Evaluation of MALDI-TOF MS, sequencing of D2 LSU rRNA and internal transcribed spacer regions (ITS) for the identification of filamentous fungi isolated from a pharmaceutical facility.

The identification of filamentous fungi through culture characterization may be hampered by phenotypic variability. Information obtained from the identification of microorganisms are important for investigation of sources of contamination of a product or process. The aim of this study was to identify filamentous fungal strains (n = 50) isolated from a pharmaceutical facility by using Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), as well as D2 domain of the large-subunit (LSU) ribosomal RNA gene and internal transcribed spacer regions (ITS) sequencing. MALDI-TOF MS system only identified five strains at the species level, while 45 were not identified. The analysis through GenBank allowed the identification of up to 19 strains at the species level, while MycoBank allowed the identification of up to nine strains at the species level. The databases identified up to 11 genera: Penicillium, Aspergillus, Cladosporium, Chaetomium, Coniochaeta, Curvularia, Diaporthe, Fusarium, Trichoderma, Rhizopus and Microdochium. MALDI-TOF MS showed an insufficient database to identify the species of fungi. DNA sequencing was the best methodology to identify to the genus level but was unable to differentiate between closely related species. Therefore further methods for the identification of filamentous fungi from pharmaceutical areas at species level need to be developed.

MALDI-TOF MS database expansion for identification of Bacillus and related genera isolated from a pharmaceutical facility.

Bacillus and related genera are among the main bacterial groups isolated from pharmaceutical production areas. The identification of Bacillus species and related genera by classical methods is particularly difficult, due to similarities between closely related species. The Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) is one of the most promising techniques for chemotaxonomic characterization of microorganisms, being an alternative to genotypic methods. This study aimed to identify Bacillus strains and related genera isolated from immunobiological production areas by phylogenetic analysis of housekeeping genes and expand the database associated with MALDI-TOF MS to improve their identification. In a previous study, 97 aerobic endospore-forming bacteria isolated from a pharmaceutical facility were analyzed by MALDI-TOF MS and 16S rRNA gene full-length sequencing. All strains were identified as Bacillus and related genera by the latest methodology. Among the 97 strains, 22 were unidentified and 2 strains were misidentified by MALDI-TOF MS. In the present study, these 24 strains were subjected to 16S rRNA gene phylogenetic analysis. Strains not identified at species level by this methodology were submitted to rpoB gene phylogenetic analysis. After identifying the strains, 19 of the 24 strains were incubated for 24, 48, and 72 h on Tryptic Soy Agar and Sheep Blood Agar and subjected to analysis by MALDI-TOF MS. A SuperSpectrum for each strain was created and entered into the equipment database. Finally, the 24 strains were again submitted to proteomic analysis by MALDI-TOF MS, and, at this time, all were correctly identified. The genotypic identification of in-house isolated strains and the introduction of these spectra in MALDI-TOF MS, in order to obtain a customized database, proved to be an extremely effective tool in the identification of Bacillus and related genera from pharmaceutical industry origin.

Assessment of VITEK® 2, MALDI-TOF MS and full gene 16S rRNA sequencing for aerobic endospore-forming bacteria isolated from a pharmaceutical facility.

VITEK®2, MALDI-TOF MS and 16S rRNA sequencing were evaluated for the identification of aerobic endospore-forming bacteria (AEB) from a pharmaceutical facility. MALDI-TOF MS demonstrated higher accuracy compared to VITEK®2, although both databases were insufficient to identify AEB species. Sequencing was the best methodology, but unable to identify closely related species.

Isolamento de bactérias potencialmente patogênicas emindivíduos da comunidade indígena Xavante do estado doMato Grosso, Brasil / Isolation of potentially pathogenic bacteria of individuals from the Xavante indigenous community of Mato Grosso state, Brazil

Neste estudo, são apresentados os resultados obtidos em uma pesquisa para bactérias entéricas conduzida entre os Xavantes, grupo indígena de Mato Grosso, Brasil, a partir de fezes conservadas em meio de transporte. Os resultados mostraram, por meio datécnica clássica de isolamento, bioquímica e sorologia, a presença de duas espécies bacterianas importantes, causadoras de diarreia: Salmonella enterica e Escherichia coli enteropatogênica (EPEC). Essas mesmas amostras, pesquisadas para outros agentes não bacterianos, indicaram também, em 40% dos casos, associação com parasitos, sugerindo uma relação direta com a baixa salubridade da comunidade e a necessidade da implementação de saneamento básico...

