RESUMO
Five mycobacterial isolates from sewage were classified as members of the genus Mycobacterium but presented inconclusive species assignments. Thus, the isolates (MYC017, MYC098, MYC101, MYC123 and MYC340) were analyzed by phenotypical, biochemical, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and genomic features to clarify their taxonomic position. Phenotypic analysis and biochemical tests did not distinguish these isolates from other non-pigmented mycobacteria. In contrast, MALDI-TOF MS analysis showed that isolates were not related to any previously described Mycobacterium species. Comparative genomic analysis showed values of ANI and dDDH between 81.59-85.56% and 24.4-28.8%, respectively, when compared to the genomes of species of this genus. In addition, two (MYC101 and MYC123) presented indistinguishable protein spectra from each other and values of ANI = 98.57% and dDDH = 97.3%, therefore being considered as belonging to the same species. Phylogenetic analysis grouped the five isolates within the Mycobacterium terrae complex (MTC) but in a specific subclade and separated from the species already described and supported by 100% bootstrap value, confirming that they are part of this complex but different from earlier described species. According to these data, we propose the description of four new species belonging to the Mycobacterium genus: (i) Mycobacterium defluvii sp. nov. strain MYC017T (= ATCC TSD-296T = JCM 35364T), (ii) Mycobacterium crassicus sp. nov. strain MYC098T (= ATCC TSD-297T = JCM 35365T), (iii) Mycobacterium zoologicum sp. nov. strain MYC101T (= ATCC TSD-298T = JCM 35366T) and MYC123 (= ATCC BAA-3216 = JCM 35367); and (iv) Mycobacterium nativiensis sp. nov. strain MYC340T (= ATCC TSD-299T = JCM 35368T).
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Polycyclic aromatic hydrocarbons (PAHs) are chemical compounds that are widespread in the environment, arising from the incomplete combustion of organic material, as well as from human activities involving petrol exploitation, petrochemical industrial waste, gas stations, and environmental disasters. PAHs of high molecular weight, such as pyrene, have carcinogenic and mutagenic effects and are considered pollutants. The microbial degradation of PAHs occurs through the action of multiple dioxygenase genes (nid), which are localized in genomic island denominate region A, and cytochrome P450 monooxygenases genes (cyp) dispersed in the bacterial genome. This study evaluated pyrene degradation by five isolates of Mycolicibacterium austroafricanum using 2,6-dichlorophenol indophenol (DCPIP assay), gas chromatography/mass spectrometry (CG/MS), and genomic analyses. Two isolates (MYC038 and MYC040) exhibited pyrene degradation indexes of 96% and 88%, respectively, over a seven-day incubation period. Interestingly, the genomic analyses showed that the isolates do not have nid genes, which are involved in PAH biodegradation, despite their ability to degrade pyrene, suggesting that degradation may occur due to the presence of cyp150 genes, or even genes that have not yet been described. To the best of our knowledge, this is the first report of isolates without nid genes demonstrating the ability to degrade pyrene.
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BACKGROUND Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis, and the number of new cases of multidrug resistant TB (MDR-TB), pre extensively drug-resistant TB (pre-XDR-TB) and extensively drug-resistant TB (XDR-TB) has increased considerably worldwide. OBJECTIVES Herein, using 156 M. tuberculosis isolates from 106 patients previously classified as MDR or pre-XDR or XDR isolates, we investigated the genetic mutation profiles associated with phenotypic resistances in patients with MDR-TB, pre-XDR-TB and XDR-TB, treatment outcomes and resistance evolution. METHODS Molecular analyses were performed by partial sequencing of the rpoB, katG, gyrA, gyrB, rrs genes and analysis of the fabG-inhA promoter region. Clinical, epidemiologic and demographic data were obtained from the TB Notification database system of São Paulo (TB-WEB) and the Information System for Special Tuberculosis Treatments (SITE-TB). FINDINGS Drug resistance was attributed to previously known mutations and a novel Asp449Val mutation in gyrB was observed in four isolates from the same patient. Ten patients had more than one isolate evaluated and eight of these patients displayed resistance progression. MAIN CONCLUSIONS The present study is the first to report the frequency of mutations related to second-line drug resistance in MDR-TB, pre-XDR-TB and XDR-TB isolates. The results could lead to the improvement of available technologies for the rapid detection of drug resistant TB.
