SepA Enhances Shigella Invasion of Epithelial Cells by Degrading Alpha-1 Antitrypsin and Producing a Neutrophil Chemoattractant.

Shigella spp. are highly adapted pathogens that cause bacillary dysentery in human and nonhuman primates. An unusual feature of Shigella pathogenesis is that this organism invades the colonic epithelia from the basolateral pole. Therefore, it has evolved the ability to disrupt the intestinal epithelial barrier to reach the basolateral surface. We have shown previously that the secreted serine protease A (SepA), which belongs to the family of serine protease autotransporters of Enterobacteriaceae, is responsible for the initial destabilization of the intestinal epithelial barrier that facilitates Shigella invasion. However, the mechanisms used by SepA to regulate this process remain unknown. To investigate the protein targets cleaved by SepA in the intestinal epithelium, we incubated a sample of homogenized human colon with purified SepA or with a catalytically inactive mutant of this protease. We discovered that SepA targets an array of 18 different proteins, including alpha-1 antitrypsin (AAT), a major circulating serine proteinase inhibitor in humans. In contrast to other serine proteases, SepA cleaved AAT without forming an inhibiting complex, which resulted in the generation of a neutrophil chemoattractant. We demonstrated that the products of the AAT-SepA reaction induce a mild but significant increase in neutrophil transepithelial migration in vitro. Moreover, the presence of AAT during Shigella infection stimulated neutrophil migration and dramatically enhanced the number of bacteria invading the intestinal epithelium in a SepA-dependent manner. We conclude that by cleaving AAT, SepA releases a chemoattractant that promotes neutrophil migration, which in turn disrupts the intestinal epithelial barrier to enable Shigella invasion. IMPORTANCEShigella is the second leading cause of diarrheal death globally. In this study, we identified the host protein targets of SepA, Shigella's major protein secreted in culture. We demonstrated that by cleaving AAT, a serine protease inhibitor important to protect surrounding tissue at inflammatory sites, SepA releases a neutrophil chemoattractant that enhances Shigella invasion. Moreover, SepA degraded AAT without becoming inhibited by the cleaved product, and SepA catalytic activity was enhanced at higher concentrations of AAT. Activation of SepA by an excess of AAT may be physiologically relevant at the early stages of Shigella infection, when the amount of synthesized SepA is very low compared to the concentration of AAT in the intestinal lumen. This observation may also help to explain the adeptness of Shigella infectivity at low dose, despite the requirement of reaching the basolateral side to invade and colonize the colonic epithelium.

Enterobacterial repetitive intergenic consensus (ERIC)-PCR analysis as a reliable evidence for suspected Shigella spp. outbreaks

Abstract Background Shigellosis remains a serious public health problem and an important cause of morbidity and mortality worldwide. The aim of this study was to characterize fliC and the genetic relatedness of Shigella spp. isolated during a one-year period from children in a suspected outbreak in Tehran, Iran. Methods and results Fifty Shigella spp. were isolated from 3779 stool samples of children with diarrhea (prevalence rate: 1.32%). Among the isolates, 92% were characterized as Shigella sonnei, while 6% and 2% were identified as S. flexneri and S. boydii, respectively. S. dysenteriae was not recovered from the patients. All isolates were negative for fliC except for Shigella standard strains. The enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) profiles allowed differentiating the 50 isolates into 5 ERIC types, which were grouped into five clusters (ET1-ET5). Computer-assisted clustering of the strains showed a high degree of similarity among the isolates. Conclusion In conclusion, given the clonal correlation of the Shigella strains isolated in this study and the lack of fliC among them, we propose that probably a single or limited fliC-defected Shigella clone spread and caused the outbreak.

Relative proportions of E. coli and Enterococcus spp. may be a good indicator of potential health risks associated with the use of roof harvested rainwater stored in tanks.

