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PubMed1977

Generalized indicator plate for genetic, metabolic, and taxonomic studies with microorganisms.

We have developed an indicator plate that works well for diverse types of substrates and microorganisms. The plates are inexpensive and easy to prepare. The essential components are agar, buffer, growth-supporting nutrients, a test substrate, and 2,3,5-triphenyl tetrazolium chloride (TTC). Using various strains of Salmonella typhimurium and Escherichia coli, we have studied and defined the contribution of each component to the satisfactory function of the plate. Colonies capable of catabolizing the test substrate reduce TTC and produce a deep red formazan, whereas colonies failing to catabolize the substrate remain uncoloured. Those with intermediate rates of catabolism differ in rate and/or extent of color formation. In all cases the color is stable because TTC reduction is essentially irreversible. Since the mode of action of these plates is fairly well understood, alternative formulations can be devised to meet specific needs. The general applicability of this TTC indicator system makes it an extremely useful tool in microbial genetics, metabolism, and taxonomy.

Acinetobacter↗
PubMed2004

Unexpected diversity in the fine specificity of monoclonal antibodies that use the same V region gene to glucuronoxylomannan of Cryptococcus neoformans.

Most mAbs to the capsular polysaccharide glucuronoxylomannan (GXM) of Cryptococcus neoformans are generated from the same VH and VL gene families. Prior Ab studies have assessed protective efficacy, Id structure and binding to capsular polysaccharides, and peptide mimetics. These data have been interpreted as indicating that most mAbs to GXM have the same specificity. A new approach to Ab specificity analysis was investigated that uses genetic manipulation to generate C. neoformans variants with structurally different capsules. C. neoformans mutants expressing GXM with defective O-acetylation were isolated and complemented by the C. neoformans gene CAS1, which is necessary for the O-acetylation of GXM. The mAbs exhibited differences in their binding to the GXM from these mutant strains, indicating previously unsuspected differences in specificity. Analysis of three closely related IgMs revealed that one (mAb 12A1) bound to an epitope that did not require O-acetylation, another (mAb 21D2) was inhibited by O-acetylation, and the third (mAb 13F1) recognized an O-acetylation-dependent conformational epitope. Furthermore, an IgG Ab (mAb 18B7) in clinical development retained binding to de-O-acetylated polysaccharide; however, greater binding was observed to O-acetylated GXM. Our findings suggest that microbial genetic techniques can provide a new approach for epitope mapping of polysaccharide-binding Abs and suggest that this method may applicable for studying the antigenic complexity of polysaccharide Ags in other capsulated microorganisms.

Antibodies, Fungal↗
PubMed2026

Metagenomic insights into biogeochemical functional potential and resistome dynamics of PM2.5 microbial communities.

Atmospheric particulate matter harbors diverse microorganisms, yet their functional potential in biogeochemical cycling and the associated risks of resistome remain poorly understood. Here, we performed metagenomic sequencing on PM2.5 samples collected across four months to unravel the microbial genetic repertoire involved in methane, nitrogen, phosphorus, and sulfur cycling, as well as the resistome, and pathogen composition. A broad range of functional genes was detected for each biogeochemical cycle, with more than 65% of gene subtypes shared across all months, indicating conserved functional signatures. In contrast, more than 80% of the resistome showed temporal variation in abundance, with the lowest richness observed in March. Temporal shifts were also observed in resistome composition, with several resistance determinants reaching higher abundances in April and May. Network analysis indicated frequent co-occurrence among several pathogenic and opportunistic taxa. Contig-based profiling identified 51 potential pathogenic taxa, including 32 human- or animal-associated taxa. In addition, both PM10 and PM2.5 concentrations were associated with pathogen abundance and functional gene richness (e.g., antibiotic resistance genes and virulence factors). Together, this metagenomic survey suggests contrasting temporal patterns between conserved biogeochemical functional potential and more variable resistome-related traits in PM2.5 microbial communities. While constrained by limited temporal coverage and sample size, this study provides preliminary insights into the ecological and potential public health relevance of airborne microbial communities in urban environments.

