Do different probing depths exhibit striking differences in microbial profiles?

AIM: To perform a thorough characterization of the subgingival microbiota of shallow, moderate and deep sites in subjects with chronic periodontitis (ChP). MATERIAL AND METHODS: Subgingival samples were collected from subjects with ChP (n = 3/category of probing depth: ≤3, 4-6 and ≥7 mm) and periodontal health (PH). Individual samples were submitted to 16S rDNA high- throughput sequencing and the analysis was made using mothur and R packages. RESULTS: Nine subjects with ChP and seven with PH were included and 101 samples were evaluated. Thirteen phyla, 118 genera and 211 OTUs were detected. Taxa from Chloroflexi and Spirochaetes phyla were associated with initial stages of disease. Fretibacterium, Eubacterium[XI][G-6], Desulfobulbus, Peptostreptococcaceae[XI][G-1] and [G-3], Bacteroidetes[G-3], Bacteroidaceae[G-1] genera and Filifactor alocis, Fretibacterium fastidiosum, Johnsonella spHOT166, Peptostreptococcaceae[XIII][G-1]HOT113, Porphyromonas endodontalis and Treponema sp. HOT258, which are not conventionally associated with disease, increased with the deepening of the pockets and/or were elevated in ChP; while Streptococcus, Corynebacterium and Bergeyella genera were associated with PH (p < .05). CONCLUSION: Striking differences were observed between the microbiota of shallow and moderate/deep sites, but not between moderate and deep sites in ChP subjects. Differences between shallow sites in PH and ChP were also observed. The characterized microbiota included known oral microorganisms and newly identified periodontal taxa, some of them not-yet-cultured.

Complete nucleotide sequences of two blaKPC-2-bearing IncN Plasmids isolated from sequence type 442 Klebsiella pneumoniae clinical strains four years apart.

We sequenced the oldest blaKPC-2-bearing plasmid isolated in Brazil and another plasmid also carried by a Klebsiella pneumoniae strain of sequence type 442 (ST442), isolated 52 months later. Both plasmids present an IncN backbone and few acquired regions. Because the 2005 plasmid presented deletions and a truncated gene within Tn4401b compared to the 2009 plasmid, we can thus infer that IncN blaKPC-2-bearing plasmids pFCF1305 and pFCF3SP had a common ancestor circulating in Brazil prior to May 2005.

Presence of bla TEM-116 Gene in Environmental Isolates of Aeromonas Hydrophila and Aeromonas Jandaei from Brazil.

It is known that Aeromonas spp. possess different chromosomal ß-lactamase genes. Presence and phenotypic expression of bla TEM, bla SHV, and bla CTX-M ESBL-encoding genes were investigated in environmental water isolates of Aeromonas hydrophila and Aeromonas jandaei. Presence of bla SHV and bla CTX-M genes was not observed, and bla TEM gene was verified in 91% of the isolates. Sequencing of 10 fragments showed the occurrence of bla TEM-116.

Emergence of carbapenem-resistant Acinetobacter pittii carrying the blaOXA-72 gene in the Amazon region, Brazil.

We sought to characterize the genetic context of blaOXA-72 gene in a carbapenem-resistant Acinetobacter pittii strain recovered from a hospitalized patient from Belém, North Brazil, in the Amazon region. We found that the blaOXA-72 gene was carried by a small plasmid, pIEC338SCox, that is 10,498 bp. The gene is flanked by XerC/XerD-like recombinase sites, which suggests that this gene was acquired onto this plasmid by recombination.

Molecular mechanisms of membrane impermeability in clinical isolates of Enterobacteriaceae exposed to imipenem selective pressure.

