Caracterização de Escherichia coli enteroagregativa entre crianças com diarreia na Amazônia ocidental brasileira / Characterization of enteroaggregative escherichia coli among diarrheal childrenin western brazilian amazon

ABSTRACT BACKGROUND: Enteroaggregative Escherichia coli (EAEC) is one of the main acute and chronic diarrhea causes both in children and adults, mainly in developing countries. OBJECTIVE: The aim of the present study is to characterize EAEC strains isolated from faecal samples and to identify genes potentially contributing to virulence, biofilm production and antimicrobial resistance in children admitted to a pediatric hospital in Porto Velho, Rondônia State. METHODS: The total of 1,625 E. coli specimens were isolated from 591 children in the age group 6 years or younger who were hospitalized in Cosme and Damião Children Hospital in Porto Velho, between February 2010 and February 2012, with acute gastroenteritis. Colonies suggestive of E. coli were subjected to polymerase chain reaction testing in order to identify the virulence factors. The in vitro adhesion assays using HEp-2 adherence were tests. Biofilm detection through spectrophotometry and antimicrobial susceptibility tests were conducted in the disk diffusion method. RESULTS: The mentioned study examined 591 stool samples from children with diarrhea. Diarrheogenic E. coli was found in 27.4% (162/591) of the children. EAEC was the diarreagenic E. coli most frequently associated with diarrhea 52.4% (85/162), which was followed by enteropathogenic E. coli 43.8% (71/162), enterotoxigenic E. coli 2.4% (4/162), and enterohemorrhagic E. coli 1.2% (2/162). The aggR gene was detected in 63.5% (54/85) of EAEC isolates; moreover, statistically significant correlation was observed among typical EAEC (aggR) and aatA (P<0.0001), irp2 (P=0.0357) and shf (P=0.0328). It was recorded that 69% (59/85) of the 85 analyzed EAEC strains were biofilm producers; 73% (43/59) of the biofilm producers carried the aggR gene versus 42.3% (11/26) of non-producers (P=0.0135). In addition, there was association between the aatA gene and biofilm production; 61% (36/59) of the samples presented producer strains, versus 19.2% (5/26) of non-producers (P<0.0004). Antibiotic sensitivity test evidenced that most EAEC were ampicillin 70.6% (60/85), sulfamethoxazole 60% (51/85), tetracycline 44.7% (38/85) and cefotaxime 22.4% (19/85) resistant. CONCLUSION: As far as it is known, the present study is pioneer in Northern Brazil to investigate EAEC virulence factors and to show the antimicrobial susceptibility of EAEC strains isolated from children with diarrhea.

RESUMO CONTEXTO: A Escherichia coli enteroagregativa (EAEC) é um dos principais agentes causadores de diarreia aguda e crônica em crianças e adultos, principalmente em países em desenvolvimento. OBJETIVO: Caracterizar cepas de EAEC isoladas de amostras fecais e identificar genes que potencialmente contribuem para a virulência, produção de biofilme e resistência antimicrobiana em crianças internadas em um hospital pediátrico em Porto Velho, Rondônia. MÉTODOS: Um total de 1.625 cepas de E. coli foram isolados de 591 crianças com gastroenterite aguda na faixa etária de 6 anos que foram internadas no Hospital Infantil Cosme e Damião na cidade de Porto Velho, entre fevereiro de 2010 e fevereiro de 2012. Colônias sugestivas de E. coli foram submetidas a reação em cadeia da polimerase para identificação de fatores de virulência. O ensaio de adesão in vitro foi desenvolvido com célula HEp-2. A detecção de biofilme foi realizada através do teste de espectrofotometria e os testes de susceptibilidade aos antimicrobiana foram realizados através do método de difusão em disco. RESULTADOS: A E. coli diarreiogênica foi encontrada em 27,4% (162/591) das crianças e a EAEC foi a E. coli diarreiogênica mais frequentemente associada à diarreia com 52,4% (85/162), seguida pela E. coli enteropatogênica 43,8% (71/162), E. coli enterotoxigênica 2,4% (4/162) e E. coli enterohemorrágica 1,2% (2/162). O gene aggR foi detectado em 63,5% (54/85) dos isolados de EAEC com correlação estatisticamente significante entre esse gene com os genes aatA (P<0,0001), irp2 (P=0,0357) e shf (P=0,0328). Neste estudo 69% (59/85) das cepas de EAEC eram produtoras de biofilme, destas 73% (43/59) possuíam o gene aggR, ao passo que entre as não produtoras 42,3% (11/26) possuíam o gene (P=0,0135). Essa associação também foi observada com o gene aatA, presente em 61% (36/59) das cepas produtoras e em 19,2% (5/26) das não produtoras (P<0,0004). O teste de sensibilidade aos antibimicrobianos evidenciou que a maioria das EAEC eram resistentes a ampicilina 70,6% (60/85), ao sulfametoxazol 60% (51/85), a tetraciclina 44,7% (38/85) e a cefotaxima 22,4% (19/85). CONCLUSÃO: Este é o primeiro estudo no Norte do Brasil sobre a investigação dos fatores de virulência de EAEC mostrando a susceptibilidade antimicrobiana de cepas de EAEC isoladas de crianças com diarreia.