Antibiotic resistance of Vibrio parahaemolyticus isolated from pond-reared Litopenaeus vannamei marketed in Natal, Brazil

Ten out of fifty fresh and refrigerated samples of shrimp (Litopenaeus vannamei) collected from retailers in Natal (Rio Grande do Norte, Northeastern Brazil) tested positive for Vibrio parahaemolyticus. The Kanagawa test and multiplex PCR assays were used to detect TDH and TRH hemolysins and the tdh, trh and tlh genes, respectively. All strains were Kanagawa-negative and tlh-positive. Antibiotic susceptibility testing was done for seven antibiotics by the agar diffusion technique. Five strains (50 percent) presented multiple antibiotic resistance to ampicillin (90 percent) and amikacin (60 percent), while two strains (20 percent) displayed intermediate-level resistance to amikacin. All strains were sensitive to chloramphenicol. Intermediate-level susceptibility and/or resistance to other antibiotics ranged from 10 to 90 percent, with emphasis on the observed growing intermediate-level resistance to ciprofloxacin. Half our isolates yielded a multiple antibiotic resistance index above 0.2 (range: 0.14-0.29), indicating a considerable risk of propagation of antibiotic resistance throughout the food chain.

Antimicrobial susceptibilities of Listeria monocytogenes human strains isolated from 1970 to 2008 in Brazil.

INTRODUCTION: Listeria monocytogenes is the causative agent of listeriosis, a foodborne illness that affects mainly pregnant women, the elderly and immunocompromised patients. The primary treatment is a combination of ampicillin with an aminoglycoside, in addition to a second-choice drug represented by chloramphenicol, erythromycin, tetracycline and rifampicin. The aim of this study was to analyze the antimicrobial susceptibility profile of strains isolated from human sources in the last four decades. METHODS: Sixty-eight strains were selected from the culture collection of the Laboratory of Bacterial Zoonoses/LABZOO/FIOCRUZ isolated in different regions of Brazil from 1970 to 2008 and primarily isolated from cerebrospinal fluid and blood culture. Susceptibility tests to antimicrobials drugs were evaluated using the criteria established by Soussy using the Kirby-Bauer method and E-Test strips were used to determine the minimum inhibitory concentration (MIC). RESULTS: Among the strains tested, serovar L4b (60.3%) was the most prevalent, followed by serovar 1/2a (20.6%), 1/2b (13.2%) and the more uncommon serovars 1/2c, 3b and 4ab (5.9%). All strains were susceptible to ampicillin, cephalothin, erythromycin, gentamicin, teicoplanin and vancomycin. Only one strain (1.5%) showed resistance to rifampin, and two (3%) were resistant to trimethoprim-sulfamethoxazole. MICs with values up to 2 µg/ml reinforce the need for microbiological surveillance. CONCLUSIONS: The study demonstrated low prevalence of strains resistant to the antimicrobial drugs indicated in the treatment of human listeriosis. Monitoring antimicrobial resistance profile is still very important to determine adequate treatment, especially in immunocompromised patients.

Antimicrobial susceptibilities of Listeria monocytogenes human strains isolated from 1970 to 2008 in Brazil / Susceptibilidade antimicrobiana de cepas humanas de Listeria monocytogenes isoladas no período de 1970 a 2008 no Brasil

INTRODUCTION: Listeria monocytogenes is the causative agent of listeriosis, a foodborne illness that affects mainly pregnant women, the elderly and immunocompromised patients. The primary treatment is a combination of ampicillin with an aminoglycoside, in addition to a second-choice drug represented by chloramphenicol, erythromycin, tetracycline and rifampicin. The aim of this study was to analyze the antimicrobial susceptibility profile of strains isolated from human sources in the last four decades. METHODS: Sixty-eight strains were selected from the culture collection of the Laboratory of Bacterial Zoonoses/LABZOO/FIOCRUZ isolated in different regions of Brazil from 1970 to 2008 and primarily isolated from cerebrospinal fluid and blood culture. Susceptibility tests to antimicrobials drugs were evaluated using the criteria established by Soussy using the Kirby-Bauer method and E-Test strips were used to determine the minimum inhibitory concentration (MIC). RESULTS: Among the strains tested, serovar L4b (60.3 percent) was the most prevalent, followed by serovar 1/2a (20.6 percent), 1/2b (13.2 percent) and the more uncommon serovars 1/2c, 3b and 4ab (5.9 percent). All strains were susceptible to ampicillin, cephalothin, erythromycin, gentamicin, teicoplanin and vancomycin. Only one strain (1.5 percent) showed resistance to rifampin, and two (3 percent) were resistant to trimethoprim-sulfamethoxazole. MICs with values up to 2μg/ml reinforce the need for microbiological surveillance. CONCLUSIONS: The study demonstrated low prevalence of strains resistant to the antimicrobial drugs indicated in the treatment of human listeriosis. Monitoring antimicrobial resistance profile is still very important to determine adequate treatment, especially in immunocompromised patients.