Intrinsic mechanisms leading to carbapenem-induced membrane impermeability and multidrug resistance are poorly understood in clinical isolates of Enterobacteriaceae. In this study, molecular behaviours during the establishment of membrane impermeability in members of the Enterobacteriaceae family under imipenem selective pressure were investigated. Clinical isolates (n = 22) exhibiting susceptibility to multiple antibiotics or characterised as extended-spectrum ß-lactamase (ESBL)- or AmpC-producers were submitted to progressive passages on Mueller-Hinton agar plates containing subclinical concentrations of imipenem [0.5 × the minimum inhibitory concentration (MIC)]. Changes in outer membrane permeability were evaluated by determination of antimicrobial MICs, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and gene expression analysis related to membrane permeability (i.e. omp35-like, omp36-like and acrA) and regulatory mechanisms (i.e. marA and ompR) by quantitative reverse transcription PCR. Following imipenem induction, 73% of isolates showed increased carbapenem MICs by ≥2 doubling dilutions. At an early stage of treatment, imipenem modulated the expression of porins and efflux pump genes, represented by a reduction of 78% in omp36-like and a two-fold increase in acrA expression. Transcriptional factors marA and ompR were also affected by imipenem induction, increasing mRNA expression by 14- and 4-fold, respectively. High marA expression levels were associated with higher values of acrA expression. These results suggest that imipenem is an important factor in the development of an adaptive response to carbapenems by regulating key genes involved in the control of efflux pumps and porins, which could lead to a multidrug-resistant profile in clinical isolates, contributing to possible treatment failure.

Staphylococcal cassette chromosome mec elements from Staphylococcus intermedius group (SIG) isolates from dogs in a center for veterinary diagnostics in Brazil / Cassetes cromossômicos estafilocócicos mec de isolados do grupo Staphylococcus intermedius (GSI) de cães em um centro veterinário de diagnósticos no Brasil

ABSTRACT: Methicillin resistance in the Staphylococcus intermedius group (SIG) has emerged in small animal practice. Methicillin-resistant SIG (MRSIG) members have been implicated as causes of infections in both companion animals and humans. Staphylococcal cassette chromosome mec (SCCmec) elements carry the mecA/C genes, which encode for the transpeptidase PBP2a (PBP2') responsible for β-lactam antibiotic resistance in staphylococci. This study examined the SCCmec types of MRSIG isolates from different clinical specimens of dogs that exhibited methicillin MIC ≥ 0.5 μg/mL by an automated identification and susceptibility system in a Center for Veterinary Diagnostics in São Paulo, Brazil. Susceptibility to methicillin was determined by broth microdilution testing, and Oxoid® M.I.C.Evaluator® strips. PBP2a production was detected using a latex agglutination assay. SCCmec typing was performed according to the International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC) guidelines. SCCmec type II (2A), SCCmec type III (3A), composite SCC structures consisting of a class A mec gene complex in addition to multiple ccr gene complexes, and non-typable SCCmec elements were reported in these MRSIG isolates. SCCmec type variants differing from those so far acknowledged by IWG-SCC were found, indicating new rearrangements in the genetic context of mecA in these canine MRSIG isolates.

RESUMO: A resistência à meticilina no grupo Staphylococcus intermedius (GSI) tem aumentado na clínica de pequenos animais. Membros GSI resistentes à meticilina (GSIRM) têm sido causas de infecções tanto em animais de companhia e humanos. Cassetes cromossômicos estafilocócicos mec (SCCmec) carregam os genes mecA/C, que codificam a transpeptidase PBP2a (PBP2') responsável pela resistência aos antibióticos β-lactâmicos em estafilococos. Nosso objetivo foi investigar os elementos SCCmec de GSIRM isolados de diferentes amostras clínicas de cães que exibiram CIM de meticilina ≥ 0,5 μg/mL por meio de um sistema automatizado em um Centro Veterinário de Diagnósticos em São Paulo, Brasil. A sensibilidade à meticilina foi determinada por meio do teste de microdiluição em caldo e fitas Oxoid® M.I.C.Evaluator®. A produção de PBP2a foi detectada usando um ensaio de aglutinação de látex. A tipagem dos elementos SCCmec foi realizada de acordo com as diretrizes do International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC). SCCmec tipo II (2A), SCCmec tipo III (3A), SCC compostos de um complexo mec de classe A com múltiplos complexos ccr, e elementos SCCmec não tipáveis foram encontrados nesses isolados GSIRM. Variantes que diferem dos elementos SCCmec reconhecidos até o momento pelo IWG-SCC foram encontradas, indicando novos rearranjos no contexto genético de mecA nesses isolados GSIRM caninos.

Complete Genome Sequence of Staphylococcus aureus FCFHV36, a Methicillin-Resistant Strain Heterogeneously Resistant to Vancomycin.