A total of 285 water samples were collected from 71 roof harvested rainwater tanks from four villages in different provinces over a two-year (2013-2014) period during the early (October to December) and late (January to March) rainy season. Water quality was evaluated based on Escherichia coli, faecal coliforms and Enterococcus spp. prevalence using the IDEXX Quanti-Tray quantification system. Real-Time PCR was used to analyse a subset of 168 samples for the presence of Shigella spp., Salmonella spp. and E. coli virulence genes (stx1, stx2 and eaeA). Escherichia coli were detected in 44.1% of the samples, Enterococcus spp. in 57.9% and faecal coliforms in 95.7%. The most prevalent E. coli concentrations in harvested rainwater were observed in 29.1% of samples and 22.5% for Enterococcus spp. and, were within 1-10 cfu/100 ml and 10-100 cfu/100 ml, respectively, whereas those for faecal coliforms (36.6%) were within 100-1000 cfu/100 ml. On average 16.8% of the samples had neither E. coli nor Enterococcus spp. detected, while 33.9% had only Enterococcus spp. and 23.7% had only E. coli. E. coli and Enterococcus spp. were detected together in 25.5% of the samples. Evaluation of samples for potential pathogenic bacteria showed all tested samples to be negative for the Shigella spp. ipaH gene, while five tested positive for Salmonella ipaB gene. None of the samples tested positive for the stx1 and stx2 genes, and only two tested positive for the eaeA gene. These findings are potentially useful in the development of a simplified risk assessment strategy based on the concentrations of indicator bacteria.

In silico analysis to identify vaccine candidates common to multiple serotypes of Shigella and evaluation of their immunogenicity.

Shigellosis or bacillary dysentery is an important cause of diarrhea, with the majority of the cases occurring in developing countries. Considering the high disease burden, increasing antibiotic resistance, serotype-specific immunity and the post-infectious sequelae associated with shigellosis, there is a pressing need of an effective vaccine against multiple serotypes of the pathogen. In the present study, we used bio-informatics approach to identify antigens shared among multiple serotypes of Shigella spp. This approach led to the identification of many immunogenic peptides. The five most promising peptides based on MHC binding efficiency were a putative lipoprotein (EL PGI I), a putative heat shock protein (EL PGI II), Spa32 (EL PGI III), IcsB (EL PGI IV) and a hypothetical protein (EL PGI V). These peptides were synthesized and the immunogenicity was evaluated in BALB/c mice by ELISA and cytokine assays. The putative heat shock protein (HSP) and the hypothetical protein elicited good humoral response, whereas putative lipoprotein, Spa32 and IcsB elicited good T-cell response as revealed by increased IFN-γ and TNF-α cytokine levels. The patient sera from confirmed cases of shigellosis were also evaluated for the presence of peptide specific antibodies with significant IgG and IgA antibodies against the HSP and the hypothetical protein, bestowing them as potential future vaccine candidates. The antigens reported in this study are novel and have not been tested as vaccine candidates against Shigella. This study offers time and cost-effective way of identifying unprecedented immunogenic antigens to be used as potential vaccine candidates. Moreover, this approach should easily be extendable to find new potential vaccine candidates for other pathogenic bacteria.

Relationship among Shigella spp. and enteroinvasive Escherichia coli (EIEC) and their differentiation

Shigellosis produces inflammatory reactions and ulceration on the intestinal epithelium followed by bloody or mucoid diarrhea. It is caused by enteroinvasive E. coli (EIEC) as well as any species of the genus Shigella, namely, S. dysenteriae, S. flexneri, S. boydii, and S. sonnei. This current species designation of Shigella does not specify genetic similarity. Shigella spp. could be easily differentiated from E. coli, but difficulties observed for the EIEC-Shigella differentiation as both show similar biochemical traits and can cause dysentery using the same mode of invasion. Sequencing of multiple housekeeping genes indicates that Shigella has derived on several different occasions via acquisition of the transferable forms of ancestral virulence plasmids within commensal E. coli and form a Shigella-EIEC pathovar. EIEC showed lower expression of virulence genes compared to Shigella, hence EIEC produce less severe disease than Shigella spp. Conventional microbiological techniques often lead to confusing results concerning the discrimination between EIEC and Shigella spp. The lactose permease gene (lacY) is present in all E. coli strains but absent in Shigella spp., whereas β-glucuronidase gene (uidA) is present in both E. coli and Shigella spp. Thus uidA gene and lacY gene based duplex real-time PCR assay could be used for easy identification and differentiation of Shigella spp. from E. coli and in particular EIEC.

Bacterial and viral etiology of childhood diarrhea in Ouagadougou, Burkina Faso.

