también son bacteriasambientales presentes en el suelo y en el agua. Pueden persistir de manera asintomática enel intestino o comportarse como patógenos oportunistas, asociados con bacteriemia primariae infecciones intraabdominales. El número de infecciones por Desulfovibrio spp. puede estarsubestimado debido a su lenta velocidad de crecimiento y a que muchos laboratorios no realizancultivos en anaerobiosis de manera rutinaria. Pruebas sencillas, como el examen de la movilidaden fresco y de la morfología celular en la coloración de Gram, sumadas a la presencia de SH2en agar SIM y a la observación de una fluorescencia roja a pH alcalino bajo luz UV, seríanindicativas de Desulfovibrio spp. Se describe el caso de una bacteriemia por Desulfovibriodesulfuricans en una mujer con cuadro clínico de sepsis abdominal por apendicitis gangrenosacon fallo multiorgánico.

Abstract Desulfovibrio spp. are strict anaerobes that are ubiquitous in nature. They can reside in the human or animal gastrointestinal tract and, as they are also environmental bacteria, may be present in soil and water. They can persist asymptomatically in the intestine or behave as opportunistic pathogens associated with primary bacteremia and intraabdominal infections. Several Desulfovibrio spp. infections may be underestimated due to their slow growth rate and because many laboratories do not routinely perform anaerobic cultures. Simple tests such as motility detection on a fresh subculture, Gram stain to confirm cell morphology, presence of H2S in SIM agar and production of a red fluorescence in alkaline pH under UV light would be indicative of Desulfovibrio spp.

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)

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 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.

Use of MALDI-TOF for identification and surveillance of gram-negative bacteria in captive wild psittacines

Abstract 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.

Resumo 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.

Molecular and proteomic identification of Arthrobacter gandavensis isolated from cows with subclinical mastitis in a dairy farm

Aims@#The genus Arthrobacter is a pleomorphic and heterogeneous Gram-positive bacteria mainly isolated from the soil, only two species of Arthrobacter have been reported worldwide as pathogens of veterinary importance. This paper aims to report the isolation and identification of the Arthrobacter gandavensis from cows with subclinical mastitis at a dairy farm in the savanna of Bogotá, Colombia.@*Methodology and results@#A total of 209 milk and skin samples were taken from cows with and without subclinical mastitis, nasal swabs from workers and the environment. All samples were cultured in blood and MacConkey agar and identified by 16S rRNA gene sequencing and mass spectrometry MALDI TOF-MS. From the isolates identified, 33 corresponded to Staphylococcus spp., nine to the Enterobacteriaceae family and seven from Arthrobacter spp. (only identified by MALDI-ToF MS). The A. gandavensis isolates were obtained from six different positive cows for the California mastitis test, all with a matching pattern corresponding to Arthrobacter gandavensis strain DSM N: 15046, isolated from milk from cows with subclinical mastitis in Belgium. Analysis of the 16S rRNA gene showed 100% genetic similarity with sequences of A. gandavensis previously reported in the NCBI databases.@*Conclusion, significance and impact of study@#The identification by MALDI-ToF-MS and molecular, as shown in this report, is important to provide data that allow us to approach the actual ecology of the opportunistic pathogens of subclinical mastitis, especially in regions where the infection is endemic.

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.

In vitro screening of siderophore-producing rice root endophytic bacteria from up-land paddies in north-western Vietnam for plant growth-promoting activities

Aims@#Endophytic bacteria (EB) living inside plant tissues possess different beneficial traits including siderophore production and other plant growth-promoting (PGP) activities. Siderophore-producing EB promote host plant growth by secreting ferrum in iron-deficient conditions. This study screened 19 siderophore producers in vitro, isolated from upland rice roots grown in mountain farms of Tung Village, Nậm Có Commune, Mù Cang Chải District, Yên Bái Province, Vietnam, for PGP traits, including phosphate solubilisation, indole-3-acetic acid (IAA), ammonia, gelatinase, amylase and catalase production.@*Methodology and results@#The bacteria were identified by Matrix assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-TOF MS). All 19 isolates were identified as genera Pseudomonas, Enterobacter, Pantoe, Bacillus, Burkholderia, Staphylococcus, Ralstonia and Cronotacter. The isolates produced catalase and ammonia. The amount of ammonia ranged from 60.74 ± 0.14 to 466.72 ± 0.18 mg/L. Out of the 19 siderophore producers, 17 (89.47%) were able to solubilise phosphate with solubilisation index (PSI) ranging from 1.12 ± 0.07 to 2.14 ± 0.15. The qualitative assays identified 12 isolates (63.15%) positive for IAA production with a tryptophan concentration of 5 mM, whereas 15 (78.94%) and 17 (89.47%) isolates were positive for gelatin and starch hydrolysis, respectively. Especially, 7 isolates were found to be positive for all tested assays in vitro including Pseudomonas rhodesiae (NC2), Enterobacter asburiae (NC50), Pantoea ananatis (NC63), Bacillus cereus (NC64), Burkholderia cenocepacia (NC110), Staphylococcus sciuri (NC112) and Ralstonia pickettii (NC122).@*Conclusion, significance and impact of study@#This study serves as crucial findings of multi-trait plant growth-promoting endophytic bacteria isolated from upland rice root in north-western Vietnam. The seven potential isolates positive for all tested assays could be effective PGP bacteria for bio-inoculants.