We report here the sequence of the entire chromosome of Staphylococcus aureus strain FCFHV36, a methicillin-resistant strain heterogeneously intermediate to vancomycin, bearing a type II staphylococcal chromosome cassette mec element (SCCmec), belonging to multilocus sequence type (MLST) 105, and isolated from a vertebra of a patient with osteomyelitis.

Complete Genome Sequence of Lactococcus lactis Strain AI06, an Endophyte of the Amazonian Açaí Palm.

We report the genome, in a single chromosome, of Lactococcus lactis strain AI06, isolated from the mesocarp of the açaí fruit (Euterpe oleracea) in eastern Amazonia, Brazil. This strain is an endophyte of the açaí palm and also a component of the microbiota of the edible food product.

Methicillin/Oxacillin-resistant Staphylococcus aureus as a hospital and public health threat in Brazil.

Methicillin-resistant Staphylococcus aureus is an established nosocomial pathogen (HA-MRSA, hospital acquired MRSA), but has recently begun to appear in the community (CA-MRSA, community acquired MRSA). The cause of resistance to methicillin and all other beta-lactam antibiotics is the mecA gene, which is situated on a mobile genetic element, the Staphylococcal Cassette Chromosome mec (SCCmec). Seven major variants of SCCmec, type I to VII are distinguished. HA-MRSA disseminated worldwide and causes the majority of S. aureus nosocomial infections with a limited number of clones disseminated including the Brazilian Epidemic Clone (BEC, ST239-MRSA-III). CA-MRSA isolates are susceptible to non-beta-lactam antibiotics, usually isolated from healthy individuals which do not possess any unknown risk factors for MRSA infection and are associated with a larger clonal diversity compared with HA-MRSA. However, during recent years distinction between HA-MRSA and CA-MRSA is beginning to fade. Actually, knowledge about MRSA disseminating clones is required to implement any strategies to control the transmission of MRSA either within hospitals or in community. For this reason, rapid identification of strains is an important issue. The rate of HA-MRSA can be reduced substantially through the implementation of interventions strategies, even in settings where MRSA is endemic as in most Brazilian hospitals. However, these policies could be quite complicated in the light of an increasing CA-MRSA prevalence in healthcare facilities, considering that distinction between HA-MRSA and CA-MRSA has started to disappear.

Type IV SCCmec found in decade old Brazilian MRSA isolates.

Methicillin-resistant Staphylococcus aureus (MRSA) commonly causes infection in hospitalized patients. Since its appearance in the 1960s, the SCCmec has evolved throughout the years into 5 different types (I-V), each bearing a different set of genes. Infection with MRSA SCCmec types I, II or III is almost exclusively restricted to hospitalised patients. However, recently, community acquired MRSA (CA-MRSA) infections have been reported with increasing frequency, usually caused by a type IV SCCmec MRSA in nosocomial settings. We studied the prevalence of SCCmec types in 50 nosocomial strains collected from 1995 to 1999. The SCCmec complex type and presence of Panton-Valentine leukocidin (PVL) were determined by PCR. Strains had been previously typed by PFGE and were now typed by MLST. We found that 3 of the isolates studied bore a type IVc SCCmec all having different PFGE and MLST profiles (ST3, ST5 and ST88). All strains bearing a type III SCCmec belonged to MLST ST239 (Brazilian/Iberian clone). Only the strain which presented the ST5 profile bore the pvl gene. The type IVc SCCmec strains presented relatively lower levels of resistance to oxacillin in comparison to the type III SCCmec strains. The pattern of dissemination of the type IV SCCmec remains to be elucidated. The finding of strains carrying a type IV SCCmec in the present study among strains isolated at least 7 years ago indicates that clones bearing a type IV SCCmec have been present in Brazil for quite some time, and must have gone by undetected.

Characterization of ndh gene of isoniazid resistant and susceptible Mycobacterium tuberculosis isolates from Brazil.