INTRODUÇÃO: Listeria monocytogenes é o agente etiológico da listeriose, doença de origem alimentar que acomete principalmente grávidas, pacientes imunodeprimidos e idosos. O tratamento primário é a associação de ampicilina a um aminoglicosídeo além de outros, em segunda escolha, representados por cloranfenicol, eritromicina, tetraciclina e rifampicina. O presente estudo teve como objetivo analisar o perfil de susceptibilidade aos antimicrobianos de amostras de origem humana isoladas nas últimas quatro décadas. MÉTODOS: Foram selecionadas 68 cepas provenientes de casos clínicos humanos ocorridos em diferentes regiões do país no período de 1970-2008. A susceptibilidade aos antimicrobianos testados foi determinada através dos critérios estabelecidos por Soussy pelo método de Kirby-Bauer e a concentração mínima inibitória realizada através do E-Test. RESULTADOS: A amostragem constituiu-se de 68 cepas, isoladas principalmente de líquido cefalorraquidiano, e hemocultura no período, pertencentes ao Laboratório de Zoonoses Bacterianas/LABZOO/Fiocruz. O sorovar L4b (60,3 por cento) foi o mais prevalente, seguido do sorovar 1/2a (20,6 por cento), 1/2b (13,2 por cento) e aqueles mais raros representados por 1/2c, 3b e 4ab (5,9 por cento). Todas as cepas foram sensíveis à ampicilina, cefalotina, eritromicina, gentamicina, teicoplanina e vancomicina. Apenas uma cepa (1,5 por cento) apresentou resistência à rifampicina, enquanto duas (3 por cento) foram resistentes à associação de sulfametoxazol-trimetoprim. CONCLUSÕES: Apesar de o estudo ter demonstrado uma baixa prevalência de amostras resistentes aos antimicrobianos indicados na terapêutica da listeriose humana, o sistema de monitoramento do perfil de resistência antimicrobiana é de extrema importância para a orientação do tratamento adequado, principalmente nas infecções em pacientes imunocomprometidos.

[Characterization of Aeromonas spp isolates from newborns hospitalized]. / Caracterização de Aeromonas spp isoladas de neonatos hospitalizados.

Aeromonas spp is recognized as pathogenic to humans after consumption of contaminated water and food. In the present investigation, 2,323 rectal swab samples from newborns hospitalized in Rio de Janeiro were evaluated with a view to isolating Aeromonas. The samples were collected and sent to the national reference laboratory for cholera and other bacterial intestinal infections, at the Oswaldo Cruz Institute of the Oswaldo Cruz Foundation. The swabs were subjected to enrichment in alkaline peptonated water with the addition of 1% sodium chloride (NaCl) and alkaline peptonated water plus 3% NaCl (37 degrees C/18-24h) and were streaked onto agar that was selective for Pseudomonas-Aeromonas (GSP Agar). Fifty-six Aeromonas strains were isolated, distributed as follows: Aeromonas caviae (42.8%), Aeromonas media (25%), Aeromonas veronii biogroup sobria (10.7%), Aeromonas hydrophila (9%), Aeromonas veronii biogroup veronii (5.3%), Aeromonas sobria (1.8%), Aeromonas jandaei (1.8%), Aeromonas schubertii (1.8%) and Aeromonas sp (1.8%). Resistance to one or more antimicrobial drugs was observed in 26.8% of the strains. Considering the importance of Aeromonas, there is an urgent need to warn about this in relation to nosocomial infection control.