Adherence and virulence genes of Escherichia coli from children diarrhoea in the Brazilian Amazon

The bacterial pathogen most commonly associated with endemic forms of childhood diarrhoea is Escherichia coli. Studies of epidemiological characteristics of HEp-2 cell-adherent E. coli in diarrhoeal disease are required, particularly in developing countries. The aim of this study was evaluate the presence and significance of adherent Escherichia coli from diarrhoeal disease in children. The prevalence of LA, AA, and DA adherence patterns were determined in HEp-2 cells, the presence of virulence genes and the presence of the O serogroups in samples obtained from 470 children with acute diarrhoea and 407 controls in Porto Velho, Rondônia, Brazil. E. coli isolates were identified by PCR specific for groups of adherent E. coli. Out of 1,156 isolates obtained, 128 (11.0%) were positive for eae genes corresponding to EPEC, however only 38 (29.6%) of these amplified bfpA gene. EAEC were isolated from 164 (14.1%) samples; of those 41(25%), 32 (19%) and 16 (9.7%) amplified eagg, aggA or aafA genes, respectively and aggA was significantly associated with diarrhoea (P = 0.00006). DAEC identified by their adhesion pattern and there were few isolates. In conclusion, EAEC was the main cause of diarrhoea in children, especially when the aggA gene was present, followed by EPEC and with a negligible presence of DAEC.

Antimicrobial activity of allylic thiocyanates derived from the Morita-Baylis-Hillman reaction

Bacterial resistance to commonly used antibiotics has been recognized as a significant global health issue. In this study, we carried out the screening of a family of allylic thiocyanates for their action against a diversity of bacteria and fungi with a view to developing new antimicrobial agents. Allylic thiocyanates bearing halogenated aryl groups, which were readily obtained in two steps from the Morita-Baylis-Hillman adducts, showed moderate-to-high activity against selective pathogens, including a methicillin-resistant S. aureus (MRSA) strain. In particular cases, methyl (Z)-3-(2,4-dichlorophenyl)-2-(thiocyanomethyl)-2-propenoate exhibited antimicrobial activity comparable to the reference antibiotic Imipenem.

