Proteomics Goes to Court: A Statistical Foundation for Forensic Toxin/Organism Identification Using Bottom-Up Proteomics.

Bottom-up proteomics is increasingly being used to characterize unknown environmental, clinical, and forensic samples. Proteomics-based bacterial identification typically proceeds by tabulating peptide "hits" (i.e., confidently identified peptides) associated with the organisms in a database; those organisms with enough hits are declared present in the sample. This approach has proven to be successful in laboratory studies; however, important research gaps remain. First, the common-practice reliance on unique peptides for identification is susceptible to a phenomenon known as signal erosion. Second, no general guidelines are available for determining how many hits are needed to make a confident identification. These gaps inhibit the transition of this approach to real-world forensic samples where conditions vary and large databases may be needed. In this work, we propose statistical criteria that overcome the problem of signal erosion and can be applied regardless of the sample quality or data analysis pipeline. These criteria are straightforward, producing a p-value on the result of an organism or toxin identification. We test the proposed criteria on 919 LC-MS/MS data sets originating from 2 toxins and 32 bacterial strains acquired using multiple data collection platforms. Results reveal a > 95% correct species-level identification rate, demonstrating the effectiveness and robustness of proteomics-based organism/toxin identification.

Microbiology of regressive autism.

This manuscript summarizes some of our earlier work on the microbiology of autism subjects' stool specimens, as compared with stools from control subjects. Our most recent data indicating that Desulfovibrio may play an important role in regressive autism is also presented. In addition, we present information on antimicrobial susceptibility patterns of Desulfovibrio using the CLSI agar dilution susceptibility technique. In addition, we summarize data from our earlier studies showing the impact of various antimicrobial agents on the indigenous bowel flora. This shows that penicillins and cephalosporins, as well as clindamycin, have a major impact on the normal bowel flora and therefore might well predispose subjects to overgrowth of such organisms as Clostridium difficile, and of particular importance for autism, to Desulfovibrio.

Desulfovibrio species are potentially important in regressive autism.

Autism is a complex disorder with no specific diagnostic test so the disease is defined by its characteristics including cognitive defects, social, communication and behavioral problems, repetitive behaviors, unusual sensitivity to stimuli such as noise, restricted interests, and self stimulation. The incidence of this disease has increased remarkably in recent years and was 110/10,000 children (∼1%) in multiple areas of the US in 2007. The financial burden on families and communities is enormous. In terms of predisposing factors, heredity plays a role in some subjects, but it is clear that environmental factors are also important. Environmental toxins can affect the immune system adversely. Intestinal bacteria are recognized by a few investigators as potentially important and we have proposed that certain antimicrobial drugs may be a key factor in modifying the intestinal bacterial flora adversely, selecting out potentially harmful bacteria that are normally suppressed by an intact normal intestinal flora. We had felt that clostridia in the gut might be involved in autism because they are virulent organisms and spore-formers; spores would resist antibacterial agents so that when antibiotics were discontinued the spores would germinate and by toxin production or another mechanism lead to autism. However, a recent study of ours employing the powerful pyrosequencing technique on stools of subjects with regressive autism showed that Desulfovibrio was more common in autistic subjects than in controls. We subsequently confirmed this with pilot cultural and real-time PCR studies and found siblings of autistic children had counts of Desulfovibrio that were intermediate, suggesting possible spread of the organism in the family environment. Desulfovibrio is an anaerobic bacillus that does not produce spores but is nevertheless resistant to aerobic and other adverse conditions by other mechanisms and is commonly resistant to certain antimicrobial agents (such as cephalosporins) often used to treat ear and other infections that are relatively common in childhood. This bacterium also produces important virulence factors and its physiology and metabolism position it uniquely to account for much of the pathophysiology seen in autism. If these results on Desulfovibrio are confirmed and extended in other studies, including treatment trials with appropriate agents and careful clinical and laboratory studies, this could lead to more reliable classification of autism, a diagnostic test and therapy for regressive autism, development of a vaccine for prevention and treatment of regressive autism, tailored probiotics/prebiotics, and important epidemiologic information.

Desulfovibrio spp. survive within KB cells and modulate inflammatory responses.