BACKGROUND: Diarrhea is the most frequent health problem among children in developing countries. This study investigated the bacterial and viral etiology and related clinical and epidemiological factors in children with acute diarrhea in Ouagadougou, Burkina Faso. METHODS: Stool specimens were collected from 283 children under 5 years of age visiting hospital due to acute diarrhea and from 60 healthy controls of similar age. Pathogens were investigated by using conventional culture techniques, PCR and immunochromatographic testing. Salmonella and Shigella strains were serotyped and their susceptibility to 23 antimicrobial agents was determined by the agar dilution method. RESULTS: At least one pathogen was detected in 64% of the 283 patients and in 8% of the 60 controls (p < 0.001). Rotavirus was found in 30% of the patients, followed by diarrheagenic Escherichia coli (24%), Salmonella enterica ssp. enterica (9%), Shigella spp. (6%), adenovirus (5%) and Campylobacter spp. (2%). Multiple pathogens were found in 11% of the patients and in 2% of the controls (p = 0.028). Viruses were found mainly in children of ≤ 2 years of age, whereas bacteria were equally prevalent among all the age groups. Viral infections occurred mostly during the cool dry season and the bacterial infections during the rainy season. Fever (64%) and vomiting (61%) were the most common symptoms associated with diarrhea. Only one Salmonella strain was resistant to nalidixic acid and ciprofloxacin. Of the Shigella strains, one was resistant to nalidixic acid but 81% to trimethoprim- sulfamethoxazole, 63% to streptomycin and 50% to ampicillin. Most of all the other Salmonella and Shigella strains were sensitive to all antimicrobials tested. CONCLUSION: Rotaviruses and diarrheal E. coli were the most predominant pathogens associated with acute diarrhea in Burkinabe children. Constant antimicrobial surveillance is warranted to observe for the emergence of enteric bacteria resistant to antimicrobials that are important in treatment also of severe infections.

Shigella in Brazilian children with acute diarrhoea: prevalence, antimicrobial resistance and virulence genes

Diarrhoeal disease is still considered a major cause of morbidity and mortality among children. Among diarrhoeagenic agents, Shigella should be highlighted due to its prevalence and the severity of the associated disease. Here, we assessed Shigella prevalence, drug susceptibility and virulence factors. Faeces from 157 children with diarrhoea who sought treatment at the Children's Hospital João Paulo II, a reference children´s hospital in Belo Horizonte, state of Minas Gerais, Brazil, were cultured and drug susceptibility of the Shigella isolates was determined by the disk diffusion technique. Shigella virulence markers were identified by polymerase chain reaction. The bacterium was recovered from 10.8% of the children (88.2% Shigella sonnei). The ipaH, iuc, sen and ial genes were detected in strains isolated from all shigellosis patients; set1A was only detected in Shigella flexneri. Additionally, patients were infected by Shigella strains of different ial, sat, sen and set1A genotypes. Compared to previous studies, we observed a marked shift in the distribution of species from S. flexneri to S. sonnei and high rates of trimethoprim/sulfamethoxazole resistance.

Antimicrobial Resistance of Shigella spp. isolated in the State of Pará, Brazil / Resistência Antimicrobiana de Shigella spp. isoladas no Estado do Pará

INTRODUCTION: Shigella spp. are Gram-negative, nonsporulating, rod-shaped bacteria that belong to the family Enterobacteriaceae and are responsible for shigellosis or bacillary dysentery, an important cause of worldwide morbidity and mortality. METHODS: We studied the antibiotic resistance profiles of 122 Shigella spp. strains (81 S. flexneri, 41 S. sonnei, 1 S. boydii) isolated from patients (female and male from 0 to 80 years of age) presenting diarrhea in different districts of the State of Pará, in the North of Brazil. The antibiotic resistance of the strains, isolated from human fecal samples, was determined by the diffusion disk method and by using the VITEK-2 system. RESULTS: The highest resistance rate found was the resistance rate to tetracycline (93.8 percent), followed by the resistance rate to chloramphenicol (63.9 percent) and to trimethoprim/sulfamethoxazole (63.1 percent). Resistance to at least three drugs was more common among S. flexneri than S. sonnei (39.5 percent vs. 10 percent). Six (4.9 percent) strains were susceptible to all the antibiotics tested. All strains were susceptible to cefotaxime, ceftazidime, ciprofloxacin, nalidixic acid and nitrofurantoin. CONCLUSIONS: High rates of multidrug resistance in Shigella spp. are a serious public health concern in Brazil. It is extremely important to continuously monitor the antimicrobial resistances of Shigella spp. for effective therapy and control measures against shigellosis.

