In silico phylogenetic and virulence gene profile analyses of avian pathogenic Escherichia coli genome sequences / Análises in silico da filogenia e do perfil de genes associados à virulência, dos genomas de linhagens de Escherichia coli de origem aviária

Avian pathogenic Escherichia coli (APEC) infections are responsible for significant losses in the poultry industry worldwide. A zoonotic risk has been attributed to APEC strains because they present similarities to extraintestinal pathogenic E. coli (ExPEC) associated with illness in humans, mainly urinary tract infections and neonatal meningitis. Here, we present in silico analyses with pathogenic E. coli genome sequences, including recently available APEC genomes. The phylogenetic tree, based on multi-locus sequence typing (MLST) of seven housekeeping genes, revealed high diversity in the allelic composition. Nevertheless, despite this diversity, the phylogenetic tree was able to cluster the different pathotypes together. An in silico virulence gene profile was also determined for each of these strains, through the presence or absence of 83 well-known virulence genes/traits described in pathogenic E. coli strains. The MLST phylogeny and the virulence gene profiles demonstrated a certain genetic similarity between Brazilian APEC strains, APEC isolated in the United States, UPEC (uropathogenic E. coli) and diarrheagenic strains isolated from humans. This correlation corroborates and reinforces the zoonotic potential hypothesis proposed to APEC.

As infecções causadas por linhagens de Escherichia coli de origem aviária (APEC) são responsáveis por perdas significativas na indústria avícola em todo mundo. Risco zoonótico tem sido atribuído às linhagens APEC, devido às semelhanças existentes entre elas e linhagens de E. coli patogênicas extraintestinais (ExPEC) de origem humana, causadoras de infecções no trato urinário e meningite neonatal. Neste trabalho, apresentamos os resultados de análises in silico feitas a partir dos genomas de linhagens patogênicas de E. coli, incluindo genomas recentemente obtidos de linhagens APEC. Uma árvore filogenética foi obtida, com base na tipagem de sequência multilocus (MLST) de sete genes essenciais, revelando alta diversidade na composição de alelos, mas ainda assim possibilitando o agrupamento dos diferentes patótipos. Foi determinado também, para cada linhagem, o perfil gênico, por meio da presença ou ausência de 83 genes associados à virulência. A árvore filogenética e o perfil gênico demonstraram que existem semelhanças genéticas entre cepas APEC brasileiras, APEC isolada nos Estados Unidos, UPEC (uropathogenic E. coli) e linhagens produtoras de diarreia em humanos. Essa correlação corrobora e reforça a hipótese de que linhagens APEC apresentam potencial risco zoonótico.

Molecular epidemiology of Shigella spp strains isolated in two different metropolitam areas of southeast Brazil

Shigella spp., the human pathogen responsible for shigellosis, is highly infectious even at low levels. The incidence rate of shigellosis varies with geographical distribution, location human development index, and age groups, being higher among children aged under 5 years. In Brazil, a few works indicate that shigellosis cases are underestimated, with S. flexneri and S. sonnei strains being the major agents responsible for the shigellosis cases. The present study used pulsed field gel electrophoresis (PFGE) to investigate the molecular epidemiology of 119 strains of S. sonnei and S. flexneri isolated from shigellosis cases that occurred in the metropolitan areas of Ribeirão Preto and Campinas Cities, São Paulo Sate, southeast Brazil. The results indicated (i) the existence of just a few strain clusters for both species, but with genotype variability with either a high speed of genetic change or constant introduction of several genotypes, considering the intense migration to these two metropolitan areas, and (ii) the prevalence of specific genotypes in each geographical area, which suggests the successful adaptation of some genotypes to the local environmental conditions. Our results indicate the need of more efficacious sanitary barriers to prevent Shigella spp. outbreaks and epidemics.

