Genetic relationships between sympatric populations of Bacillus cereus and Bacillus thuringiensis, as revealed by rep-PCR genomic fingerprinting

The bacterial strain Bacillus cereus is closely related to Bacillus thuringiensis, although any genetic relationship between the two strains is still in debate. Using rep-PCR genomic fingerprinting, we established the genetic relationships between Brazilian sympatric populations of B. cereus and B. thuringiensis simultaneously collected from two geographically separate sites. We observed the formation of both B. thuringiensis and B. cereus clusters, as well as strains of B. cereus that are more closely related to B. thuringiensis than to other B. cereus strains. In addition, lower genetic variability was observed among B. thuringiensis clusters compared to B. cereus clusters, indicating that either the two species should be categorized as separate or that B. thuringiensis may represent a clone from a B. cereus background.

Molecular characterization of Brazilian strains of Xanthomonas campestris pv. viticola by rep-PCR fingerprinting

Bacterial canker of grapevine (Vitis vinifera), caused by Xanthomonas campestris pv. viticola was first detected in Brazil in 1998, affecting grapevines in the São Francisco river basin, state of Pernambuco. The disease was also reported in Juazeiro, Bahia and later in Piauí and Ceará. Due to its limited geographical distribution and relatively recent detection in Brazil, very little is known about the pathogen's biology and diversity. Repetitive DNA based-PCR (rep-PCR) profiles were generated from purified bacterial DNA of 40 field strains of X. campestris pv. viticola, collected between 1998 and 2001 in the states of Pernambuco, Bahia and Piauí. Combined analysis of the PCR patterns obtained with primers REP, ERIC and BOX, showed a high degree of similarity among Brazilian strains and the Indian type strain NCPPB 2475. Similar genomic patterns with several diagnostic bands, present in all strains, could be detected. Fingerprints were distinct from those of strains representing other pathovars and from a yellow non-pathogenic isolate from grape leaves. The polymorphism observed among the Brazilian strains allowed their separation into five subgroups, although with no correlation with cultivar of origin, geographic location or year collected.

Virulence and rep-PCR analysis of Pyricularia grisea isolates from two Brazilian upland rice cultivars

The phenotypic and genetic diversity of 77 isolates of Pyricularia grisea collected from two upland rice cultivars, Maravilha and Primavera, was studied. Isolates exhibiting compatible reaction to cv.Primavera were incompatible to cv.Maravilha and vice versa, with the exception of six isolates that were compatible to both cultivars. The virulence of isolates from cv. Maravilha on 32 test genotypes of rice was significantly higher (t = 9.09, p < 0.0001) than the isolates from cv.Primavera. A phenogram constructed from virulence data showed two main groups, one constituted mainly of isolates from cv.Primavera (97.6%) and the other of isolates from cv.Maravilha (91.17%). Rep-PCR analysis of isolates using two primers designed from sequences of Pot2 showed that isolates could be clustered broadly into two groups. The average similarity within a cluster of isolates from cv.Primavera was significantly greater than the average similarity among the isolates of cv.Maravilha (t = 5.37, p < 0.0001). There was close correspondence between clusters based on PCR and virulence data (r = 0.48, p < 0.011). The results showed that isolates of P. grisea were cultivar specific and had low phenotypic and genetic diversity.

Typing of avian pathogenic Escherichia coli strains by REP-PCR

In the present study the repetitive extragenic palindromic (REP) polymerase chain reaction (PCR) technique was used to establish the clonal variability of 49 avian Escherichia coli (APEC) strains isolated from different outbreak cases of septicemia (n=24), swollen head syndrome (n=14) and omphalitis (n=11). Thirty commensal strains isolated from poultry with no signs of these illnesses were used as control strains. The purified DNA of these strains produced electrophoretic profiles ranging from 0 to 15 bands with molecular sizes varying from 100 bp to 6.1 kb, allowing the grouping of the 79 strains into a dendrogram containing 49 REP-types. Although REP-PCR showed good discriminating power it was not able to group the strains either into specific pathogenic classes or to differentiate between pathogenic and non-pathogenic strains. On the contrary, we recently demonstrated that other techniques such as ERIC-PCR and isoenzyme profiles are appropriate to discriminate between commensal and APEC strains and also to group these strains into specific pathogenic classes. In conclusion, REP-PCR seems to be a technique neither efficient nor universal for APEC strains discrimination. However, the population clonal structure obtained with the use of REP-PCR must not be ignored particularly if one takes into account that the APEC pathogenic mechanisms are not completely understood yet.