Assuntos
Antituberculosos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Mutação/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Adolescente , Adulto , Brasil , Tuberculose Extensivamente Resistente a Medicamentos/microbiologia , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mycobacterium tuberculosis/isolamento & purificação , Fatores Socioeconômicos , Adulto JovemRESUMO
We investigated the species diversity of Mycobacteriaceae in surface water samples from six environments at the zoological park in São Paulo, Brazil. Three hundred and eighty isolates were cultivated and identified by phenotypic characteristics (growth rate and pigmentation) and sequencing of hsp65, rpoB and 16S rRNA genes. The results revealed that almost 48% of the isolates could be identified at the species level; about 50% were classified at the genus level, and only less than 2% of the isolates showed an inconclusive identification. The isolates classified at the genus level and not identified were then evaluated by phylogenetic analyses using the same three concatenated target genes. The results allowed us to identify at the genus level some isolates that previously had inconclusive identification, and they also suggested the presence of putative candidate species within the sample, demonstrating that this zoological park is an important source of diversity.
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Mycobacteriaceae/genética , Microbiologia da Água , Brasil , DNA Bacteriano/genética , Genes Bacterianos , Genômica , Mycobacteriaceae/classificação , Mycobacteriaceae/isolamento & purificação , Parques Recreativos , Filogenia , RNA Ribossômico 16S/genéticaRESUMO
BACKGROUND Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis, and the number of new cases of multidrug resistant TB (MDR-TB), pre extensively drug-resistant TB (pre-XDR-TB) and extensively drug-resistant TB (XDR-TB) has increased considerably worldwide. OBJECTIVES Herein, using 156 M. tuberculosis isolates from 106 patients previously classified as MDR or pre-XDR or XDR isolates, we investigated the genetic mutation profiles associated with phenotypic resistances in patients with MDR-TB, pre-XDR-TB and XDR-TB, treatment outcomes and resistance evolution. METHODS Molecular analyses were performed by partial sequencing of the rpoB, katG, gyrA, gyrB, rrs genes and analysis of the fabG-inhA promoter region. Clinical, epidemiologic and demographic data were obtained from the TB Notification database system of São Paulo (TB-WEB) and the Information System for Special Tuberculosis Treatments (SITE-TB). FINDINGS Drug resistance was attributed to previously known mutations and a novel Asp449Val mutation in gyrB was observed in four isolates from the same patient. Ten patients had more than one isolate evaluated and eight of these patients displayed resistance progression. MAIN CONCLUSIONS The present study is the first to report the frequency of mutations related to second-line drug resistance in MDR-TB, pre-XDR-TB and XDR-TB isolates. The results could lead to the improvement of available technologies for the rapid detection of drug resistant TB.
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Humanos , Masculino , Feminino , Adolescente , Adulto , Adulto Jovem , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , Mutação/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Antituberculosos/farmacologia , Fatores Socioeconômicos , Brasil , Testes de Sensibilidade Microbiana , Tuberculose Extensivamente Resistente a Medicamentos/microbiologia , Pessoa de Meia-Idade , Mycobacterium tuberculosis/isolamento & purificaçãoRESUMO
Hydrocarbons are important environmental pollutants, and the isolation and characterization of new microorganisms with the ability to degrade these compounds are important for effective biodegradation. In this work we isolated and characterized several bacterial isolates from compost, a substrate rich in microbial diversity. The isolates were obtained from selective culture medium containing n-hexadecane, aiming to recover alkane-degraders. Six isolates identified as Gordonia by MALDI-TOF and 16S rRNA sequencing had the ability to degrade n-hexadecane in three days. Two isolates were selected for genomic and functional characterization, Gordonia paraffinivorans (MTZ052) and Gordonia sihwensis (MTZ096). The CG-MS results showed distinct n-hexadecane degradation rates for MTZ052 and MTZ096 (86% and 100% respectively). The genome sequence showed that MTZ052 encodes only one alkane degrading gene cluster, the CYP153 system, while MTZ096 harbors both the Alkane Hydroxylase (AH) and the CYP153 systems. qPCR showed that both gene clusters are induced by the presence of n-hexadecane in the growth medium, suggesting that G. paraffinivorans and G. sihwensis use these systems for degradation. Altogether, our results indicate that these Gordonia isolates have a good potential for biotransformation of hydrocarbons.
