Purification, characterization, mode of action, and enhanced production of Salivaricin mmaye1, a novel bacteriocin from Lactobacillus salivarius SPW1 of human gut origin

Background: Emergence of antibiotic resistance among pathogenic and food spoilage bacteria such as Staphylococcus aureus, Micrococcus luteus, Streptococcus pyogenes, Streptococcus sanguinis, Streptococcus mutans, Bacillus cereus, and Listeria monocytogenes triggered the search for alternative antimicrobials. An investigation aimed at purifying, characterizing, elucidating the mode of action, and enhancing the production of salivaricin from Lactobacillus salivarius of human gut origin was conducted. Results: Salivaricin mmaye1 is a novel bacteriocin purified from L. salivarius isolated from human feces. It is potent at micromolar concentrations and has a molecular weight of 1221.074 Da as determined by MALDI-TOF mass spectrometry. It has a broad spectrum of antibacterial activity. Salivaricin mmaye1 showed high thermal and chemical stability and moderate pH stability. The proteinaceous nature of salivaricin mmaye1 was revealed by the complete loss of activity after treatment with pepsin, trypsin, α-chymotrypsin, protease, and proteinase. Salivaricin mmaye1 is cell wall associated, and adsorption­desorption of the bacteriocin from the cell wall of the producer by pH modification proved successful. It exhibited a bactericidal mode of action mediated by pore formation. Its biosynthesis is regulated by a quorum sensing mechanism. Enhanced production of salivaricin mmaye1 was achieved in a newly developed growth medium. Conclusions: A novel, cell wall adhering, highly potent bacteriocin with a broad spectrum of inhibitory activity, membrane-permeabilizing ability, and enhanced production in a newly constituted medium has been isolated. It has a quorum sensing regulatory system and possesses interesting physicochemical characteristics favoring its future use in food biopreservation. These findings pave the way for future evaluation of its medical and food applications.

Ocorrência de Campylobacter em carne de frango, fezes de frango e humanas e pesquisa dos genes cdt / Occurrence of Campylobacter in poultry, meat chicken and human feces, and cdt GENES research

Foram coletadas 100 amostras de conteúdo fecal de aves de corte, 100 de produtos de frango (coxa, sobrecoxa, asa, dorso, carne moída e fígado) e 100 de fezes de humanos, e analisadas para pesquisa de Campylobacter. Realizou-se a determinação da espécie e da presença dos genes cdt, responsáveis pela codificação da toxina citoletal distensiva (CDT), através da técnica da PCR. A bactéria foi isolada de 61% das amostras de fezes de frango, 20% de produtos de frango e 3% de fezes de humanos. A maioria dos isolados foi determinada como C. jejuni . Destes, 93,5% apresentaram os genes para a toxina CDT. Apesar de a ocorrência de Campylobacter em fezes de humanos ter sido baixa, a prevalência em frangos de corte e produtos de frango foi elevada, fato que, aliado à presença dos genes cdt na maioria dos isolados, representa risco potencial para os consumidores. Esses resultados são indicativos da necessidade de medidas preventivas no sistema de produção e de boas práticas de fabricação na indústria, de forma a minimizar a contaminação dos produtos e diminuir o risco para os consumidores.

A hundred chicken fecal samples, a hundred samples of retail poultry products and a hundred samples of human feces were collected and tested for the presence of Campylobacter. The species identification and the analysis for the presence of cdt genes, responsible for encoding the cytolethal distending toxin, were performed by PCR. Campylobacter was found in 61% of the chicken fecal samples, in 20% of the poultry products and in 3% of the human feces. Most isolates were identified as C. jejuni. In 93.5% of these isolates, the cdt genes have been detected. Despite the occurrence of Campylobacter in feces of humans has been low, the prevalence in broilers and poultry products was high, which, combined with the presence of cdt genes in most isolates, represents a potential risk to consumers. These results suggest there is a need for preventive measures in the production system and good manufacturing practices in the industry so as to minimize contamination of products and reduce the risk to consumers.

Establishment and Application of a Real-Time Quantitative PCR Assay for Detection of Campylobacter Jejuni in Human Feces / 现代检验医学杂志

Objective To establish a real-time quantitative PCR assay for the identification of Campylobacter jejuni in fecal samples.Methods Specific primers of the PCR were designed according to the conserved sequences of Campylobacterjeju-ni,and the real-time quantitative PCR assay was established.150 cases of fecal samples were tested by both culture and PCR methods.With the culture testing results as the reference standard,the sensitivity,specificity,accuracy and repetition of the real-time quantitative PCR were validated.Kappa test was used to estimate the difference between the two detection meth-ods.Results The standard carve of the real-time quantitative PCR assay fitted the equationY=-3.51Log(X)+37.09 (R2=0.996)well.The sensitivity,specificity,and accuracy of the established method were 92.4%,95.8% and 94%,respective-ly.The theoretical detection limit of the PCR method was 102 CFU/ml,and its reproducibility was good (CV<5%).Statisti-cal analysis demonstrated that the results of the two methods were consistent,and the consistent strength was very strong (Kappa=0.88,P<0.05).Conclusion The established real-time PCR method can assay the Campylobacterjejuni in human fecal samples rapidly and accurately.