Resistance in Mycobacterium tuberculosis to isoniazid (INH) is caused by mutations in the catalase-peroxidase gene (katG), and within the inhA promoter and/or in structural gene. A small percentage (approximately 10%) of INH-resistant strains do not present mutations in both of these loci. Other genes have been associated with INH resistance including the gene encoding for NADH dehydrogenase (ndh). Here we report the detection of two ndh locus mutations (CGT to TGT change in codon 13 and GTG to GCG change in codon 18) by analyzing 23 INH-resistant and in none of 13 susceptible isolates from Brazilian tuberculosis patients. We also detected two isolates without a mutation in ndh, or any of the other INH resistance-associated loci examined, suggesting the existence of additional, as yet to be described, INH resistance mechanisms.

Methicillin/Oxacillin-resistant Staphylococcus aureus as a hospital and public health threat in Brazil

Methicillin-resistant Staphylococcus aureus is an established nosocomial pathogen (HA-MRSA, hospital acquired MRSA), but has recently begun to appear in the community (CA-MRSA, community acquired MRSA). The cause of resistance to methicillin and all other â-lactam antibiotics is the mecA gene, which is situated on a mobile genetic element, the Staphylococcal Cassette Chromosome mec (SCCmec). Seven major variants of SCCmec, type I to VII are distinguished. HA-MRSA disseminated worldwide and causes the majority of S. aureus nosocomial infections with a limited number of clones disseminated including the Brazilian Epidemic Clone (BEC, ST239-MRSA-III). CA-MRSA isolates are susceptible to non-â-lactam antibiotics, usually isolated from healthy individuals which do not possess any unknown risk factors for MRSA infection and are associated with a larger clonal diversity compared with HA-MRSA. However, during recent years distinction between HA-MRSA and CA-MRSA is beginning to fade. Actually, knowledge about MRSA disseminating clones is required to implement any strategies to control the transmission of MRSA either within hospitals or in community. For this reason, rapid identification of strains is an important issue. The rate of HA-MRSA can be reduced substantially through the implementation of interventions strategies, even in settings where MRSA is endemic as in most Brazilian hospitals. However, these policies could be quite complicated in the light of an increasing CA-MRSA prevalence in healthcare facilities, considering that distinction between HA-MRSA and CA-MRSA has started to disappear.

Presence of blaTEM-116 gene in environmental isolates of Aeromonas hydrophila and Aeromonas jandaei from Brazil

It is known that Aeromonas spp. possess different chromosomal â-lactamase genes. Presence and phenotypic expression of blaTEM, blaSHV, and blaCTX-M ESBL-encoding genes were investigated in environmental water isolates of Aeromonas hydrophila and Aeromonas jandaei. Presence of blaSHV and blaCTX-M genes was not observed, and blaTEM gene was verified in 91 percent of the isolates. Sequencing of 10 fragments showed the occurrence of blaTEM-116.

Type IV SCCmec found in decade old Brazilian MRSA isolates

Methicillin-resistant Staphylococcus aureus (MRSA) commonly causes infection in hospitalized patients. Since its appearance in the 1960s, the SCCmec has evolved throughout the years into 5 different types (I-V), each bearing a different set of genes. Infection with MRSA SCCmec types I, II or III is almost exclusively restricted to hospitalised patients. However, recently, community acquired MRSA (CA-MRSA) infections have been reported with increasing frequency, usually caused by a type IV SCCmec MRSA in nosocomial settings. We studied the prevalence of SCCmec types in 50 nosocomial strains collected from 1995 to 1999. The SCCmec complex type and presence of Panton-Valentine leukocidin (PVL) were determined by PCR. Strains had been previously typed by PFGE and were now typed by MLST. We found that 3 of the isolates studied bore a type IVc SCCmec all having different PFGE and MLST profiles (ST3, ST5 and ST88). All strains bearing a type III SCCmec belonged to MLST ST239 (Brazilian/Iberian clone). Only the strain which presented the ST5 profile bore the pvl gene. The type IVc SCCmec strains presented relatively lower levels of resistance to oxacillin in comparison to the type III SCCmec strains. The pattern of dissemination of the type IV SCCmec remains to be elucidated. The finding of strains carrying a type IV SCCmec in the present study among strains isolated at least 7 years ago indicates that clones bearing a type IV SCCmec have been present in Brazil for quite some time, and must have gone by undetected.