Antimicrobial activity of honeys from two stingless honeybee species and Apis mellifera (Hymenoptera: Apidae) against pathogenic microorganisms / Atividade antimicrobiana de méis de duas espécies de abelhas sem ferrão e Apis mellifera (Hymenoptera, Apidae) contra micro-organismos patogênicos

Honeys are described possessing different properties including antimicrobial. Many studies have presented this activity of honeys produced by Apis mellifera bees, however studies including activities of stingless bees honeys are scarce. The aim of this study was to compare the antimicrobial activity of honeys collected in the Amazonas State from Melipona compressipes, Melipona seminigra and Apis mellifera against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Chromobacterium violaceum, and Candida albicans. Minimum inhibitory concentrations were determined using the agar dilution method with Müller-Hinton agar (for bacteria) or Saboraud agar (for yeast). Staphylococcus aureus and E. faecalis were inhibited by all honeys at concentrations below 12%, while E. coli and C. violaceum were inhibited by stingless bee honeys at concentrations between 10 and 20%. A. mellifera honey inhibited E. coli at a concentration of 7% and Candida violaceum at 0.7%. C. albicans were inhibited only with honey concentrations between 30 and 40%. All examined honey had antimicrobial activity against the tested pathogens, thus serving as potential antimicrobial agents for several therapeutic approaches.

Méis são descritos possuindo diferentes propriedades, incluindo a antimicrobiana. Muitos estudos têm apresentado essa atividade de méis produzidos por abelhas Apis mellifera, no entanto estudos incluindo atividades de méis de abelhas sem ferrão são escassos. O objetivo deste estudo foi comparar a atividade antimicrobiana de méis de Melipona compressipes, Melipona seminigra e A. mellifera, coletados no Estado do Amazonas, contra Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Chromobacterium violaceum, e Candida albicans. As concentrações inibitórias mínimas foram determinadas usando o método de diluição em ágar, com ágar Muller-Hinton (para bactérias) ou ágar Sabouraud (para a levedura). S. aureus e E. faecalis foram inibidos por todos os méis em concentrações inferiores a 12%, enquanto E. coli e C. violaceum foram inibidos por méis de abelhas sem ferrão em altas concentrações entre 10 e 20%. A. mellifera inibiu E. coli na concentração de 7% e C. violaceum em baixa concentração (0,7%). C. albicans foi inibida apenas em concentrações entre 30 e 40% dos méis. Assim, todas as variedades de mel testadas apresentaram atividade antimicrobiana sobre os patógenos testados, servindo assim como agente antimicrobiano potencial para diversas abordagens terapêuticas.

Rotavirus genotyping in gastroenteritis cases of an infantile population from Western Brazilian Amazonia / Genotipagem de rotavírus em casos de gastrenterites de uma população infantil do oeste da Amazônia brasileira

INTRODUCTION: During the period from 2000 to 2002, 79 rotavirus-positive stool samples were collected from children presenting diarrhea in the Western Brazilian Amazon. METHODS: Molecular characterization of the G and P genotypes was performed using RT-PCR and electropherotyping analysis by polyacrylamide gel electrophoresis. RESULTS: A total of 59 samples were confirmed as group A rotavirus. A long electrophoretic profile was exhibited by the G1P[8], G3P[8], and G4P[8] genotypes. The G1P[8] genotype was found in greater proportion. The short electropherotype was exhibited only by G2 genotype strains. CONCLUSIONS: The proportion of the rotavirus genotypes observed was not different from that in other areas of Brazil. This study is the first genotyping of rotavirus in the Western Brazilian Amazon.

INTRODUÇÃO: Entre 2000 e 2002, 79 amostras positivas para rotavírus foram coletadas de crianças com diarreia na Amazônia ocidental brasileira. MÉTODOS: Para a caracterização molecular dos genótipos G e P foram realizadas as reações de RT-PCR e a análise dos eletroferotipos por eletroforese em gel de poliacrilamida (PAGE). RESULTADOS: 59 amostras foram confirmadas como pertencentes ao rotavírus grupo A. Os genótipos G1P[8], G3P[8] e G4P[8] apresentaram perfis eletroforéticos longos. O genótipo G1P[8] foi encontrado em maior proporção. O eletroferotipo curto ocorreu apenas em genótipos G2. CONCLUSÕES: A proporção dos genótipos de rotavírus observada não foi diferente de outras áreas do Brasil. Este estudo é a primeira genotipagem de rotavírus na Amazônia ocidental brasileira.