Two sequential PCR amplifications for detection of Schistosoma mansoni in stool samples with low parasite load / Duas amplificações sequenciais por PCR para detecção de Schistosoma mansoni em amostras de fezes com baixa carga parasitária

Schistosomiasis constitutes a major public health problem, with an estimated 200 million individuals infected worldwide and 700 million people living in risk areas. In Brazil there are areas of high, medium and low endemicity. Studies have shown that in endemic areas with a low prevalence of Schistosoma infection the sensitivity of parasitological methods is clearly reduced. Consequently diagnosis is often impeded due to the presence of false-negative results. The aim of this study is to present the PCR reamplification (Re-PCR) protocol for the detection of Schistosoma mansoni in samples with low parasite load (with less than 100 eggs per gram (epg) of feces). Three methods were used for the lysis of the envelopes of the S. mansoni eggs and two techniques of DNA extraction were carried out. Extracted DNA was quantified, and the results suggested that the extraction technique, which mixed glass beads with a guanidine isothiocyanate/phenol/chloroform (GT) solution, produced good results. PCR reamplification was conducted and detection sensitivity was found to be five eggs per 500 mg of artificially marked feces. The results achieved using these methods suggest that they are potentially viable for the detection of Schistosoma infection with low parasite load.

A esquistossomose constitui grande problema de saúde pública, sendo que estimativas apontam para 200 milhões de pessoas infectadas no mundo e 700 milhões de pessoas em áreas de risco. No Brasil, existem áreas de alta, média e baixa endemicidade. Estudos demonstram que nas áreas endêmicas de baixa prevalência da infecção, a reduzida sensibilidade dos métodos parasitológicos torna-se evidente. Isto dificulta o diagnóstico, pela presença de resultados falso-negativos. O objetivo deste estudo foi a padronização de um protocolo de reamplificação da PCR (Re-PCR) para a detecção de Schistosoma mansoni em amostras com menos de 100 ovos por grama (opg) de fezes. Foram utilizados três métodos para ruptura dos envoltórios dos ovos de S. mansoni e duas técnicas de extração de DNA foram aplicadas. O DNA extraído foi quantificado e os resultados sugerem que a técnica de extração de melhor produtividade foi a que associa esferas de vidro a uma solução de isotiocianato de guanidina/fenol/clorofórmio (GT). Aplicou-se a Re-PCR, que demonstrou sensibilidade para a detecção de cinco ovos/500 mg de fezes artificialmente marcadas. Assim, essas novas ferramentas são potencialmente aplicáveis nas infecções por S. mansoni com baixa carga parasitária.

Determination of Lanthanide in Human Feces by ICP-MS / 环境与健康杂志

Objective To establish a method for the determination of 14 kinds of lanthanide (La) series elements in human feces at the same time by ICP-MS.Methods In January 2009,human feces were collected for three consecutive days from 30 subjects in Tianjin area,the fecal samples for the three-day period were weighed and homogenized;then dried;grinded and ashed,the samples were digested by HNO3 before determination.The solution was directly analyzed by ICP-MS to determine the concentrations of the 14 kinds of La series elements in human faces with rhenium (Re) internal standard calibration.Results The linear ranges were 0-10 ?g/L with a correlation coefficient for each element of more than 0.999.The detection limits of 139La,140Ce,141Pr,143Nd,147Sm,151Eu,158Gd,159Tb,163Dy,165Ho,166Er,169Tm,174Yb and 175Lu in human feces were 0.48,0.49,0.47,1.6,1.9,0.46,0.79,0.18,0.65,0.39,0.38,0.17,0.09 and 0.09 ng/L respectively.The recoveries of this method were 93.42%-108.21%,and RSDs were 2.91%-9.20%.The analytical values of the certified reference material of human hair GBW 09101a by this method showed closed agreement with the reference values.Conclusion This method has relatively higher sensitivity and less interference,and is applicable to the rapid determination of 14 kinds of La series elements in human faces at the same time.

Frequência de amebas de vida livre em fezes de indivíduos de uma creche da cidade de São Paulo / Frequency free-living amebas in the feces of persons from a nursery in Säo Paulo city

A ocorrência de amebas de vida livre foi pesquisada em 90 amostras de fezes de indivíduos pertencentes a uma creche da cidade de São Paulo. As amostras foram semeadas em placas de Petri contendo ágar não nutriente, ou ágar não nutriente - sal, com um tapete de Ent.erobacter aerogenes, morto pelo calor. As placas foram incubadas a temperaturas de 28, 37 e 400 C, e observadas diariamente durante sete dias. A positividade foi de 22,71% em 66 crianças, e de 8,33% em 24 adultos. Foram isoladas 21 amostras do gênero Acanthamoeba, 4 de Naegleria, 1 de Echinamoeba, e 12 de amebídeos não identificados. Foram examinadas mais 3 amostras fecais de cada indivíduo com exame positivo, com intervalo de um mês entre as coletas. Amostras de Acanthamoeba sp. foram reisoladas em dois indivíduos, o que nos leva a supor que amebídeos estavam sendo por eles albergados, e não em simples trânsito intestinal (AU).