Shigella spp., o patógeno humano responsável pela shiguelose, apresenta grande poder infeccioso, mesmo em pequenas doses. A incidência de shiguelose varia de acordo com a distribuição geográfica, o índice de desenvolvimento humano local e a faixa de idade, sendo alto entre crianças com menos de 5 anos de idade. No Brasil, alguns trabalhos indicam que os casos de shiguelose são subnotificados sendo, S. flexneri e S. sonnei os principais agentes responsáveis pelos casos ocorridos. O presente estudo usou a técnica de eletroforese em campo pulsado (PFGE) para investigar a epidemiologia molecular de 119 linhagens de S. flexneri e S. sonnei, isoladas de casos de shiguelose que ocorreram nas regiões metropolitanas das cidades de Ribeirão Preto e Campinas, estado de São Paulo, na região Sudeste do Brasil. Os resultados indicam (i) a existência de apenas alguns grupos clonais para ambas as espécies, mas com variabilidade de genótipos indicando ou um alto índice de variação genética, ou a constante introdução de vários genótipos, considerando o movimento migratório intenso de pessoas nestas duas áreas metropolitanas, e (ii) a prevalência de genótipos específicos em cada área geográfica, o que sugere a existência de genótipos com maior capacidade de adaptação às condições ambientais locais. Os resultados indicam a necessidade de barreiras sanitárias mais eficientes para prevenir surtos e epidemias de Shigella spp.

Virulence factors of avian pathogenic Escherichia coli (APEC) / Fatores de virulência de Escherichia coli aviária patogênica (APEC)

Avian pathogenic Escherichia coli (APEC) strains cause a great diversity of diseases in birds and are responsible for great economic losses in the avian industry. To date, several studies have been carried out to better understand the APEC pathogenesis for a possible development of tools which could prevent the economics losses caused by these strains. This review discusses the virulence factors described do date to be expressed by these strains and the advances made to understand and identify virulence determinants present in APEC.

Linhagens de Escherichia coli patogênicas para aves (APEC) causam uma grande diversidade de doenças em aves e são responsáveis por grandes prejuízos na indústria aviária. Nos últimos anos, vários estudos foram realizados para melhor entender a patogênese de linhagens APEC e para desenvolver ferramentas que podem prevenir as perdas econômicas causadas por estas linhagens. Esta revisão discute os fatores de virulência descritos nestas linhagens e os avanços realizados para entender e identificar os determinantes de virulência presentes em APEC.

Molecular typing of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in two metropolitan areas of São Paulo State, southeast Brazil

One hundred and fifty-one methicillin-resistant z (MRSA) strains have been isolated from patients admitted in tertiary care hospitals in two metropolitan areas (Campinas City and Ribeirão Preto City) in the southeast region of Brazil and analyzed through PCR-based techniques [(PCR amplification of spa, coa, and housekeeping genes (arcC, aroE, gmk, pta, tpi, yqiL)] and further restriction fragment typing of coa and of housekeeping genes. The heterogeneity of spa gene was determined directly by agarose gel electrophoresis migration. The results obtained indicate the existence of three (A, B, C) main clusters. Since the strain distribution in these three clusters is much characteristic, it denotes the existence of three main clones. All strains isolated in Campinas were grouped in clusters A and B, while most of the strains isolated in Ribeirão Preto were grouped in cluster C. This distribution denotes the existence of different founder strains that undergo independent genetic variability. The strains considered representative of the Brazilian Epidemic Clone (BEC) were categorized as cluster A. These results indicate a possible higher variability among Brazilian MRSA strains than currently described and indicate that the techniques herein used can be used as an alternative to Pulsed Field Gel Electrophoresis (PFGE).

Ribotyping, biotyping and capsular typing of Haemophilus influenzae strains isolated from patients in Campinas, southeast Brazil

Forty-five Haemophilus influenzae strains isolated from patients were characterized based on biochemical characteristics. Their capsular types were determined by polymerase chain reaction (PCR); they were compared, using two molecular methods [ribotyping with a specific DNA probe amplified from the 16S rDNA region from H. influenzae and through restriction fragment length polymorphism (RLFP) of an amplified 16S DNA region]. The strains were better discriminated by the ribotyping technique that used the 16S probe and by the combination of both techniques. Biotypes I and IV were the most common, followed by biotypes VI, VIII and III. Biotypes II and VII were not found. Most of the capsular samples were nontypable (89 percent), with capsular types a and b found in 2 and 9 percent of the samples, respectively. We concluded that there is a very close genetic identity among pathogenic and non-pathogenic strains.