Identification of new isolates of Bacillus thuringiensis using rep-PCR products and delta-endotoxin electron microscopy

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Discriminação de sorovares de Salmonella spp. isolados de carcaças de frango por REP e ERIC-PCR e fagotipagem do sorovar Enteriditis

Salmonelose é a infecção bacteriana de origem alimentar mais freqüente no Paraná, Brasil, e os surtos estão associados, principalmente, ao consumo de ovos, carne de aves e derivados. Os objetivos deste trabalho foram identificar os sorovares de Salmonella isolados de carcaças de frango e caracterizá-los molecularmente por REP e ERIC-PCR, assim como identificar os fagotipos de Salmonella Enteriditis. Dos 25 isolados de Salmonella spp. analisados, 18 foram identificados como Enteriditis, 4 como Braenderup, 2 como Worthington e 1 como infantis. Dos 18 isolados de Enteriditis, 14 foram PT4, 2 PT4a, 1 PT7 e 1 RDNC, por se tratar de colônia rugosa. REP-PCR forneceu padrão eletroforético distinto de 10 a 13 bandas distribuídas entre 120 e 2072 pb para cada sorovar diferente testado. A ERIC-PCR mostrou um padrão de 4 a 5 bandas entre 180 e 1000 pb e foi menos discriminativa quando comparada à REP-PCR. Os resultados encontrados confirmaram que a fagotipagem é uma ferramenta útil e discriminativa para o sorovar Enteriditis. Apesar do pequeno número de sorovares testados, os resultados sugerem que a REP-PCR parece ser um método atrativo a ser utilizado no futuro para a discriminação preliminar de sorovares de Salmonella.

Sensibilidade do método de obtenção das células bacterianas e da técnica de PCR para detecção de Curtobacterium flaccumfaciens pv. flaccumfaciens em sementes de feijão

Atualmente, existe a necessidade de se desenvolver métodos sensíveis, baratos, reproduzíveis e rápidos para a detecção de fitobactérias em sementes. O objetivo deste trabalho foi otimizar um método de obtenção das células bacterianas e a técnica de PCR para detecção de Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff), em sementes de feijão. Foi utilizado o primer CffFOR2-REV4, desenhado a partir do fragmento amplificado via PCR baseado na seqüência repetitiva (Rep-PCR). Avaliaram-se também quatro métodos de preparação dos extratos de sementes de feijão para a obtenção células de Cff : 1) extrato bruto de sementes; 2 ) extrato concentra do por filtração em membra na milipore (0,22Mu de diâmetro) e ressuspensão em água; 3) extrato concentrado por centrifugação a 10 .00 0 xg por 15 minutos no volume de 20 ou de 80 mL e 4) Bio-PCR. Dentre esses métodos, tanto a Bio-PCR quanto a concentração do extrato por centrifugação, seja no volume de 20 ou de 80 mL, possibilitaram amplificar o segmento de DNA de 306 pb, característico de Cff. Essas duas técnicas, além de detectar a bactéria, apresentam alta sensibilidade, detectando até 1 semente inoculada artificialmente artificialmente com Cff em 999 sementes sadi as. Analisaram-se dezessete lotes comerciais de sementes de feijão pelo método de concentração de extrato por centrifugação, sendo qu e em doze detectou-se a bactéria Cff, observado pela presença de bandas com 306 pb. Portanto, foi possível otimizar um método de obtenção das células bacterianas e técnica de PCR para detecção de Cff em sementes de feijão, que seja sensível, reproduzível e de fácil execução, que poderá ser utilizado rotineiramente em laboratórios de análises de sementes.

Tipificação molecular de amostras de Brucella spp. utilização a técnica de BOX-PCR