Characterization of ndh gene of isoniazid resistant and susceptible Mycobacterium tuberculosis isolates from Brazil

Resistance in Mycobacterium tuberculosis to isoniazid (INH) is caused by mutations in the catalase-peroxidase gene (katG) , and within the inhA promoter and/or in structural gene. A small percentage (~ 10 percent) of INH-resistant strains do not present mutations in both of these loci. Other genes have been associated with INH resistance including the gene encoding for NADH dehydrogenase (ndh) . Here we report the detection of two ndh locus mutations (CGT to TGT change in codon 13 and GTG to GCG change in codon 18) by analyzing 23 INH-resistant and in none of 13 susceptible isolates from Brazilian tuberculosis patients. We also detected two isolates without a mutation in ndh, or any of the other INH resistance-associated loci examined, suggesting the existence of additional, as yet to be described, INH resistance mechanisms.

DOD/AMB: in vivo activity of a novel amb formulation with synthetic cationic bilayer fragments

A habilidade do brometo de dioctadecildimetilamônio (DOBAB), em formar bicamada de lipídio sintético e a demonstração prévia do forte poder solubilizante de anfotericina B (ANB) , incentivou-nos a realizar a avaliação da atividade de DODAB/AMB in vivo contra candidíase sistêmica em modelo de camundongos para verificar a sua sobrevida bem como a recuperação das leveduras de C. albicans dos órgãos colonizados (baço e rins). O AMB foi simplesmente adicionado à DODAB em pó previamente disperso em água e sonicado com auxílio de ponteiras, nas concentrações de <0,1e 10 mg/mL, respectivamente, assegurando-se a completa ausência de solventes orgânicos nesta formulação. O estado de agregação do AMB foi avaliado por meio do espectro de absorção da luz UV-visível e a distribuição de tamanhos das formulações estudadas, determinadas por meio de analisador ZetaPlus Brookhaven Instruments Corporation, Holtsville, NY) equipado com um laser de 570 nm e dynamic light scattering (90ºC) para a medição dos tamanhos (Grabowski & Morrison, 1983). AMB foi estabilizada na bicamada de DODAB em forma monomérica, eliminando-se os grandes agregados de AMB insolúveis em água. Tanto a sobrevida dos animais como os experimentos com recuperação das leveduras dos órgãos colonizados (baço e rins) mostraram eficácia equivalente à demonstrada por Fungizona (DOC/AMB) - a sobrevida foi de 100 e 70% respectivamente nas concentrações de 0.4 mg/kg/dia via i.p. por 10 dias (P>0.05), em relação a eliminação da colonização de C. albicans dos rins e baço. Em resumo, DOD/AMB foi efetivo no tratamento de candidíase sistêmica em modelo animal.

Microbiota indígena do meato acústico externo de gatos hígidos / Normal microflora of the ear canal in healthy cats

Cinqüenta felinos hígidos, adultos - vinte e seis machos e vinte e quatro fêmeas - sem definiçäo racial, com distintas idades, reunidos em dois grupos (GRUPO 1 - animais domiciliados e GRUPO 2 - animais querenciados) de vinte e cinco animais cada. Após anamnese e prévia sedaçäo, os gatos foram submetidos a exame físico e à otoscopia, comprovando-se a ausência de lesöes meatais e timpânicas, com cureta de Buck e zaragatoas estéreis, foi colhido material meatal, que foi entäo submetido a exames bacteriológico e micológico. No Grupo 1, foi evidenciado presença de Otodectes cynotis (4 porcento das amostras), em 72 porcento dos casos de bolores e leveduras (Cladosporium sp 66,6 porcento, Malassezia sp 40 porcento, Penicillium sp 33 porcento, Aspergillus sp 33,3 porcento, Rhodotorula sp 20,0 porcento, Mycelia sp 13,3 porcento e Alternaria sp, Aureobasidium sp, Rysopus sp, Trichosporon sp, todos com 6,6 porcento) e, finalmente, em 64 porcento da amostragem, bactérias dos gêneros Staphylococcus spp (81,2 porcento), Pseudomonas sp (12,5 porcento), Klebsiella sp (12,5 porcento), Acinetobacter sp, Bacilos difteróides, Enterobacter sp, Lactobacillus spp (todos com 6,2 porcento). No grupo 2, o Otodectes sp foi identificado em 36 porcento das amostras, em 96 porcento daquelas isolaram-se fungos dos gêneros: Malassezia sp - 54,1 porcento, Aspergillus e Penicillium sp, ambos com 33,3 porcento, Microsporum sp - 29,1 porcento, Cladosporium sp - 16,6 porcento, Trichoderma sp - 12,5 porcento, Alternaria e Phoma sp, ambos com 8,3 porcento e Epicoccum sp, Neurospora sp, Mycelia sp, Rhodotorula sp, todos com 4,1 porcento e, por fim, em 20 das 25 amostras (80 porcento) isolaram-se pelo menos uma cepa bacteriana (Staphylococcus spp 75 porcento, Klebsiella sp 20,8 porcento, Bacilos difteróides 12,5 porcento, Pseudomonas sp, 8,3 porcento) e Acinetobacter sp, Enterobacter sp e Escherichia sp, todos com 4,1 porcento cada um em cultivo monoespecífico ou em associaçäo.