Characterization of Shigella spp. by antimicrobial resistance and PCR detection of ipa genes in an infantile population from Porto Velho (Western Amazon region), Brazil

The incidence of Shigella spp. was assessed in 877 infants from the public hospital in Rondônia (Western Amazon region, Brazil) where Shigella represents the fourth cause of diarrhea. Twenty-five isolates were identified: 18 were Shigella flexneri, three Shigella sonnei, three Shigella boydii and one Shigella dysenteriae. With the exception of S. dysenteriae, all Shigella spp. isolated from children with diarrhea acquired multiple antibiotic resistances. PCR detection of ipa virulence genes and invasion assays of bloody diarrhea and fever (colitis) were compared among 25 patients testing positive for Shigella. The ipaH and ipaBCD genes were detected in almost all isolates and, unsurprisingly, all Shigella isolates associated with colitis were able to invade HeLa cells. This work alerts for multiple antibiotic resistant Shigella in the region and characterizes presence of ipa virulence genes and invasion phenotypesin dysenteric shigellosis.

Improving the production of the denatured recombinant N-terminal domain of rhoptry-associated protein 2 from a Plasmodium falciparum target in the pathology of anemia in falciparum malaria

Rhoptry-associated protein 2 (RAP2) is known to be discharged from rhoptry onto the membrane surface of infected and uninfected erythrocytes (UEs) ex vivo and in vitro and this information provides new insights into the understanding of the pathology of severe anemia in falciparum malaria. In this study, a hexahistidine-tagged recombinant protein corresponding to residues 5-190 of the N-terminal of Plasmodium falciparum RAP2 (rN-RAP2) was produced using a new method of solubilization and purification. Expression was induced with D-lactose, a less expensive alternative inducer to the more common isopropyl-²-D-thio-galactopyranosidase. The recombinant protein was purified using two types of commercially-available affinity columns, iminodiacetic and nitrilotriacetic. rN-RAP2 had immunogenic potential, since it induced high titers of anti-RAP2 antibodies in mice. These antibodies recognized full-length RAP2 prepared from Triton X-100 extracts from two strains of P. falciparum. In fact, the antibody recognized a 29-kDa product of RAP2 cleavage as well as 82 and 70-kDa products of RAP1 cleavage. These results indicate that the two antigens share sequence epitopes. Our expressed protein fragment was shown to contain a functional epitope that is also present in rhoptry-derived ring surface protein 2 which attaches to the surface of both infected and UEs and erythroid precursor cells in the bone marrow of malaria patients. Serum from malaria patients who developed anemia during infection recognized rN-RAP2, suggesting that this protein fragment may be important for epidemiological studies investigating whether immune responses to RAP2 exacerbate hemolysis in falciparum malaria patients.

Rotavirus and adenovirus in Rondônia

Acute gastroenteritis is one of the most common diseases in humans worldwide. Viral gastroenteritis is a global problem in infants and young children. In this study the incidence of diarrhea was assessed in 877 hospitalized children under five years old, over a period of 24 months and distributed in 470 cases of diarrhea and 407 age-matched group with other pathologies, as control group. Two antigen detection techniques based on enzyme immunoassay (EIA) and latex particles were used for detection of rotavirus and adenovirus. Rotavirus A was a major cause of gastroenteritis with 23.6 percent of cases, being 90 percent of these cases in young children. Adenovirus infections was detected by EIA with frequency of 6.4 percent. Rotavirus and adenovirus were detected in 10.1 and 1.7 percent of stools from control group, respectively. Interestingly, the frequency of the youngest children in the control group excreting Rotavirus A was comparable to that detected in stools from diarrheic children. We cannot rule out the existence of other enteric viruses because the etiology of 171 cases of diarrhea was not determined and active search for astrovirus and calicivirus was not done. This is the first study that shows the presence of enteric viruses in the infantile population from Western Brazilian Amazonia and it was important to help physicians in the treatment of viral gastroenteritis.