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Actinobacteria , Alcanos/metabolismo , Compostagem , Microbiologia do Solo , Actinobacteria/genética , Actinobacteria/isolamento & purificação , Actinobacteria/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Genoma BacterianoRESUMO
BACKGROUND: The nontuberculous mycobacteria (NTM) are widely spread. In Brazil, 2,520 cases of rapidly growing mycobacteria (RGM) infections after medical procedures were reported, with 5.4% of cases related to nonsurgical invasive procedures and with an occurrence of 1 clone (BRA100) of Mycobacterium abscessus subsp bolletii. OBJECTIVE: To describe a pseudooutbreak of M abscessus subsp bolletii in an endoscopy and bronchoscopy unit. METHODS: The alert for a pseudooutbreak was given when 3 patients, in the same week, had a positive bronchoalveolar lavage culture for M abscessus subsp bolletii. The patients had no symptoms/signs of mycobacterial infection; thus, contamination of bronchoscopes was suspected. Samples for culturing were collected from bronchoscopes, digestive endoscopes, automated disinfection machines, and the water supply. Clinical samples were identified by polymerase chain reaction restriction-enzyme analysis (PRA) of the hsp65 gene and their pulsed-field gel electrophoresis pattern was compared with environmental samples. RESULTS: The investigation demonstrated a contamination of bronchoscopes, digestive endoscopes, and disinfection machines. Molecular typing demonstrated that all strains belonged to the same clone (MAB01), identical to clone BRA100. DISCUSSION: Cross-transmission due to poor disinfection as well as resistance to glutaraldehyde may play roles in the spread of MAB01 M abscessus subsp bolletii, which may have a unique resistance to the environment and adaption to human hosts. However the water supply may have played a role. Attention is needed to ensure the quality of water used to rinse disinfected equipment.
Assuntos
Líquido da Lavagem Broncoalveolar/microbiologia , Broncoscópios/microbiologia , Broncoscopia/efeitos adversos , Contaminação de Equipamentos , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium abscessus/isolamento & purificação , Brasil , Eletroforese em Gel de Campo Pulsado , Hospitais , Humanos , Epidemiologia Molecular , Tipagem Molecular , Mycobacterium abscessus/classificação , Mycobacterium abscessus/genética , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de RestriçãoRESUMO
BACKGROUND: A large collection of sequenced mycobacteriophages capable of infecting a single host strain of Mycobacterium smegmatis shows considerable genomic diversity with dozens of distinctive types (clusters) and extensive variation within those sharing evident nucleotide sequence similarity. Here we profiled the mycobacterial components of a large composting system at the São Paulo zoo. RESULTS: We isolated and sequenced eight mycobacteriophages using Mycobacterium smegmatis mc(2)155 as a host. None of these eight phages infected any of mycobacterial strains isolated from the same materials. The phage isolates span considerable genomic diversity, including two phages (Barriga, Nhonho) related to Subcluster A1 phages, two Cluster B phages (Pops, Subcluster B1; Godines, Subcluster B2), three Subcluster F1 phages (Florinda, Girafales, and Quico), and Madruga, a relative of phage Patience with which it constitutes the new Cluster U. Interestingly, the two Subcluster A1 phages and the three Subcluster F1 phages have genomic relationships indicating relatively recent evolution within a geographically isolated niche in the composting system. CONCLUSIONS: We predict that composting systems such as those used to obtain these mycobacteriophages will be a rich source for the isolation of additional phages that will expand our view of bacteriophage diversity and evolution.
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Micobacteriófagos/genética , Micobacteriófagos/isolamento & purificação , Mycobacterium/genética , Mycobacterium/virologia , Microbiologia do Solo , Solo , Bacteriófagos/genética , Sequência de Bases , Brasil , DNA Bacteriano/genética , DNA Viral/genética , Evolução Molecular , Genes Bacterianos , Variação Genética , Genoma Viral , Família Multigênica , Micobacteriófagos/classificação , Mycobacterium/classificação , Mycobacterium/isolamento & purificação , Mycobacterium smegmatis/classificação , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/isolamento & purificação , Mycobacterium smegmatis/virologia , FilogeniaRESUMO
An epidemic of surgical-site infections by a single strain of Mycobacterium abscessus subsp. bolletii affected >1,700 patients in Brazil from 2004 to 2008. The genome of the epidemic prototype strain M. abscessus subsp. bolletii INCQS 00594, deposited in the collection of the National Institute for Health Quality Control (INCQS), was sequenced.
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Composting operations are a rich source for prospection of biomass degradation enzymes. We have analyzed the microbiomes of two composting samples collected in a facility inside the São Paulo Zoo Park, in Brazil. All organic waste produced in the park is processed in this facility, at a rate of four tons/day. Total DNA was extracted and sequenced with Roche/454 technology, generating about 3 million reads per sample. To our knowledge this work is the first report of a composting whole-microbial community using high-throughput sequencing and analysis. The phylogenetic profiles of the two microbiomes analyzed are quite different, with a clear dominance of members of the Lactobacillus genus in one of them. We found a general agreement of the distribution of functional categories in the Zoo compost metagenomes compared with seven selected public metagenomes of biomass deconstruction environments, indicating the potential for different bacterial communities to provide alternative mechanisms for the same functional purposes. Our results indicate that biomass degradation in this composting process, including deconstruction of recalcitrant lignocellulose, is fully performed by bacterial enzymes, most likely by members of the Clostridiales and Actinomycetales orders.