Campylobacter termofílico em fezes de crianças com diarréia: estudo com controles / Thermophilic Campylobacter in feces of children with diarrhea: study of controls

Foram determinadas as frequências de isolamento de Campylobacter termofílico e de outros enteropatógenos das fezes de 208 crianças com diarréia aguda (Grupo diarréico) e de 133 crianças sem sintomas gastrintestinais (Grupo controle). Campylobacter sp. foi recuperado das fezes de 29 crianças do grupo diarréico, tendo sido o agente bacteriano isolado com maior frequência nesse grupo de crianças - 13,9%, seguido por EPEC - 6,8%, Shigella sp. - 4,3% e Salmonella sp. - 2,9%. Näo foi isolada nenhuma cepa de Campylobacter termofílico das fezes das crianças do grupo controle. Baseados nestes dados salientamos a necessidade da pesquisa de Campylobacter termofílico de forma rotineira em crianças com diarréia aguda, a fim de melhorar o diagnóstico etiológico dessa afecçäo

Leucócitos fecais na diarréia aguda por Campylobacter sp termofílico / Fecal leucocytes in Campylobacter associated acute diarrhea

Com o intuito de se comparar as porcentagens de positividade para leucócitos fecais obtidas em esfregaços de fezes corados pelo método de Gram, de acordo com o agente enteropatogênico identificado, foram estudadas, em Säo Paulo - SP, 184 amostras de fezes provenientes de crianças com diarréia aguda. Dessas amostras, 29 eram positivas para Campylobacter sp termofílico, 12 para agentes invasivos e 103 para agentes näo invasivos, sendo que nas 40 amostras restantes nenhum agente enteropatogênico. Leucócitos fecais foram encontrados nos casos de campilobacteriose em proporçöes estatisticamente semelhante às encontradas em casos de diarréias causadas por patógenos intestinais invasivos e significativamente maiores do que em infecçöes por agentes näo invasivos ou naquelas onde nenhum agente enteropatogênico foi identificado

Diagnóstico rápido (presuntivo) da diarréia aguda causada por Campylobacter sp termofílico / Rapid (presumptive) diagnosis of acute diarrhea caused by thermophilic Campylobacter sp

Com o objetivo de se verificar o valor do exame direto de fezes coradas pelo método de Gram, no diagnóstico rápido (presuntivo) da diarréia aguda por Campylobacter sp termofílico, foram estudadas, em Säo Paulo - SP, amostras provenientes de 208 crianças com diarréia aguda e de 46 sem sintomas gastrintestinais, nas quais pesquisaram-se microrganismos delgados, Gram-negativos, espiralados ou em forma de "S", ou ainda de "asa de gaivota". Os resultados foram comparados com os da cultura das fezes em meio de Skirrow modificado. No grupo de crianças com diarréia, microrganismos com as características citadas foram observadas em 28 das 29 lâminas de fezes (96,5%) com cultura positivas para o microrganismo e em 19 lâminas de fezes do total de 179 examinadas (19,6%), cujas culturas foram negativas para essa bactéria. Assim, o método da coloraçäo de Gram apresentou sensibilidade de 96,5%, especificidade de 89%, valores preditivos de um resultado positivo 60% e de um resultado negativo 99,4%, tendo sido sua eficiência calculada em 90,4%