Caracterização de Aeromonas spp isoladas de neonatos hospitalizados / Characterization of Aeromonas spp isolates from newborns hospitalized

Aeromonas spp é reconhecida como patogênica para o homem após o consumo de água e alimentos contaminados. Na presente investigação, foram avaliadas 2.323 amostras de swabs retais de neonatos hospitalizados no Rio de Janeiro objetivando o isolamento de Aeromonas. As amostras foram coletadas e enviadas ao Laboratório de Referência Nacional de Cólera e outras enteroinfecções bacterianas, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz. Os swabs foram submetidos ao enriquecimento em água peptonada alcalina adicionada de 1 por cento de cloreto de sódio (NaCl) e água peptonada alcalina adicionada de 3 por cento de NaCl (37ºC/18-24h) e semeadas em agar seletivo para Pseudomonas aeromonas (Agar GSP). Foram isoladas 56 cepas de Aeromonas assim distribuídas: Aeromonas caviae (42,8 por cento), Aeromonas media (25 por cento), Aeromonas veronii biogrupo sobria (10,7 por cento), Aeromonas hydrophila (9 por cento), Aeromonas veronii biogrupo veronii (5,3 por cento), Aeromonas sobria (1,8 por cento), Aeromonas jandaei (1,8 por cento), Aeromonas schubertii (1,8 por cento) e Aeromonas sp (1,8 por cento). Foi observada resistência a uma ou mais drogas antimicrobianas em 26,8 por cento das cepas. Considerando a relevância de Aeromonas torna-se urgente alertar sobre sua importância para o controle de infecções hospitalares.

Aeromonas spp is recognized as pathogenic to humans after consumption of contaminated water and food. In the present investigation, 2,323 rectal swab samples from newborns hospitalized in Rio de Janeiro were evaluated with a view to isolating Aeromonas. The samples were collected and sent to the national reference laboratory for cholera and other bacterial intestinal infections, at the Oswaldo Cruz Institute of the Oswaldo Cruz Foundation. The swabs were subjected to enrichment in alkaline peptonated water with the addition of 1 percent sodium chloride (NaCl) and alkaline peptonated water plus 3 percent NaCl (37°C/18-24h) and were streaked onto agar that was selective for Pseudomonas-Aeromonas (GSP Agar). Fifty-six Aeromonas strains were isolated, distributed as follows: Aeromonas caviae (42.8 percent), Aeromonas media (25 percent), Aeromonas veronii biogroup sobria (10.7 percent), Aeromonas hydrophila (9 percent), Aeromonas veronii biogroup veronii (5.3 percent), Aeromonas sobria (1.8 percent), Aeromonas jandaei (1.8 percent), Aeromonas schubertii (1.8 percent) and Aeromonas sp (1.8 percent). Resistance to one or more antimicrobial drugs was observed in 26.8 percent of the strains. Considering the importance of Aeromonas, there is an urgent need to warn about this in relation to nosocomial infection control.

[Aeromonas associated with an acute diarrhea outbreak in São Bento do Una, Pernambuco]. / Envolvimento de Aeromonas em surto de doença diarréica aguda em São Bento do Una, Pernambuco.

An acute diarrhea outbreak, with 2170 cases, was described during January to July, 2004, in São Bento do Una, Pernambuco. 582 stools were examined and an enteric pathogen was recovered in 25% (145 patients). Aeromonas species were the most frequent (114-19.5%) and the main isolates were Aeromonas caviae (57-9.8%), Aeromonas veronii biovar sobria (23-3.9%), Aeromonas veronii biovar veronii (15-2.6%) and other species (19-3.2%). The other isolated enteropathogens were Vibrio cholerae O1-Ogawa toxigenic (18-3.1%), Salmonella spp (8-1.4%), Shigella spp (3-0.5%) and Vibrio cholerae non-O1/non-O139 (2-0.3%).

Envolvimento de Aeromonas em surto de doenca diarréica aguda em São Bento do Una, Pernambuco / Aeromonas associated with an acute diarrhea outbreak in São Bento do Una, Pernambuco

No primeiro semestre de 2004, ocorreu um surto de diarréia em São Bento do Una, Pernambuco, registrando-se 2.170 casos. Nas 582 coproculturas realizadas, 145 (25 por cento) revelaram um enteropatógeno bacteriano, destacando 114 casos (19,5 por cento) com a participacão de Aeromonas, representadas por Aeromonas caviae (57/9,8 por cento), Aeromonas veronii biovar sobria (23/3,9 por cento), Aeromonas veronii biovar veronii (15/2,6 por cento) e outras espécies (19/3,2 por cento). Nos 31 episódios restantes (5,3 por cento), foram detectados: V. cholerae O1 Ogawa toxigênico (18/3,1 por cento), Salmonella spp (8/1,4 por cento), Shigella spp (3/0,5 por cento) e Vibrio cholerae não O1/não O139 (2/0,3 por cento).