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Biodiversidade , Biomassa , Metagenômica , Microbiologia do Solo , Bactérias/classificação , Bactérias/genética , Composição de Bases , Brasil , Análise por Conglomerados , Ordem dos Genes , Lactobacillus/classificação , Lactobacillus/genética , Lactobacillus/metabolismo , Lignina/metabolismo , Anotação de Sequência Molecular , Pectinas/metabolismo , RNA Ribossômico 16S , Análise de Sequência de DNARESUMO
AIM: Our aim is to investigate if the clusters of postsurgical mycobacterial infections, reported between 2004 and 2008 in seven geographically distant states in Brazil, were caused by a single mycobacterial strain. MATERIALS & METHODS: Available information from 929 surgical patients was obtained from local health authorities. A total of 152 isolates from surgical patients were identified by PCR restriction enzyme analysis of the hsp65 gene (PRA-hsp65) and sequencing of the rpoB gene. Isolates were typed by pulsed-field gel electrophoresis (PFGE) using two restriction enzymes, DraI and AseI. A total of 15 isolates not related to surgical cases were analyzed for comparison. RESULTS: All isolates were identified as Mycobacterium abscessus ssp. massiliense. Isolates from surgical patients and one sputum isolate grouped in a single PFGE cluster, composed of two closely related patterns, with one band difference. A total of 14 other isolates unrelated to surgical cases showed distinctive PFGE patterns. CONCLUSION: A particular strain of M. abscessus ssp. massiliense was associated with a prolonged epidemic of postsurgical infections in seven Brazilian states, suggesting that this strain may be distributed in Brazilian territory and better adapted to cause surgical-site infections.
Assuntos
Técnicas de Tipagem Bacteriana , Infecção Hospitalar/epidemiologia , Impressões Digitais de DNA , Infecções por Mycobacterium/epidemiologia , Mycobacterium/classificação , Mycobacterium/isolamento & purificação , Infecção da Ferida Cirúrgica/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Brasil/epidemiologia , Criança , Análise por Conglomerados , Eletroforese em Gel de Campo Pulsado , Feminino , Genótipo , Geografia , Humanos , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Mycobacterium/genética , Adulto JovemRESUMO
OBJECTIVES: A rapid-growing mycobacteria biological prosthetic valve (BPV) endocarditis related to prosthetic manufacturing process is described in Brazil. METHODS: From 1999 to 2008, thirty-nine patients underwent BPV replacement due to culture-negative suspected endocarditis. All these cases had histological sections stained by Ziehl-Neelsen method. Clinical and microbiological data were reviewed in all acid-fast bacilli (AFB) positive cases. The 16S-23S internal transcribed sequence (ITS) was amplified using DNA extracted from paraffin-embedded samples, digested with restrictions enzymes and/or sequenced. RESULTS: Eighteen AFB positive BPV (18/39)(46%) were implanted in 13 patients and were from the same manufacturer. Four of them were implanted in other hospitals. Thirteen BPV were histologically proven endocarditis and five showed a colonization pattern. The examination of six non-implanted "sterile" BPV from this manufacturer resulted in 5 AFB positive. Mycobacterium chelonae was the AFB identified by ITS restriction analysis and sequencing. CONCLUSIONS: Rapid-growing mycobacteria infections must be suspected and Ziehl-Neelsen stain always performed on histology of either early or late BPV endocarditis, particularly when blood cultures are negative.