Particulate Matter↗
PubMed2003

Severity of nonbullous Staphylococcus aureus impetigo in children is associated with strains harboring genetic markers for exfoliative toxin B, Panton-Valentine leukocidin, and the multidrug resistance plasmid pSK41.

Nonbullous impetigo is a common skin infection in children and is frequently caused by Staphylococcus aureus. Staphylococcal toxins and especially exfoliative toxin A are known mediators of bullous impetigo in children. It is not known whether this is also true for nonbullous impetigo. We set out to analyze clonality among clinical isolates of S. aureus from children with nonbullous impetigo living in a restricted geographical area in The Netherlands. We investigated whether staphylococcal nasal carriage and the nature of the staphylococcal strains were associated with the severity and course of impetigo. Bacterial isolates were obtained from the noses and wounds of children suffering from impetigo. Strains were genetically characterized by pulsed-field gel electrophoresis-mediated typing and binary typing, which was also used to assess toxin gene content. In addition, a detailed clinical questionnaire was filled in by each of the participating patients. Staphylococcal nasal carriage seems to predispose the patients to the development of impetigo, and 34% of infections diagnosed in the Rotterdam area are caused by one clonal type of S. aureus. The S. aureus strains harbor the exfoliative toxin B (ETB) gene as a specific virulence factor. In particular, the numbers (P = 0.002) and sizes (P < 0.001) of the lesions were increased in patients infected with an ETB-positive strain. Additional predictors of disease severity and development could be identified. The presence of a staphylococcal plasmid encoding multiple antibiotic resistance traits, as detected by binary typing, was associated with a reduction in the cure rate. Our results recognize that a combination of staphylococcal virulence and resistance genes rather than a single gene determines the development and course of nonbullous impetigo. The identification of these microbial genetic markers, which are predictive of the severity and the course of the disease, will facilitate guided individualized antimicrobial therapy in the future.

Anti-Bacterial Agents↗
PubMed2005

Characterization of a forest soil metagenome clone that confers indirubin and indigo production on Escherichia coli.

A microbial community analysis of forest soil from Jindong Valley, Korea, revealed that the most abundant rRNA genes were related to Acidobacteria, a major taxon with few cultured representatives. To access the microbial genetic resources of this forest soil, metagenomic libraries were constructed in fosmids, with an average DNA insert size of more than 35 kb. We constructed 80,500 clones from Yuseong and 33,200 clones from Jindong Valley forest soils. The double-agar-layer method allowed us to select two antibacterial clones by screening the constructed libraries using Bacillus subtilis as a target organism. Several clones produced purple or brown colonies. One of the selected antibacterial clones, pJEC5, produced purple colonies. Structural analysis of the purified pigments demonstrated that the metagenomic clone produced both the pigment indirubin and its isomer, indigo blue, resulting in purple colonies. In vitro mutational and subclonal analyses revealed that two open reading frames (ORFs) are responsible for the pigment production and antibacterial activity. The ORFs encode an oxygenase-like protein and a putative transcriptional regulator. Mutations of the gene encoding the oxygenase canceled both pigment production and antibacterial activity, whereas a subclone carrying the two ORFs retained pigment production and antibacterial activity. This finding suggests that these forest soil microbial genes are responsible for producing the pigment with antibacterial activity.

Bacteria↗
PubMed2005

Biologic therapy for inflammatory bowel disease.