O gênero Brucella é formado por coco-bacilos Gram negativos patogênicos ao homem e animais, sendo classificado como patógeno de grupo de risco III. A identificação dessas bactérias apresenta várias limitações como: exigência de inoculação em vários meios, tempo de incubação longo e necessidade de soros imunes e bacteriófagos. Devido à sua alta patogenicidade e ao longo tempo de exposição dos laboratoristas à bactéria, a brucelose é uma das infecções mais freqüentemente adquiridas em laboratório. Além da contaminação em laboratório, a transmissão ao homem pode ocorrer através de animais infectados e ingestão de produtos derivados, como o leite cru. A procura de métodos rápidos de identificação das espécies e biovares pode ser útil para diminuir os riscos do manuseio desta bactéria e na tomada de medidas de controle epidemiológico. O principal objetivo deste trabalho foi facilitar a classificação de cepas de referência de Brucella spp. e isoladas no Brasil utilizando a técnica de rep-PCR com oligonucleotídeos do elemento BOX, uma seqüência repetida presente no genoma de várias bactérias. Foram analisados 38 isolados representando diferentes espécies e biovares de Brucella sp. e 13 isolados de gêneros relacionados como controle da especificidade da reação. Foi realizada uma confirmação prévia dos isolados de brucela por testes bioquímicos e PCR gênero-específica. A técnica de BOX-PCR agrupou todas as espécies e biovares de Brucella em um único grupo com nível de similaridade entre 100 e 74%. Diferenças entre os isolados, quanto a presença ou ausência de bandas, puderam ser observadas. Entretanto, essas divergências não caracterizam uma espécie ou biovar. Bandas comuns a todos os isolados de Brucella sp. podem caracterizar o gênero.

Rapid genotyping of Pseudomonas aeruginosa isolates harboured by adult and paediatric patients with cystic fibrosis using repetitive-element-based PCR assays

In this study, the suitability of two repetitive-element-based PCR (rep-PCR) assays, enterobacterial repetitive intergenic consensus (ERIC)-PCR and BOX-PCR, to rapidly characterize Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis (CF) was examined. ERIC-PCR utilizes paired sequence-specific primers and BOX-PCR a single primer that target highly conserved repetitive elements in the P. aeruginosa genome. Using these rep-PCR assays, 163 P. aeruginosa isolates cultured from sputa collected from 50 patients attending an adult CF clinic and 50 children attending a paediatric CF clinic were typed. The results of the rep-PCR assays were compared to the results of PFGE. All three assays revealed the presence of six major clonal groups shared by multiple patients attending either of the CF clinics, with the dominant clonal group infecting 38% of all patients. This dominant clonal group was not related to the dominant clonal group detected in Sydney or Melbourne (pulsotype 1), nor was it related to the dominant groups detected in the UK. In all, PFGE and rep-PCR identified 58 distinct clonal groups, with only three of these shared between the two clinics. The results of this study showed that both ERIC-PCR and BOX-PCR are rapid, highly discriminatory and reproducible assays that proved to be powerful surveillance screening tools for the typing of clinical P. aeruginosa isolates recovered from patients with CF.

PCR detection and analysis of Pasteurella multocida from the tonsils of slaughtered pigs in Vietnam

A total of 36 tonsil swab samples were collected from healthy swine prior to slaughter at the abattoirs in Can tho and Tien giang provinces of Southern Vietnam, The presence of Pasteurella multocida in these samples was detected by the combination of direct cultivation and isolation, mouse inoculation and the polymerase chain reaction (PM-PCR). P. multocida was detected in 16 samples by PCR, with 17 strains ultimately isolated. All samples were negative for serogroup B by HSB-PCR and conventional serotyping, with isolates identified as A:3, D:1 or D:3. In addition, all samples were determined to be negative for the P. multocida toxin (PMT). Characterisation of isolated P, multocida by REP-PCR and biotyping revealed nine distinct REP profiles and seven biotypes among the 17 isolates. Some correlation was seen with P. multocida isolated from a previous Australian outbreak of acute swine pasteurellosis, and those isolated from fowl cholera outbreaks in Vietnamese poultry. (C) 2000 Elsevier Science B.V. All rights reserved.

Analysis of the clonal relationship among clinical isolates of Salmonella enterica serovar Infantis by different typing methods

Salmonella Infantis has been the second most common serovar in Argentina in the last two years, being isolated mostly from paediatric hospitalised patients. In order to determine the clonal relationship among Salmonella Infantis strains, we examined 15 isolates from paediatric patient faeces in Argentina (12 geographically related and 3 geographically non-related) by using antimicrobial susceptibility, plasmid profiling, repetitive extragenic palindromic (REP) PCR, enterobacterial repetitive intergenic consensus (ERIC) PCR, and low-frequency restriction analysis of chromosomal DNA by pulsed field gel electrophoresis (PFGE). Four Spanish strains were included as controls of clonal diversity in molecular techniques. Antibiotype and plasmid profile was not useful as epidemiological tools. PFGE and REP-PCR were able to discriminate between Argentinean and Spanish isolates of Salmonella Infantis allowing to detect genetically related strains in three different cities. This finding indicates that a possible spread of a clone of this serovar in the North-eastern Region of Argentina has taken place in 1998.