Occurrence of virulence-related sequences and phylogenetic analysis of commensal and pathogenic avian Escherichia coli strains (APEC) / Ocorrência de seqüências relacionadas com a virulência e análise filogenética de estirpes comensais e patogênicas de Escherichia coli aviário (APEC)

The presence of iron uptake (irp-2, fyuA, sitA, fepC, iucA), adhesion (iha, lpfA O157/O141, lpfA O157/O154, efa, toxB) and invasion (inv, ial-related DNA sequences and assignment to the four main Escherichia coli phylogenetic groups (A, B1, B2 e D) were determined in 30 commensal E. coli strains isolated from healthy chickens and in 49 APEC strains isolated from chickens presenting clinical signs of septicemia (n=24) swollen head syndrome (n=14) and omphalitis (n=11) by PCR. None of the strains presented DNA sequences related to the inv, ial, efa, and toxB genes. DNA sequences related to lpfA O157/O154, iucA, fepC, and irp-2 genes were significantly found among pathogenic strains, where iucA gene was associated with septicemia and swollen head syndrome and fepC and irp-2 genes were associated with swollen head syndrome strains. Phylogenetic typing showed that commensal and omphalitis strains belonged mainly to phylogenetic Group A and swollen head syndrome to phylogenetic Group D. Septicemic strains were assigned in phylogenetic Groups A and D. These data could suggest that clonal lineage of septicemic APEC strains have a multiple ancestor origin; one from a pathogenic bacteria ancestor and other from a non-pathogenic ancestor that evolved by the acquisition of virulence related sequences through horizontal gene transfer. Swollen head syndrome may constitute a pathogenic clonal group. By the other side, omphalitis strains probably constitute a non-pathogenic clonal group, and could cause omphalitis as an opportunistic infection. The sharing of virulence related sequences by human pathogenic E. coli and APEC strains could indicate that APEC strains could be a source of virulence genes to human strains and could represent a zoonotic risk.(AU)

A presença de seqüências de DNA associadas à capacidade de captação de ferro (irp-2, fyuA, sitA, fepC, iucA), adesão (iha, lpfA O157/O141, lpfA O157/O154, efa, toxB) e de invasão (inv, ial) e a classificação dentro dos quatro grupos filogenéticos principais de Escherichia coli (Grupos A, B1, B2 e D) foram determinadas, através de PCR, em 30 amostras comensais de E. coli isoladas de frangos e de 49 linhagens APEC (24 isoladas de frangos com septicemia, 14 isoladas de frangos com síndrome da cabeça inchada e 11 isoladas de embriões de galinhas com onfalite). Nenhuma das linhagens apresentou os genes inv, ial, efa, e toxB. Os genes lpfA O157/O154, iucA, fepC e irp-2 foram encontrados em freqüências significativas entre as amostras patogênicas. O gene iucA foi associado com amostras causadoras de septicemia e de síndrome da cabeça inchada. Os genes fepC e irp-2 foram associados a amostras causadoras de síndrome da cabeça inchada. A análise filogenética demonstrou que linhagens comensais e causadoras de onfalite pertenceram principalmente ao Grupo filogenético A, não patogênico. Amostras causadoras de síndrome da cabeça inchada pertenceram, em sua maioria, ao Grupo patogênico D. Linhagens causadoras de septicemia pertenceram aos Grupos A e D. Estes dados sugerem que linhagens APEC causadoras de septicemia provavelmente têm uma origem ancestral múltipla: uma derivada de uma linhagem patogênica e outra de uma linhagem não patogênica que possivelmente evoluiu através da aquisição horizontal de genes de virulência. Amostras causadoras de síndrome da cabeça inchada possivelmente constituem um grupo clonal patogênico. Por outro lado, amostras causadoras de onfalite possivelmente constituem um grupo clonal não patogênico, que, possivelmente causam onfalite devido a uma infecção oportunista. A presença de genes de virulência também encontrados em E. coli de origem humana pode indicar a possível ocorrência de zoonoses causadas por APEC.(AU)