INTRODUÇÃO: Shigella spp. são bactérias Gram-negativas, não esporuladas, em forma de bastonete, pertencentes a família Enterobacteriaceae responsáveis pela shigelose ou disenteria bacilar, uma importante causa de mortalidade e morbidade mundial. MÉTODOS: Foi estudado o perfil de resistência a antimicrobianos de 122 amostras de Shigella spp. (81 S. flexneri, 41 sonnei, 1 S. boydii) isoladas de pacientes (sexo feminino e masculino com faixa etária de 0 a 80 anos) com distúrbios gastrointestinais em diferentes municípios no Estado do Pará, Brasil. A resistência antimicrobiana das amostras isoladas de coprocultura, foi determinada pelo método de difusão em disco e pelo sistema Vitek II. RESULTADOS: A maior resistência foi observada em relação à tetraciclina (93,8 por cento), seguida de cloranfenicol (63,9 por cento), e trimetoprimsulfametoxazol (63,1 por cento). Multirresistência a pelo menos três antimicrobianos foi mais comum em S. flexneri comparada a S. sonnei (39,5 por cento vs. 10 por cento). Seis (4,9 por cento) amostras foram sensíveis a todos antimicrobianos testados. Todas as amostras apresentaram sensibilidade a cefotaxima, ceftazidima, ciprofloxacina, ácido nalidixico e nitrofurantoína. CONCLUSÕES: As altas taxas de multirresistência de Shigella spp. são um sério problema de saúde pública no Brasil. Sendo assim, torna-se extremamente importante um monitoramento contínuo da resistência antimicrobiana de Shigella spp. para uma terapia efetiva e medidas de controle contra shigelose.

Frequency and antimicrobial susceptibility of Shigella species isolated in Children Medical Center Hospital, Tehran, Iran, 2001-2006

Appropriate antimicrobial treatment of shigellosis depends on identifying its changing resistance pattern over time. We evaluated 15,255 stool culture submitted from July 2001 to June 2006 to the Laboratory of Children Medical Center Hospital. Specimen culture, bacterial identification, and disk diffusion susceptibility testing were performed according to National Committee for Clinical Laboratory Standards guidelines. From 15,255 stool samples, 682 (4.5 percent) were positive for Shigella species. The most common species of Shigella were S. flexneri (48 percent) and S. sonnei (45 percent); other results were S. dysenteriae (5 percent) and S. boydii (2 percent). The rate of Sensitivity to ceftriaxone (95 percent), ceftizoxime (94 percent), and nalidixic acid (84 percent) were among our isolates. Resistance to co-trimoxazole and ampicillin was 87 percent and 86 percent, respectively. S. flexneri was more multiresistant than other species (47.9 percent). Our isolates are overall most sensitive to ceftriaxone, ceftazidime, and nalidixic acid (> 84 percent). They were most resistant to co-trimoxazole and ampicillin (> 86 percent). Because resistance varies according to specific location, continuous local monitoring of resistance patterns is necessary for the appropriate selection of empirical antimicrobial therapy.

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.

Frequency of serovars and antimicrobial resistance in Shigella spp. from Brazil

A total of 296 Shigella spp. were received from State Public Health Laboratories, during the period from 1999 to 2004, by National Reference Laboratory for Cholera and Enteric Diseases (NRLCED) - IOC/Fiocruz, Rio de Janeiro, Brazil. The frequency of Shigella spp. was: S. flexneri (52.7 percent), S. sonnei (44.2 percent), S. boydii (2.3 percent), and S. dysenteriae (0.6 percent). The most frequent S. flexneri serovars were 2a and 1b. The highest incidence rates of Shigella isolation were observed in the Southeast (39 percent) and Northeast (34 percent) regions and the lowest rate in the South (3 percent) of Brazil. Strains were further analyzed for antimicrobial susceptibility by disk diffusion method as part of a surveillance program on antimicrobial resistance. The highest rates of antimicrobial resistance were to trimethoprim-sulfamethozaxole (90 percent), tetracycline (88 percent), ampicillin (56 percent), and chloramphenicol (35 percent). The patterns of antimicrobial resistance among Shigella isolates pose a major difficulty in the determination of an appropriate drug for shigellosis treatment. Continuous monitoring of antimicrobial susceptibilities of Shigella spp. through a surveillance system is thus essential for effective therapy and control measures against shigellosis.

The use of transepithelial models to examine host-pathogen interactions.