Assuntos
Bioprótese/microbiologia , Endocardite Bacteriana/microbiologia , Próteses Valvulares Cardíacas/microbiologia , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium chelonae/isolamento & purificação , Infecções Relacionadas à Prótese/microbiologia , Adulto , Animais , Contaminação de Equipamentos , Feminino , Histocitoquímica , Humanos , Masculino , Pessoa de Meia-Idade , SuínosRESUMO
An outbreak of postsurgical infections caused by rapidly growing mycobacteria has been ongoing in Brazil since 2004. The degrees of similarity of the rpoB and hsp65 sequences from the clinical isolates and the corresponding sequences from both the Mycobacterium massiliense and the M. bolletii type strains were above the accepted limit for interspecies variability, leading to conflicting identification results. Therefore, an extensive characterization of members of the M. chelonae-M. abscessus group was carried out. The M. abscessus, M. chelonae, M. immunogenum, M. massiliense, and M. bolletii type strains and a subset of clinical isolates were analyzed by biochemical tests, high-performance liquid chromatography, drug susceptibility testing, PCR-restriction enzyme analysis of hsp65 (PRA-hsp65), rpoB, and hsp65 gene sequencing and analysis of phylogenetic trees, DNA-DNA hybridization (DDH), and restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene (RFLP-16S rRNA). The clinical isolates and the M. abscessus, M. massiliense, and M. bolletii type strains could not be separated by phenotypic tests and were grouped in the phylogenetic trees obtained. The results of DDH also confirmed the >70% relatedness of the clinical isolates and the M. abscessus, M. massiliense, and M. bolletii type strains; and indistinguishable RFLP-16S rRNA patterns were obtained. On the contrary, the separation of clinical isolates and the M. abscessus, M. massiliense, and M. bolletii type strains from M. chelonae and M. immunogenum was supported by the results of PRA-hsp65, DDH, and RFLP-16S rRNA and by the rpoB and hsp65 phylogenetic trees. Taken together, these results led to the proposition that M. abscessus, M. massiliense, and M. bolletii represent a single species, that of M. abscessus. Two subspecies are also proposed, M. abscessus subsp. abscessus and M. abscessus subsp. massiliense, and these two subspecies can be distinguished by two different PRA-hsp65 patterns, which differ by a single HaeIII band, and by differences in their rpoB (3.4%) and hsp65 (1.3%) sequences.
Assuntos
Surtos de Doenças , Infecções por Mycobacterium não Tuberculosas/epidemiologia , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium/classificação , Mycobacterium/isolamento & purificação , Infecção da Ferida Cirúrgica/epidemiologia , Infecção da Ferida Cirúrgica/microbiologia , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Brasil/epidemiologia , Cromatografia Líquida de Alta Pressão , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium/química , Mycobacterium/genética , Mycobacterium chelonae/classificação , Mycobacterium chelonae/isolamento & purificação , Ácidos Micólicos/análise , Filogenia , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
An epidemic of infections after video-assisted surgery (1,051 possible cases) caused by rapidly growing mycobacteria (RGM) and involving 63 hospitals in the state of Rio de Janeiro, Brazil, occurred between August 2006 and July 2007. One hundred ninety-seven cases were confirmed by positive acid-fast staining and/or culture techniques. Thirty-eight hospitals had cases confirmed by mycobacterial culture, with a total of 148 available isolates recovered from 146 patients. Most (n = 144; 97.2%) isolates presented a PRA-hsp65 restriction pattern suggestive of Mycobacterium bolletii or Mycobacterium massiliense. Seventy-four of these isolates were further identified by hsp65 or rpoB partial sequencing, confirming the species identification as M. massiliense. Epidemic isolates showed susceptibility to amikacin (MIC at which 90% of the tested isolates are inhibited [MIC(90)], 8 microg/ml) and clarithromycin (MIC(90), 0.25 microg/ml) but resistance to ciprofloxacin (MIC(90), >or=32 microg/ml), cefoxitin (MIC(90), 128 microg/ml), and doxycycline (MIC(90), >or=64 microg/ml). Representative epidemic M. massiliense isolates that were randomly selected, including at least one isolate from each hospital where confirmed cases were detected, belonged to a single clone, as indicated by the analysis of pulsed-field gel electrophoresis (PFGE) patterns. They also had the same PFGE pattern as that previously observed in two outbreaks that occurred in other Brazilian cities; we designated this clone BRA100. All five BRA100 M. massiliense isolates tested presented consistent tolerance to 2% glutaraldehyde. This is the largest epidemic of postsurgical infections caused by RGM reported in the literature to date in Brazil.
Assuntos
Infecção Hospitalar/epidemiologia , Surtos de Doenças , Infecções por Mycobacterium/epidemiologia , Mycobacterium/isolamento & purificação , Infecção da Ferida Cirúrgica/epidemiologia , Adulto , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Brasil/epidemiologia , Chaperonina 60 , Chaperoninas/genética , Análise por Conglomerados , Infecção Hospitalar/microbiologia , Impressões Digitais de DNA , DNA Bacteriano/química , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/genética , Eletroforese em Gel de Campo Pulsado , Feminino , Genótipo , Humanos , Masculino , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Dados de Sequência Molecular , Mycobacterium/classificação , Infecções por Mycobacterium/microbiologia , Análise de Sequência de DNA , Infecção da Ferida Cirúrgica/microbiologiaRESUMO
Microbiologistas clínicos devem estar atentos à possibilidade debacteremia relacionada com cateter causada por infecçãodifteróide, como possivelmente foi o caso neste relato, no qual um menino de dois anos, portador de um cateter venoso central paratratamento de leucemia, apresentou pneumonia. A cultura dosangue obtido no cateter revelou Curtobacterium spp.