Diversity of Vibrionaceae bacteria isolated from the haemolymph of the spider crab Maja brachydactyla

Aims: The aim of this study was to characterise and identify vibrios isolated from the haemolymph of apparently healthy adult spider crabs (Maja brachydactyla) wild-caught in the Spanish localities of Galician coast and in the Canary Islands and also from captive animals held at IRTA’s facilities in the Ebro Delta of Catalonia, north-west Spanish Mediterranean coast. Methods and Results: A total of 277 bacterial isolates were obtained, and of these, 171 were characterised with rep-PCR, resulting electrophoretic bands were analysed and clusters formed. Identification of representative strains of each cluster was made by sequencing the 16S rRNA. Samples from animals caught in Galicia and captive at IRTA (around 15–18 C) rendered mostly species belonging to the Splendidus clade (72Æ2 and 76Æ6% respectively), commonly found in cold waters (below 20 C). Higher species diversity was found in the haemolymph of the captive animals. In the warmer Canary Islands waters (around 21 C), the diversity of vibrios is dominated by three clades, Harveyi (Vibrio core group, 39Æ3%), Orientalis (23Æ2%) and Splendidus (21Æ4%) with a species diversity that equals that of the colder captive animals. Conclusions: Differences in the vibrios populations were found in the haemolymph extracted from animals collected from the three localities. Potential new species were found, and their description is under way. Significance and Impact of Study: As with other invertebrates, spider crabs also contain a diverse population of vibrios. These findings should help researchers to diagnose when a crab is infected.

Genetic diversity and virulence pattern in field populations of Pyricularia grisea from rice cultivar Metica-1

Rice blast is a major yield constraint of the irrigated rice in the State of Tocantins, Brazil. The objective of this investigation was to study the phenotypic and genetic diversity within the pathogen population of Pyricularia grisea in samples collected from four individual farms of rice cultivar Metica-1, under epidemic conditions of leaf blast. A set of 87 isolates was tested on 32 rice genotypes including eight international differentials. Considering 80% similarity in virulence, two groups comprising a total of 81 isolates were recognized, independently of the farms from which they were collected. Eighty percent of the isolates pertained to pathotype ID-14, indicating high cultivar specificity and narrow diversity of virulence in the sample population. The virulence in pathogen population on rice cultivars BR-IRGA 409 and Rio Formoso was low. Analysis of P. grisea isolates using rep-PCR with two primer sequences from Pot2 generated fingerprint profiles of one to nine bands. Cluster analysis revealed the occurrence of six fingerprint groups with similarities ranging from 0.09 to 1. There was no straight relationship between virulence of the isolates based on reaction pattern on 32 genotypes and grouping based on Pot2 rep-PCR analysis of P. grisea isolates collected from 'Metica-1'.

Diversidade genética de isolados de Ralstonia solanacearum e caracterização molecular quanto a filotipos e sequevares

O objetivo deste trabalho foi identificar isolados brasileiros de Ralstonia solanacearum quanto a filotipos e sequevares, determinar sua diversidade genética, realizar a associação da estrutura genética do patógeno com sua classificação e origem geográfica e identificar um marcador molecular para a diagnose do moko-da-bananeira. Um grupo de 33 isolados de R. solanacearum, da coleção da Embrapa Tabuleiros Costeiros, coletado de diversos hospedeiros, foi caracterizado por meio de PCR em sequência palindrômica extragênica repetitiva (rep-PCR) e RAPD. Deste grupo, 19 perteciam à raça 2 do patógeno e 14 à raça 1, e 15 isolados eram associados à cultura da bananeira. Os filotipos e sequevares foram caracterizados e determinados por PCR Multiplex. Verificou-se que 82% dos isolados pertencem ao filotipo II, e 12% ao filotipo III. Todos os isolados da bananeira pertencem ao filotipo II. Atécnica de RAPD foi eficiente em agrupar os isolados de acordo com sua origem geográfica; entretanto, ela requer um número elevado de marcas moleculares. Foi possível relacionar os isolados pela análise rep-PCR. O iniciador com sequências repetitivas enterobacterianas intergênicas de consenso (ERIC) possibilitou a separação dos isolados de acordo com a raça, eoiniciador REP permitiua discriminação entre os filotipos. Estas duas análises foram as mais informativas.