Prevalência de Streptococcus pyogenes em orofaringe de crianças que freqüentam creches: estudo comparativo entre diferentes regiões do país / Prevalence of Streptococcus pyogenes as an oropharynx colonizer in children attending daycare: a comparative study of different regions in Brazil

Trinta por cento das faringotonsilites agudas são de etiologia estreptocócica com potencial de complicações como a glomerulonefrite difusa aguda e febre reumática. Crianças de creches apresentam maior incidência destas infecções. OBJETIVO: Identificar e comparar a prevalência do Streptococcus pyogenes na orofaringe entre crianças que freqüentam creches e crianças não-institucionalizadas, em duas regiões do Brasil. CASUíSTICA E MÉTODO: Estudo prospectivo com 200 crianças, provenientes da cidade de Porto Velho/RO e São Paulo/SP, em quatro grupos, freqüentadoras ou não de creches. Realizou-se swab de orofaringe e cultura para identificação do Streptococcus pyogenes. RESULTADOS: Prevalência de 8 por cento e 2 por cento entre as crianças de São Paulo que atendem a creches e do grupo controle, respectivamente, apresentando valor estatístico (p=0,02). Prevalência de 24 por cento e 16 por cento nos grupos de Porto Velho/RO que freqüentam creche e controle respectivamente, não caracterizando diferença estatisticamente significante (p=0,18). Observou-se diferença estatisticamente significante entre os grupos creche e controle de São Paulo/SP aos seus correspondentes de Porto Velho/RO (p < 0,01). CONCLUSÃO: Os resultados deste estudo nos permitem sugerir que freqüência em creches representa um fator de risco para a colonização de orofaringe pelo Streptococcus pyogenes, fato observado em populações distintas, porém com significância estatística em apenas uma das duas amostras.

Thirty percent of acute pharyngotonsillitis is caused by Streptococcus pyogenes, which increased the risk of glomerulonephritis and rheumatic fever. Children attending daycare centers have a higher incidence of these infections. AIM: to identify and compare the prevalence of Streptococcus pyogenes in the oropharynx of children who are enrolled and who are not enrolled in daycare centers in different regions of Brazil. MATERIALS AND METHODS: A prospective study of two hundred children from Sao Paulo/SP and Porto Velho/RO. Children from each city were divided into two groups: those attending, and those not attending daycare centers. Swabs of the oropharynx were taken for bacteriological culture and identification. RESULTS: The prevalence of Streptococcus pyogenes in the Sao Paulo groups were 8 percent and 2 percent for daycare and control groups, which was statistically significant (p=0.02). The prevalence in children from Porto Velho/RO was 24 percent and 16 percent for daycare and control groups, which was statistically significant (p=0.015). Statistical analysis also showed a significant difference between the corresponding groups in the two locations (p<0.01). CONCLUSION: These results show that daycare attendance is a risk factor for oropharyngeal streptococcal colonization; this was seen in different populations, but was statistically significance in only one of the two samples.

Enteropathogens associated with diarrheal disease in infants of poor urban areas of Porto Velho, Rondônia: a preliminary study

One hundred and thirty cases of diarrhea and 43 age-matched controls, 0 to 5 years old, were studied in a pediatric outpatient unit from a poor peri urban area of Porto Velho, Rondônia. Eighty percent of diarrheal cases were observed in the groups under 2 years of age. Rotavirus (19.2 percent) was the most frequent enteropathogen associated with diarrhea, followed by Shigella flexneri (6.15 percent) and S. sonnei (1.5 percent) and Salmonella sp. (6.9 percent). Four cases of E. coli enterotoxigenic infections (3.1 percent), E. coli enteropathogenic (EPEC)(2.3 percent) one case of E. coli enteroinvasive infection (0.8 percent) and one case of Yersinia enterocolitica (0.8 percent) were also identified. Mixed infections were frequent, associating rotavirus, EPEC and Salmonella sp. with Entamoeba histolytica and Giardia lamblia