[Serovars of Listeria monocytogenes and related species isolated from human clinical specimens]. / Sorovares de Listeria monocytogenes e espécies relacionadas, isoladas de material clínico humano.

Using phenotype techniques, characterization was realized of species and serovars of 255 strains of Listeria isolated from human material: 220-86.3% were from patients with possible invasive disease, while 35-13.7% were from colonized healthy individuals. The strains were collected in several regions of Brazil from 1969-2000. In individuals aged 0-10 or 41-60 years old Listeria monocytogenes was isolated more often in cerebral spinal fluid than in blood cultures, including samples from renal transplant recipients. All Listeria monocytogenes serovars were detected in blood culture strains. The predominant serovars characterized were 4b (154-60.3%) and 1/2a (74-29%). In this study, Listeria monocytogenes causing invasive diseases, such as meningitis or septicemia, or colonizing individuals, were identified. Consequently further studies focusing on clinical and pathological as well as epidemiological issues, including risk factors associated with foodborne transmission should be pursued.

Sorovares de Listeria monocytogenes e espécies relacionadas, isoladas de material clínico humano / Serovars of Listeria monocytogenes and related species isolated from human clinical specimens

A análise fenotípica de 255 amostras do gênero Listeria isoladas de material clínico humano, tanto de indivíduos doentes (220-86,3 por cento), como de aparentemente normais (35-13,7 por cento) de várias regiões do país e colecionadas no período de 1969 a 2000, permitiu caracterizar a distribuicão de sorovares de Listeria monocytogenes. Nas faixas etárias de 0 a 10 e de 41 a 60 anos, predominaram os isolamentos de líquido cefalorraquidiano sobre os de sangue, incluindo dos transplantados renais. Somente dos hemocultivos foi possível detectar os sete sorovares de Listeria monocytogenes. No cômputo geral, o sorovar 4b foi o mais incidente (154-60,3 por cento) secundado por /2 a (74-29 por cento) nos três decênios considerados, além de ocorrerem em quase todas as regiões do país. Os dados deste estudo evidenciaram a circulacão de L. monocytogenes na espécie humana, provocando quadros graves de meningite e septicemia, bem como, revelando a figura do portador assintomático, razão pela qual são recomendadas novas investigacões bacteriológicas, subsidiadas por análises clínico-patológicas e epidemiológicas.

Espécies e sorovares de Listeria isolados de animais doentes e portadores no Brasil / Species and serovars of Listeria isolated from sick and clinically healthy animals in Brazil

A análise fenotípica de 246 amostras do gênero Listeria isolados de animais portadores e doentes, provenientes de três regiões do país, colecionadas no período de 1971 a 2000, permitiu caracterizar as espécies e sorovares prevalentes. Dentre os animais predominaram os espécimes fecais de bovinos normais (217 amostras, 88,2 por cento), em contraposição aos 29 isolados (11,7 por cento) de Listeria de animais doentes, apresentando comprometimento do sistema nervoso central (15 amostras, 6,0 por cento) e outras localizações sistêmicas (14 amostras, 5,6 por cento). Quanto às espécies e sorovares, predominaram L. innocua 6a e não tipável (140 amsotras, 56,9 por cento) e L.monocytogenes 4a (37 amostras, 15,0) e 4b (22 amostras, 8,9 por cento) principalmente nas fezes de bovinos hígidos e nos animais doentes, L. monocytogenes sorovares 4b (14 amostras, 5,6 por cento) com destaque nos ruminantes e 1a (8 amostras, 3,2 por cento) incidindo nas outras espécies animais (roedores e canídeos) e tendo localizações prevalentes em áreas distintas ao sistema nervoso central.

Burkholderia pseudomallei: a case report of a human infection in Ceará, Brazil

O isolamento de Burkholderia pseudomallei, de meio ambiente e de espécimes clínicos, foi raramente registrado na América do Sul, particularmente no Brasil. Este relato descreve o caso de melioidose em um paciente de 10 anos de idade, de área rural do estado do Ceará (Tejuçuoca). As hemoculturas foram positivas e as análises dos testes bioquímicos e de susceptibilidade aos antimicrobianos do isolado foram indicativos para a identificação de B. pseudomallei. A fonte de contaminação foi obscura, uma vez que as análises microbiológicas de solo e água no tanque foram negativas.