Desulfovibrio are sulfate-reducing anaerobic gram-negative rods that have been proposed as potential periodontopathogens. We investigated the capacity of Desulfovibrio to invade epithelial cells and induce cytokine secretion from these cells. Desulfovibrio strains were co-cultured with KB cells and counts of intracellular bacteria evaluated up to 3 days after infection. Desulfovibrio desulfuricans and Desulfovibrio fairfieldensis were able to survive within epithelial cells. Intracytoplasmic location of both bacterial species was confirmed by confocal laser scanning microscopy and transmission electron microscopy. Invasion was sensitive to nocodazole, an inhibitor of microtubule polymerization, but not to cytochalasin D, a microfilament inhibitor, suggesting that microtubule rearrangements were involved in the internalization of Desulfovibrio strains by KB cells. Infection by Desulfovibrio resulted in increased production of IL-6 and IL-8 by KB cells. The ability of D. desulfuricans and D. fairfieldensis to survive within oral epithelial cells and to modulate the epithelial immune response may contribute to the initiation and progression of periodontal diseases.

Processos fisiológicos e patológicos das bactérias redutoras de sulfato do gênero desulfovibrio sp / Physiologic and pathological processes of the sulphate-reducing bacteria of the genus desulfovobrio sp

As bactérias redutoras de sulfato do gênero Desulfovibrio sp. podem ser encontradas normalmente formando parte da biota intestinal e oral de seres humanos saudáveis, participando, direta ou indiretamente, com seus produtos metabólicos, de diversas afecções como: periodontites, câncer colorretal, infecções e sepsemias. Propõe-se com esta revisão avaliar os aspectos normais e as possíveis alterações patológicas correlacionadas com as bactérias redutoras de sulfato no organismo humano. As conclusões levam a crer que o desequilíbrio na biota oral e intestinal pode levar a aumento no número de bactérias redutoras de sulfato e na produção de sulfeto, como produto metabólico final, podendo representar um fator adicional no desenvolvimento daquelas afecções. Além disso, por haver forte propensão para formar associações bacterianas, aumentando seu potencial patológico, pode ser difícil a identificação do seu verdadeiro papel nas morbidades em que estão envolvidas assim como o seu potencial virulento.

Sulfate-reducing bacteria of the genus Desulfovibrio spp. can be routinely detected as member of the normal intestinal and oral microbiota in health individuals. This bacterial group produces metabolic byproducts, which participate, direct or indirect, in several diseases, such as periodontitis, colorectal cancer, infections and sepsis. The purpose of the present study was to assess the association between sulfate-reducing bacteria and normal conditions and pathology alterations in human. In conclusion, it is possible that alteration of the oral and intestinal flora can result in increase of sulfate-reducing bacteria levels and products of sulfide as final metabolic. Therefore, these conditions can represent an important fact associated with those diseases. In addition, this bacterial group presents a great tendency in to associate with other microorganisms. Like that, it can increase the pathologic potential and can difficult the identification of the true involvement with several diseases as well as its virulent potential.

[Clinical significance of sulfate-reducing bacteria for ulcerative colitis].

Ulcerative colitis(UC) is colon localized disease. Broad epithelial cell damage, crypt abscesses and accumulation of neutrophils are recognized for UC. Although the cause of UC is indistinct at this time, there is a growing consensus that abnormal intestinal microflora would be related with UC. There have been several evidences that excessive production of hydrogen sulfide by bacteria in colon would be associated with UC. Sulfate reducing bacteria are able to utilize sulfate as an electron receptor for dissimilation of organic substrate and hydrogen gas, resulting in generating toxic hydrogen sulfide. This review is dealt with the association between sulfate reducing bacteria and UC in aetiology and bacterial pathogenesis.

Isolation of the provisionally named Desulfovibrio fairfieldensis from human periodontal pockets.

Sulfate-reducing bacteria have recently been associated with periodontitis and proposed to play a role in the pathogenesis of this chronic inflammatory process. Eight isolates of sulfate-reducing bacteria belonging to the genus Desulfovibrio were obtained from the periodontal pockets of five out of seven patients presenting with active periodontitis. A multiplex PCR was devised for their identification at the species level. All isolates were identified as Desulfovibrio fairfieldensis, a recently proposed new species. This finding reinforces the suggestion that Desulfovibrio fairfieldensis is a human bacterium that may present a pathogenic potential.