Many pathogens must overcome an epithelial barrier in order to establish an infection. Unsurprisingly, such pathogens have evolved different mechanisms to overcome this obstacle, targeting specific epithelial structures or functions. These include disruption of epithelial barrier function, transcytosing from the apical to the basolateral membrane domain or inducing cell movement such as neutrophil recruitment. When studying these processes in vivo, animal models often fail to mimic the disease observed in humans and present a complex system in which many variables cannot be controlled. Therefore, in vitro transepithelial models that permit the study of a relevant biological surface have been developed, to integrate not only interactions between bacteria and epithelial cells but also, under certain conditions, to integrate a third cell type, such as neutrophils or dendritic cells. Such models are particularly useful for studying the bacteria-host relationship as it would occur in the microenvironment of the human epithelium and have enhanced our understanding of the unique strategies by which pathogenic bacteria exploit host cells to overcome the initial epithelial hurdle.

Susceptivilidad in vitro de azitromicina frente a cepas de Shigella sp. provenientes deniños con diarrea disentéria / In vitro susceptibility of azitromicine to shigella sp strain in clhildren with dysenteric diarrhea

Shigela sp., probablemente representa el principal agente etiológico de la diarrea enteroinvasiva en nuestro paías y se caracteriza por exhibir un elevado perfil de resistencia a diferentes antimicrobianos. la azitromicina, constituye na nueva clase de antibióticos denominada azálido, con espectro de actividad ampliada contra Gram negativos y Gram positivos. Nosotros estudiamos la sensibilidad in vitro, de la azitromicina frente a las cepas de Shigella sp. aisladas de niños con diarrea enteroinvasiva en nuestro país. 150 cepas de Shigella sp fueron estudiadas mediante la técnica de difusión con discos (Kirby-Bauer), 125 (83 porciento9 fueron S. fexneri, 20 (14) S sonnei y 5 (3 porciento) S. dysenteriae. El 100 porciento de las cpas de Shigelli sp. estudiadas demostraron ser sensibles in vitro a la azitroicina. se concluye en la necesidad de realizar estudios clínicos prospectivos a fin de determinar el rol de la azitromicina en niños con shigellosis.

Estudo da resistencia antimicrobiana in vitro das coproculturas positivas para Shigella sp / Study of in vitro antimicrobial resistance of positive coprocultures to Shigella sp

Introduçäo gastroenterite causada pela Shigella sp (shigelose) representa importante causa de morbimortalidade, especialmente em crianças abaixo de 2 anos de idade. Sabe-se que o tratamento antimicrobiano adequado pode melhorar o quadro clínico e diminuir a disseminaçäo da doença. Têm sido descritos níveis crescentes de resistência aos antimicrobianos comumente usados para o tratamento, e a sensibilidade da Shigella näo foi recentemente avaliada em nosso meio. Objetivo: avaliar a resitência antimicrobiana das espécies de Shigella em nosso meio. Método: Delineou-se um estudo retrospectivo, utilizando-se 106 coproculturas de pacientes, hospitalizados ou näo, que tenham procurado atendimento no HCPA ou no HPV durante o período de 1994 a 1996 com diagnóstico de shigelose firmado por coprocultura...

Shiguelose: diagnóstico e tratamento / Shiguellosis: diagnosis and therapy

É uma doença diarréica aguda infecciosa mundialmente distribuída, causada por um bacilo gram-negativo da família das enterobactérias, transmitido por contato inter-humano, por água e alimentos contaminados, causando diarréia mucopiossanguinolenta, associada à febre, cólicas abdominais, urgência e tenesmo retal. O diagnóstico é baseado na história clínica e exame físico, sendo confirmado principalmente pela coprocultura e, mais raramente, pela hemocultura. Outros exames inespecíficos podem ser usados, como o hemograma e a análise do mucofecal. A sorologia e o PCR podem ser úteis, porém nem sempre facilmente disponíveis. O tratamento visa a correçÝo dos distúrbios hidroeletrolíticos já que a shiguelose costuma ser autolimitada, sendo porém, eventualmente necessário o uso de antimicrobianos. Quando a shiguela isolada se mostra sensível à ampicilina, esta droga tem sido utilizada como primeira escolha; entretanto, nÝo se isolando o agente causal, o sulfametoxazoltrimethopim pode ser indicado. atualmente, os melhores resultados (e com baixos ídices de resistência) têm sido obtidos com o uso das quinolonas, devendo, porém, ser usadas com critério devido ao aparecimento de cepas multirresistentes.

Shigellosis in children from north India: a clinicopathological study.