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Humanos , Masculino , Pré-Escolar , Infecções Bacterianas , SepseRESUMO
A cluster of surgical site infection cases after arthroscopic and laparoscopic procedures occurred between 2005 and 2007 in Goiânia, in the central region of Brazil. Nontuberculous mycobacteria (NTM) were isolated from samples (exudates from cutaneous abscesses) from 18 patients of seven private hospitals. There were no reports of post-surgical arthroscopic and laparoscopic mycobacterial infections in Goiânia apart from this period. The 18 isolates were identified as Mycobacterium massiliense by PCR-restriction digestion of the hsp65 gene, pulsed-field gel electrophoresis (PFGE) comparisons, and rpoB partial gene sequencing. All isolates were typed as a single clone, indicating that they have the same origin, which suggests a common source of infection for all patients.
Assuntos
Infecção Hospitalar/epidemiologia , Infecções por Mycobacterium/epidemiologia , Mycobacterium/isolamento & purificação , Infecção da Ferida Cirúrgica/epidemiologia , Abscesso/epidemiologia , Abscesso/microbiologia , Adulto , Idoso , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Brasil/epidemiologia , Chaperonina 60 , Chaperoninas/genética , Análise por Conglomerados , Infecção Hospitalar/microbiologia , Impressões Digitais de DNA , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/genética , Eletroforese em Gel de Campo Pulsado , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium/genética , Infecções por Mycobacterium/microbiologia , Polimorfismo de Fragmento de Restrição , Dermatopatias Bacterianas/epidemiologia , Dermatopatias Bacterianas/microbiologia , Infecção da Ferida Cirúrgica/microbiologiaRESUMO
An outbreak of infections affecting 311 patients who had undergone different invasive procedures occurred in 2004 and 2005 in the city of Belém, in the northern region of Brazil. Sixty-seven isolates were studied; 58 were from patients who had undergone laparoscopic surgeries, 1 was from a patient with a postinjection abscess, and 8 were from patients who had undergone mesotherapy. All isolates were rapidly growing nonpigmented mycobacteria and presented a pattern by PCR-restriction enzyme analysis of the hsp65 gene with BstEII of bands of 235 and 210 bp and with HaeIII of bands of 200, 70, 60, and 50 bp, which is common to Mycobacterium abscessus type 2, Mycobacterium bolletii, and Mycobacterium massiliense. hsp65 and rpoB gene sequencing of a subset of 20 isolates was used to discriminate between these three species. hsp65 and rpoB sequences chosen at random from 11 of the 58 isolates from surgical patients and the postinjection abscess isolate presented the highest degrees of similarity with the corresponding sequences of M. massiliense. In the same way, the eight mesotherapy isolates were identified as M. bolletii. Molecular typing by pulsed-field gel electrophoresis (PFGE) grouped all 58 surgical isolates, while the mesotherapy isolates presented three different PFGE patterns and the postinjection abscess isolate showed a unique PFGE pattern. In conclusion, molecular techniques for identification and typing were essential for the discrimination of two concomitant outbreaks and one case, the postinjection abscess, not related to either outbreak, all of which were originally attributed to a single strain of M. abscessus.