Clonal study of avian Escherichia coli strains by fliC conserved-DNA-sequence regions analysis / Estudo clonal de Escherichia coli aviário por análise de seqüências de DNA conservadas do gene fliC

The clonal relationship among avian Escherichia coli strains and their genetic proximity with human pathogenic E. coli, Salmonela enterica, Yersinia enterocolitica and Proteus mirabilis, was determined by the DNA sequencing of the conserved 5' and 3'regions fliC gene (flagellin encoded gene). Among 30 commensal avian E. coli strains and 49 pathogenic avian E. coli strains (APEC), 24 commensal and 39 APEC strains harbored fliC gene with fragments size varying from 670bp to 1,900bp. The comparative analysis of these regions allowed the construction of a dendrogram of similarity possessing two main clusters: one compounded mainly by APEC strains and by H-antigens from human E. coli, and another one compounded by commensal avian E. coli strains, S. enterica, and by other H-antigens from human E. coli. Overall, this work demonstrated that fliC conserved regions may be associated with pathogenic clones of APEC strains, and also shows a great similarity among APEC and H-antigens of E. coli strains isolated from humans. These data, can add evidence that APEC strains can exhibit a zoonotic risk.(AU)

A relação clonal entre linhagens de Escherichia coli de origem aviária e sua proximidade genética com E. coli patogênica para humanos, Salmonella enterica, Yersinia enterocolitica e Proteus mirabilis foi determinada através da utilização das seqüências conservadas 5' e 3' do gene fliC (responsável pela codificação da flagelina). Entre as 30 linhagens comensais de E. coli aviária e as 49 linhagens patogênicas de E. coli para aves (APEC), 24 linhagens comensais e 39 APEC apresentaram o gene fliC, que foi encontrado em tamanhos que variam de 670pb a 1900pb. Um dendrograma representando similaridade genética foi obtido a partir do seqüenciamento das regiões 5' e 3' conservadas do gene fliC das linhagens de E. coli de origem aviária, das seqüências dos antígenos H de E. coli de origem humana, de S. enterica, Y. enterocolitica e de P. mirabilis. A análise do dendrograma demonstrou que este apresenta dois grupos principais: um composto principalmente por isolados APEC e por antígenos H de E. coli de origem humana e outro formado por isolados comensais de E. coli aviária, S. enterica e por antígenos H de E. coli. No geral, o presente trabalho demonstrou que as regiões conservadas do gene fliC podem estar associadas à diferenciação clonal de linhagens de E. coli aviária, e que existe uma grande similaridade genética entre estas linhagens e antígenos H de E. coli humana. Estes dados podem adicionar evidências de que linhagens APEC podem apresentar riscos zoonóticos.(AU)

Study of biological characteristics of Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis and from patients with extra-pulmonary infections

A total of 120 strains of Pseudomonas aeruginosa, isolated from cystic fibrosis (CF) patients (n = 80) and from patients having extra-pulmonary infections (n = 40) were studied regarding the presence of some virulence factors (hemolysin, gelatinase and elastase production) and presence of the algD and algU genes as detected by polymerase chain reaction-PCR. There was not a significant difference for the production of gelatinase and hemolysin between non-mucoid strains from CF patients and other isolates from extra-pulmonary infections and mucoid strains. The production of elastase was found to be significant among these strains. The algD gene was detected by PCR in all studied strains but the algU gene was detected only in 25 percent of the mucoid strains. Conclusion withdrawn from the results were: (i) hemolysin and gelatinase production although present in many strains of P aeruginosa should not be considered as general virulence factors for the mucoid phenotype but could help in the pathogenic process; (ii) elastase production could be a necessary virulence factor for the initial pathogenesis process; (iii) mucoid and non-mucoid phenotypes could also be expressed according to the host's tissues or environment, and finally, (iv) more than one regulator system for alginate production is probably present in each strain.