Stool samples of 1488 children suffering from acute diarrhoea were studied for bacterial culture and sensitivity. Shigella culture was positive in 143 (10 per cent) children and 53 hospitalized children could be studied in detail. Thirty-six (68 per cent) children were under 2 years of age and peak prevalence was observed in summer months. Fever and diarrhoea were universal features; 96 per cent had blood and mucus in the stools, but 32 per cent started with watery diarrhoea lasting 1-3 days followed by dysentery. Two cases (4 per cent) had watery diarrhoea. Abdominal pain dehydration, and malnutrition were present in more than two-thirds of the cases. Central nervous systemic (CNS) manifestations, renal failure, respiratory manifestations, and subacute intestinal obstruction were seen in 45, 25, 17, and 5 per cent of cases, respectively. Shigella dysenteriae was the commonest organism grown in 57 per cent, followed by Shigella flexneri in 36 per cent, Shigella boydii in 4 per cent, and Shigella sonnei in 4 per cent cases. In the majority, the organisms were sensitive to neomycin (83 per cent), furazolidine (86 per cent), and cephaloridine (87 per cent), whereas Shigella strains were resistant to tetracycline in 93 per cent, ampicillin in 83 per cent, chloramphenicol in 91 per cent and cotrimoxazole in 66 per cent cases. Proctosigmoidoscopy was useful in defining the nature of mucosal lesion, to collect swabs for culture and biopsy specimen for histopathology. Four (8 per cent) cases had pseudomembrane and in two cases Clostridium difficile could be identified. Eight (15 per cent) cases died and two of them had shigellaemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of Shigella and enteroinvasive Escherichia coli strains by a virulence-specific, monoclonal antibody-based enzyme immunoassay.

BALB/c mice were immunised with water extracts made from an Escherichia coli K-12 strain harbouring the shigella invasion plasmid, and hybridomas secreting antibodies specific to invasion plasmid-coded antigens were selected. On Western blots, antibodies produced by one of these clones (MAIC-1) recognised a protein of 43 kDa, which is the molecular mass of invasion plasmid coded antigen C (IpaC). When used in enzyme immunoassay against whole bacterial cells or against proteins secreted by actively growing bacteria, MAIC-1 clearly differentiated between invasive and non-invasive strains. Testing 123 enteroinvasive and 139 non-enteroinvasive strains the MAIC-1 based assay proved to be highly specific and sensitive in recognising enteroinvasive isolates. This test could be an inexpensive and rapid alternative to cumbersome virulence assays and a helpful technique in identifying Shigella or enteroinvasive Escherichia coli isolates.

Alkaline phosphatase capture test for the rapid identification of Escherichia coli and Shigella species based on a specific monoclonal antibody.

A specific monoclonal antibody for Escherichia coli and Shigella sp. alkaline phosphatase was used in an immunocapture assay and allowed identification of E. coli either in culture isolates or directly in clinical specimens. The assay was easy and required only four steps: (i) alkaline phosphatase was released within 10 min by using a gentle lysis procedure, (ii) cell lysates were transferred to antibody-coated tubes for 45 min, (iii) p-nitrophenyl phosphate substrate was added, and (iv) alkaline phosphatase activity was detected in a microsample spectrophotometer at 410 nm. This immunocapture assay was highly specific: only one false-positive reaction was observed with a Klebsiella pneumoniae lysate among the 205 non-E. coli strains tested. The assay was sensitive, detecting 10(7) CFU/ml from culture isolates or 10(5) CFU/ml from urine specimens which had first been grown in phosphate-limiting medium for 2 h. At these bacterial concentrations, the percentages of detected E. coli were high: 91% for blood cultures, 95.4% for culture isolates, and 96.8% for urine specimens.

Frecuencia etiológica de la shigelosis por especie y serotipo en Venezuela / Etiological frequency for species and serotypes of Shigella in Venezuela

Se determina la frecuencia etiológica por especie y por serotipo de 1369 cepas de Shigella aisladas durante el período 1968-1982 de coprocultivos procedentes principalmente de niños con clínica de disentería bacilar. De ellas, 870 (63.55%) son Shigella flexneri y 414 (30.24%) son Shigella sonnei. El predominio de Shigella flexneri se hace en base a los serotipos B2, B1, B3 y B6. Un incremento en los aislamientos de Shigella sonnei ocurre en el transcurso del estudio. A Shigella dysenteriae corresponden 77 (5.62%) cepas, 15 (19.48%) de ellas son Shigella dysenteriae A1 ocurriendo el último aislamiento de este serotipo en 1975. Shigella dysenteriae A2 se ha hecho endémico en la región y es responsable en los últimos 8 años del 96.6% de los aislamientos de Shigella dysenteriae. Shigella boydii es infrecuentemente aislada en nuestro medio (0.58%)