Assuntos
Técnicas de Tipagem Bacteriana , Surtos de Doenças , Laparoscopia/efeitos adversos , Infecções por Mycobacterium/epidemiologia , Mycobacterium/classificação , Mycobacterium/genética , Procedimentos de Cirurgia Plástica/efeitos adversos , Proteínas de Bactérias/genética , Brasil/epidemiologia , Chaperonina 60 , Chaperoninas/genética , Eletroforese em Gel de Campo Pulsado , Humanos , Dados de Sequência Molecular , Mycobacterium/isolamento & purificação , Infecções por Mycobacterium/microbiologia , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
PURPOSE: To evaluate different methods of oxacillin susceptibility testing of ocular isolates, considering polymerase chain reaction (PCR) as the 'gold standard', and to compare the in vitro susceptibility to oxacillin with that of other antimicrobials used in ophthalmologic practice. METHODS: The Vitek gram-positive identification card was used to identify ocular coagulase negative Staphylococcus species. The presence of the mecA gene was determined by the polymerase chain reaction assay with a combination of two primer sets (mecA and 16S rRNA) in a single region. Results were analyzed and compared with other oxacillin susceptibility methods: PBP2a detection by rapid slide latex agglutination test (SLA); oxacillin E-test; the Vitek automated gram-positive susceptibility card (GPS-105); the oxacillin salt agar screening test (OSAS) at a concentration of 6.0, 1.0 and 0.75 microg oxacillin per ml and the cefoxitin disk diffusion test (CDD). Automated susceptibility was also determined to other antimicrobial agents (fluoroquinolones, penicillin G, amoxicillin-ampicillin, cefazolin, ampicillin-sulbactam, erythromycin, clindamycin, gentamicin, tetracycline, trimethoprim-sulfamethoxazole, vancomycin and rifampin. RESULTS: Of the 69 CoNS isolates tested, 71% were mecA-positive and 29% mecA-negative. All methods tested had a statistically significant agreement with polymerase chain reaction. There was a tendency of positive polymerase chain reaction predomination among the S. epidermidis isolates in comparison to non-epidermidis isolates, although this was not statistically significant (78.3% vs. 56.5%; chi2= 2.54; P= 0.11). The oxacillin salt agar screening test (0.75 microg oxacillin/ml) showed the best performance, with 100% sensitivity and negative predictive value; 95% specificity and 98% positive predictive value. Using the E-test, the mecA-positive isolates were statistically significantly more resistant to ciprofloxacin, ofloxacin, gatifloxacin and moxifloxacin (P= 0.002; P= 0.008; P= 0.002 and P= 0.003, respectively). There was a statistically significant higher proportion of resistance of the coagulase negative Staphylococcus mecA-positives for: penicillin G, amoxicillin-ampicillin, cefazolin, ampicillin-sulbactam, erythromycin, clindamycin, gentamicin and tetracycline (P< or =0.05). All coagulase negative Staphylococcus species were susceptible to vancomycin and there was no statistically significant correlation between the mecA-positive isolates and resistance to trimethoprim-sulfamethoxazole or to rifampin. CONCLUSIONS: In the present study, we found that the E-test and the oxacillin salt agar screening test S (0.75 microg oxacillin per ml), when compared with polymerase chain reaction, were the most accurate currently available methods to phenotypically detect oxacillin resistance of coagulase negative Staphylococcus species. This study demonstrated that a good option for screening of ocular isolates for oxacillin resistance in the microbiology laboratory is the cefoxitin disk diffusion test and the automated Vitek system. We believe it is important to have available methods that accurately detect methicillin resistance of the less commonly encountered species, chiefly because of their increasing importance as opportunistic pathogens.
Assuntos
Antibacterianos/farmacologia , Resistência a Meticilina , Testes de Sensibilidade Microbiana/métodos , Staphylococcus/efeitos dos fármacos , Ágar , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Cefoxitina/farmacologia , Coagulase , Humanos , Testes de Fixação do Látex , Meticilina/farmacologia , Resistência a Meticilina/efeitos dos fármacos , Resistência a Meticilina/genética , Oxacilina/farmacologia , Proteínas de Ligação às Penicilinas , Fenótipo , Reação em Cadeia da Polimerase , Infecções Estafilocócicas/microbiologia , Staphylococcus/enzimologia , Staphylococcus/genéticaRESUMO
PURPOSE: To evaluate different methods of oxacillin susceptibility testing of ocular isolates, considering polymerase chain reaction (PCR) as the 'gold standard', and to compare the in vitro susceptibility to oxacillin with that of other antimicrobials used in ophthalmologic practice. METHODS: The Vitek gram-positive identification card was used to identify ocular coagulase negative Staphylococcus species. The presence of the mecA gene was determined by the polymerase chain reaction assay with a combination of two primer sets (mecA and 16S rRNA) in a single region. Results were analyzed and compared with other oxacillin susceptibility methods: PBP2a detection by rapid slide latex agglutination test (SLA); oxacillin E-test; the Vitek automated gram-positive susceptibility card (GPS-105); the oxacillin salt agar screening test (OSAS) at a concentration of 6.0, 1.0 and 0.75 æg oxacillin per ml and the cefoxitin disk diffusion test (CDD). Automated susceptibility was also determined to other antimicrobial agents (fluoroquinolones, penicillin G, amoxicillin-ampicillin, cefazolin, ampicillin-sulbactam, erythromycin, clindamycin, gentamicin, tetracycline, trimethoprim-sulfamethoxazole, vancomycin and rifampin. RESULTS: Of the 69 CoNS isolates tested, 71 percent were mecA-positive and 29 percent mecA-negative. All methods tested had a statistically significant agreement with polymerase chain reaction. There was a tendency of positive polymerase chain reaction predomination among the S. epidermidis isolates in comparison to non-epidermidis isolates, although this was not statistically significant (78.3 percent vs. 56.5 percent; chi2= 2.54; P= 0.11). The oxacillin salt agar screening test (0.75 æg oxacillin/ml) showed the best performance, with 100 percent sensitivity and negative predictive value; 95 percent specificity and 98 percent positive predictive value. Using the E-test, the mecA-positive isolates were statistically significantly more...