Infecciones abdominales por Actinomyces odontolyticus: comunicación de dos casos

Resumen Actinomyces spp es una familia de bacilos grampositivos saprofíticos que rara vez producen infecciones en el ser humano. Actinomyces odontolyticus forma parte de la microbiota oral y existen escasos reportes de casos de infecciones asociadas a este microorganismo, principalmente de localización oral, torácica, pélvica y bacteremias. Estas infecciones se caracterizan por ser recidivantes y causar abscesos y trayectos fistulosos. Su aislamiento microbiológico es difícil ya que la mayoría de los equipos automatizados no identifican la especie de Actinomyces, por lo que técnicas como MALDI-TOF MS resulta de gran ayuda en el diagnóstico definitivo. Finalmente, el tratamiento antibacteriano debe ser prolongado, acompañado del drenaje quirúrgico de las colecciones. Presentamos dos casos de infección abdominal recurrente por A. odontolyticus, en pacientes inmunocompetentes, con tratamiento exitoso.

Abstract Actinomyces spp is a family of saprophytic gram-positive rods that rarely cause infections in humans. Actinomyces odontolyticus is part of the oral microbiota and there are few case reports of infections associated, mainly oral, thoracic, pelvic involvement and bacteremia. These infections are characterized by being recurrent and causing abscesses and fistulous tracts. Microbiological isolation of the microorganism is difficult because most of the automated identification equipment does not detect the Actinomyces species. The use of identification techniques such as MALDI-TOF MS is a great help in the definitive diagnosis. Finally, antibacterial treatment should be prolonged, and accompanied by surgical drainage of the collections. We report two cases of recurrent abdominal infection by A. odontolyticus, in immunocompetent patients, with successful treatment.

Identificación de especies de micobacterias mediante espectrometría de masas (MALDI-TOF) / Identification of mycobacteria species through mass spectrometry (MALDI-TOF)

Resumen Introducción: Las enfermedades producidas por micobacterias son de gran importancia clínica y epidemiológica presentando el complejo Mycobacterium tuberculosis (MTBc) una morbi-mortalidad mayor que la producida por micobacterias no tuberculosas (MNTB). La identificación tradicional está basada en sus características fenotípicas mediante procesos laboriosos e incapaces en algunos casos de distinguir entre especies. Actualmente, la mayoría de las técnicas utilizadas se basan en métodos moleculares que tienen alta veracidad, pero son complejas y de alto costo. La espectrometría de masas con desorción/ionización láser asistida por una matriz asociada a tiempo de vuelo (MALDI-TOF MS) se basa en la comparación del espectro proteico producido con respecto al de una base de datos de referencia. Objetivo: Evaluar el rendimiento de MALDI-TOF MS en la identificación de micobacterias comparado con métodos moleculares: Material y Métodos: Se analizaron 28 aislados de nueve especies distintas mediante MALDI-TOF MS. Resultados: Se identificó correctamente 78,5% de las aislados (22/28), concordante en 100% (9/9) de MNTB de crecimiento rápido, 60% (9/15) en las MNTB de crecimiento lento y 100% (4/4) de MTBc. Todas las especies no identificadas (6/6) pertenecen al complejo M. avium/intracellulare. Conclusión: MALDI-TOD MS es una metodología rápida, fácil y de bajo costo, con adecuada veracidad respecto a los métodos moleculares.

Abstract Background: Mycobacterial diseases are very important both clinically and epidemiologically. Mycobacterium tuberculosis complex (MTBc) infections confer higher morbidity and mortality rate than non-tuberculous mycobacteria (NTM) infections. Traditional species identification techniques are based on phenotypic characteristics which take a long time by laborious processes and in occasions are no conclusive. Currently, most used techniques are based on molecular methods, which are accurate but are expensive and complex. Matrix Assisted Laser Desorption/Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS) is a simple, cheap and fast identification method based on comparing protein spectra with a reference database. Aim: To assess the performance of MALDI-TOF MS in the identification of MTBc and NTM, compared with molecular methods. Methods: For that purpose, 28 isolates of 9 different species were analyzed through MALDI-TOF MS. Results: 78.5% (22/28) of isolates were correctly identified, 100% (9/9) of rapidly growers NTM, 60% (9/15) of slow growing NTM and 100% (4/4) of MTBc. Every unidentified isolate (6/6) corresponded to M. avium/intracellulare complex. Conclusion: MALDI-TOF MS is fast, simple and cheaper than molecular methods and also has adequate accuracy.

Evaluation of different samples pretreatment method of MALDI-TOF MS on the identification of common foodborne pathogens / 上海预防医学

Objective To evaluate the effects of different sample pretreatment methods of MALDI-TOF MS on the identification of common foodborne pathogens. Methods A total of 51 common foodborne pathogens were collected, covering Salmonella, Staphylococcus, Listeria, Vibrio, Escherichia, and Shigella.Then the effects of three sample treatment methods including direct smear method, in-situ formic acid extraction method and formic acid acetonitrile extraction method of MALDI-TOF MS on the identification of common foodborne pathogens were evaluated. Results There were no significant differences in the identification results of Salmonella, Shigella, and Escherichia by three sample pretreatment methods; Staphylococcus captis, Staphylococcus cohnii, Vibrio parahaemolyticus and Vibrio vulnificus could not be identified effectively by direct smear method, but these strains could be accurately identified by other methods.Non-listeria monocytogenes strains of listeria genus were often mistakenly identified by using direct smear method and in-situ formic acid extraction method, and the use of formic acid and acetonitrile extraction could improve the identification accuracy for different species of listeria. Conclusion Different sample pretreatment methods of MALDI-TOF MS have certain influence on the identification of common foodborne pathogenic bacteria.Therefore, selecting appropriate sample pretreatment methods is important to obtain reliable and accurate identification results.