Study of the adhesion and invasion capacities of an invasive avian pathogenic escherichia coli strain (APEC) to in vitro cultivated HEP-2 cells

An avian pathogenic Escherichia coli strain (APEC), designated strain sep17, was isolated from the liver of a chicken suffering from septicaemia. Its biological characteristics, including antimicrobial drug resistances, colicin production, plasmid profile, adhesion and invasion capacities to in vitro cultivated HEp-2 cells, and PCR detection of DNA sequences related to pathogenicity genes (fimA, tsh, papA, crl, csgA, afa, sfa, eae, lpfAO157/OI-141, lpfAO157/OI-154, toxB, iha, ial, efa, inv, invA/invE and ibeA) were determined. This strain (Lac+, TcR, fimA, csgA, crl, lpfAO157/OI-154) harbored a 70 MDa plasmid and was able to adhere to and invade in vitro cultured HEp-2 cells. Transference of this 70 MDa plasmid by conjugation rendered a non-pathogenic recipient strain HB101 (Lac-, SmR, csgA) capable of adhering to and invading the same type of cell. Scanning electron microscopy and light microscopy confirmed the adhesion capacity of the wild and the transconjugant strains, while the in vitro invasion technique and light microscopy confi rmed the invasion capacity of these strains. The FAS technique, which is used to visualize actin accumulation on cells where the adhesion process occurs, was negative for all those strains. None of the genes detected in strain sep17 were transferred by conjugation, which indicates that they are chromosomally located and are not related to the adhesion and invasion processes. The presence and absence of pathogenicity-related genes are discussed.

Correlation between Helicobacter pylori infection, gastric diseases and life habits among patients treated at a university hospital in Southeast Brazil

Helicobacter pylori is considered a significant agent in the development of various gastric diseases. However, the diseases caused by this bacterium are known as being multi-factorial, with the genotype, immune system and life habits of the host playing important roles in the establishment of the clinical outcome. Also, H. pylori exhibit a high degree of genetic variability, contributing to the complexity of the host-pathogen relationship. These observations, considered together with the widely-varying origins and social habits of the Brazilian population, lead us to speculate about the influence of these life habits on H. pylori infection and the clinical outcome. Therefore, in this study we evaluated the relationship between H. pylori infection and certain diseases in 172 patients treated at the Hospital das Clínicas of Ribeirão Preto (HCRP), Brazil, taking into account their different life habits, such as non-steroidal anti-inflammatory drugs and alcohol ingestion, and smoking habit. Our analysis indicated that H. pylori infection is not affected by any of the life habits evaluated but is associated with the development of peptic ulcers (gastric and duodenal ulcer) and inverse correlate with gastroesophageal reflux disease (GERD). No correlation was found between the infection with this bacterium and gastritis or intestinal metaplasia. However, gastritis and erosive gastritis were directly correlated with non-steroidal anti-inflammatory drugs (NSAID) ingestion. Moreover, ingestion of alcohol beverages exhibited a protective effect on gastritis development in men. Our data also indicated that to achieve reliable detection of this bacterium in biopsies, two or three detection methods should be used.

Biological and molecular characterization of Streptococcus crista strains isolated from human dental biofilm by means of arbitrary primers - PCR (AP-PCR)

Streptococcus spp são importantes componentes do biofilme dental sendo Streptococus crista considerado um interessante modelo de interações bacterianas que nele ocorrem. No presente trabalho linhagens de S. crista, foram isoladas do biofilme dental de indivíduos brasileiros, e estudadas em relação a suas características biológicas e ao seu perfil molecular através da técnica do AP-PCR, usando-se os iniciadores RR2, 434, OPR2, OPR8 e OPR13. Os resultados nos permitiram construir um dendrograma de similaridade. A análise do dendrograma de similaridade permitiu a separação das linhagens estudadas em grupos de similaridade. Todos os isolados apresentaram tufo de fibrilas, quando estudados por Microscopia Eletrônica de Transmissão (MET). Estes isolados foram capazes de se ligar à amilase salivar e de se aderir a células epiteliais bucais. Algumas linhagens, que apresentam tufo de fibrilas e aderência positiva, não foram capazes de coagregar com a Fusobacterium nucleatum, sugerindo que diferentes grupos de adesinas estão presentes nestas amostras.