OBJETIVOS: Avaliar os diferentes métodos de suscetibilidade à oxacillina, em isolados oculares, considerando a reação em cadeia da polimerase (PCR) como "padrão-ouro" e comparar a suscetibilidade in vitro para outros antimicrobianos de uso oftalmológico. MÉTODOS: O sistema automatizado Vitek foi utilizado para identificar as diferentes espécies de Staphylococcus coagulase negativo (SCoN). A presença do gene mecA foi determinado pela reação em cadeia da polimerase com a combinação de 2 "primer" sets (mecA e 16S rRNA) em uma única região. Estes resultados foram analisados e comparados com outros métodos de suscetibilidade à oxacilina: detecção da proteína PBP2a pelo teste de aglutinação em látex (SLA); E-test oxacilina; o sistema automatizado Vitek (GPS-105); o teste de triagem em ágar (OSAS) com oxacilina nas concentrações de 6,0, 1,0 e 0,75 æg oxacilina por ml e o teste de disco difusão com cefoxitina (CDD). A suscetibilidade automatizada foi obtida para os seguintes agentes antimicrobianos: fluorquinolonas, penicilina G, amoxicilina-ampicilina, cefazolina, ampicilina-sulbactam, eritromicina, clindamicina, gentamicina, tetraciclina, sulfametoxazol-trimetoprima, vancomicina e rifampicina. RESULTADOS: Dos 69 Staphylococcus coagulase negativo testados, 71 por cento foram mecA-positivos e 29 por cento, mecA-negativos. Todos os métodos testados apresentaram concordância estatisticamente significante com a reação em cadeia da polimerase. Houve tendência à predominância da positividade da reação em cadeia da polimerase entre os S. epidermidis comparado aos não-epidermidis, embora sem significância estatistica (78,3 por cento vs. 56,5 por cento; chi2= 2,54; p=0,11). O teste de triagem em ágar (0,75 æg oxacilina/ml) apresentou a melhor performance com resultados de: 100 por cento de sensibilidade e valor preditivo negativo, 95 por cento de especificidade e 98 por cento de valor preditivo positivo. Os isolados mecA-positivos foram estatisticamente...
Assuntos
Humanos , Antibacterianos/farmacologia , Resistência a Meticilina , Testes de Sensibilidade Microbiana/métodos , Staphylococcus/efeitos dos fármacos , Ágar , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Coagulase , Cefoxitina/farmacologia , Testes de Fixação do Látex , Resistência a Meticilina/efeitos dos fármacos , Resistência a Meticilina/genética , Meticilina/farmacologia , Oxacilina/farmacologia , Fenótipo , Reação em Cadeia da Polimerase , Infecções Estafilocócicas/microbiologia , Staphylococcus/enzimologia , Staphylococcus/genéticaRESUMO
Outbreaks of rapidly growing mycobacterium (RGM) infections are increasingly being reported worldwide. Information about genetic relatedness of isolates obtained during outbreaks can provide opportunities for prompt intervention. Pulsed-field gel electrophoresis (PFGE) is expensive, time consuming, and labor intensive. Other than that, Mycobacterium abscessus isolates can suffer DNA degradation during electrophoresis. Polymerase chain reaction (PCR)-based methods are cheaper, faster, and easier to perform, but discriminatory power varies depending on the primer used. In this study, we tested the competence of enterobacterial repetitive intergenic consensus (ERIC) PCR in comparison with PFGE to distinguish unrelated isolates (24 Mycobacterium chelonae and 24 M. abscessus) obtained from human and/or environmental samples and to group 56 isolates from 6 outbreaks confirmed epidemiologically, caused by M. chelonae and M. abscessus after ophthalmologic refractive surgery and mesotherapy. Enterobacterial repetitive intergenic consensus PCR presented discriminatory power, calculated using Simpson's index of diversity, of 0.989 for M. abscessus and 0.975 for M. chelonae and grouped outbreak isolates in distinct groups showing epidemiologic concordance. Pulsed-field gel electrophoresis also grouped outbreak isolates and presented discriminatory power of 0.972 and 0.993 for M. abscessus and M. chelonae, respectively. DNA from 8 (22%) of 36 M. abscessus isolates analyzed showed degradation during electrophoresis. Compared with PFGE and epidemiologic information as the gold standard, ERIC PCR is a simple, high throughput, affordable, reproducible, and discriminatory molecular typing method for inference of genetic relatedness of RGMs of the M. chelonae-abscessus group.