Bacteria-induced apoptosis: an approach to bacterial pathogenesis

Several pathogenic or opportunistic bacteria can induce or inhibit host cell apoptosis. The modulation of cellular pathways that results in the induction or delay of host cell apoptosis is an important mechanism of bacterial virulence. These processes can be mediated by various host cell signaling pathways that are subverted by the bacteria. Pathogens can activate apoptotic proteins such as caspases, inactivate anti-apoptotic proteins such as NFêB and mitogen-activated protein kinases, or up-regulate the endogenous receptor/ligand system that induces apoptosis, generally when the bacteria are bound to the host cell surface. Bacteria-induced apoptotic or anti-apoptotic processes are often related to the ability of the bacteria to reach the host tissues. However, since apoptosis is also involved in host defense mechanisms against infectious agents, this phenomenon apparently plays a central role in host-pathogen interactions.

Biological and genetic characteristics of uropathogenic Escherichia coli strains

The aim of the present study was to determine biological characteristics such as expression of fimbriae, Congo red binding, production of hemolysin and aerobactin, adhesion to HeLa and uroepithelial cells and invasion of HeLa cells by Escherichia coli isolates obtained from patients showing clinical signs of urinary tract infection (UTI). Also, the presence of genes (apa, afa, spa) for fimbria expression and cytotoxic necrotizing factors (CNF1, CNF2) was assayed using specific primers in PCR. The data obtained were compared with the clonal relationships obtained by analysis of multilocus enzyme electrophoresis (MLEE), restriction fragment length polymorphism (RFLP) of the rDNA (ribotyping) and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). All isolates but one presented a combination of at least two of the characteristics studied, a fact suggesting the presence of pathogenicity islands (PAIs). Diffuse adherence type to HeLa cells was observed to occur in most of the strains, but adhesion to uroepithelial cells seems to be a more reliable test to verify pathogenicity. Although four strains seemed to be able to invade HeLa cells when assayed by light microscopy, electron microscopy studies demonstrated that these strains were not invasive. MLEE, RFLP and ERIC-PCR were able to group the isolates differently into main clusters that were not correlated with the presence of pathogenic traits

Studies of the genetic expression of 31A finbriae by two bovine septicaemic Escherichia coli strains

Using transposon tnphoA, classical bacterial genetic assays, SDS-PAGE and Western blot of superficial proteins, we have studied the expression of the 31A fimbriae of two bovine wild-type septicaemic Escherichia coli strains. The genes responsible for encoding colonization factor 31A were located in both the plasmid (strain BZ2468) and the chromosome (strain BZ43). The results obtained using HeLa cell cultures led us to believe that the BZ43 strain could have another colonization factor besides 31A, since one mutatnt, which did not express fiinbriae, was still able to adhere to HeLa cells.

Plasmid mediated production of a new colonization factor (F42) in enterotoxigenic Escherichia coli

A amostra 567/7 de Escherichia coli enterotoxigênica (ETEC) isolada em Säo Paulo, SP, Brasil, de um leitäo com diarréia produz enterotoxina termoestável (STa) e um novo fator de colonizaçäo, provisoriamente denominado F42 (Yano et al., 1986). Neste trabalho, a presença de plasmídios na amostra 567/7 foi correlacionada com a resistência a vários antibióticos, produçäo de uma colicina (V), adesina F42 e enterotoxina STa. Através de técnica de eletroforese em gel de agarose, determinou-se a presença de seis plasmídios nesta amostra. Um plasmídio de 65,1 megadaltons (Md) é o responsável pela resistência à tetraciclina enquanto que as resistências para estreptomicina, canamicina e produçäo de colicina V säo codificadas por um plasmídio conjugativo de 48,8 Md embora um plasmídio de 5,8 Md é o responsável também pela resistência à canamicina. Através de transformaçäo foi demonstrado que um plasmídio de 21,1 Md provavelmente näo conjugativo é responsável tanto pela presença de adesina F42 quanto pela enterotoxina STa. Dois outros plasmídios (2,6 Md e 2,3 Md) foram considerados plasmídios crípticos