Despite all of the advances in our understanding of the pathophysiology of inflammatory bowel disease (IBD), we still do not know its cause. Some of the most recently available data are discussed in this review; however, this field is changing rapidly and it is increasingly becoming accepted that immunogenetics play an important role in the predisposition, modulation and perpetuation of IBD. The role of intestinal milieu, and enteric flora in particular, appears to be of greater significance than previously thought. This complex interplay of genetic, microbial and environmental factors culminates in a sustained activation of the mucosal immune and non-immune response, probably facilitated by defects in the intestinal epithelial barrier and mucosal immune system, resulting in active inflammation and tissue destruction. Under normal situations, the intestinal mucosa is in a state of 'controlled' inflammation regulated by a delicate balance of proinflammatory (tumour necrosis factor [TNF]-alpha, interferon [IFN]-gamma, interleukin [IL]-1, IL-6, IL-12) and anti-inflammatory cytokines (IL-4, IL-10, IL-11). The mucosal immune system is the central effector of intestinal inflammation and injury, with cytokines playing a central role in modulating inflammation. Cytokines may, therefore, be a logical target for IBD therapy using specific cytokine inhibitors. Biotechnology agents targeted against TNF, leukocyte adhesion, T-helper cell (T(h))-1 polarisation, T-cell activation or nuclear factor (NF)-kappaB, and other miscellaneous therapies are being evaluated as potential therapies for IBD. In this context, infliximab is currently the only biologic agent approved for the treatment of inflammatory and fistulising Crohn's disease. Other anti-TNF biologic agents have emerged, including CDP 571, certolizumab pegol (CDP 870), etanercept, onercept and adalimumab. However, ongoing research continues to generate new biologic agents targeted at specific pathogenic mechanisms involved in the inflammatory process. Lymphocyte-endothelial interactions mediated by adhesion molecules are important in leukocyte migration and recruitment to sites of inflammation, and selective blockade of these adhesion molecules is a novel and promising strategy to treat Crohn's disease. Therapeutic agents that inhibit leukocyte trafficking include natalizumab, MLN-02 and alicaforsen (ISIS 2302). Other agents being investigated for the treatment of Crohn's disease include inhibitors of T-cell activation, peroxisome proliferator-activated receptors, proinflammatory cytokine receptors and T(h)1 polarisation, and growth hormone and growth factors. Agents being investigated for treatment of ulcerative colitis include many of those mentioned for Crohn's disease. More controlled clinical trials are currently being conducted, exploring the safety and efficacy of old and new biologic agents, and the search certainly will open new and exciting perspectives on the development of therapies for IBD.

Animals↗
PubMed1992

Use of the HPRT gene and the HAT selection technique in DNA-mediated transformation of mammalian cells: first steps toward developing hybridoma techniques and gene therapy.

In 1956, I decided to apply my experience in microbial genetics to developing analogous systems for human cell lines, including the selection of mutants with either a loss or gain of a biochemical function. For instance, mutants resistant to azahypoxanthine showed a loss of the HPRT enzyme (hypoxanthine phosphoribosyl transferase), whereas gain of the same enzyme was accomplished by blocking de novo purine biosynthesis with aminopterin, while supplying hypoxanthine and thymine (HAT selection). Using HAT selection, we: (i) genetically transformed HPRT- mutant cells to HPRT+ wild type by using DNA extracted from HPRT+ cells, and (ii) selected HPRT+ hybrid cells by fusing HPRT- D98/AH2 cells with skin cells. These approaches, which we dubbed in 1962 as a 'first step toward gene therapy', contributed to the later development of (i) cell fusion techniques, (ii) the development of monoclonal antibodies, (iii) routine transformation of mammalian cells with cloned genes, and (iv) methods for creating transgenic organisms.

Animals↗
PubMed1999

The cellular immunology of multiple sclerosis.

Multiple sclerosis (MS) is a neurological disease that affects the central nervous system (brain and spinal cord) resulting in debilitating motor and sensory dysfunction. Its mean age of onset is 30 years and, with the exception of trauma, MS remains the most frequent cause of neurological disabilities for young adults. The disease is highly variable in its onset and progression. It may not be easily diagnosed, at least in its earliest stages. Significant disability is a hallmark of MS. Indeed, up to 50% of patients require walking aids and 10% are wheelchair-bound at 15 years after an initial diagnosis. Clinical features include deficits in sensory (parasthesias and numbness), motor (difficulties with fine movements and gait), balance, bladder, and sexual functions. Although the etiology for MS is not yet known, it is thought to be related to microbial, genetic, and/or environmental factors. Pathologically, MS is characterized by inflammation. An influx of mononuclear cells occurs through a disrupted blood-brain barrier into an immune-privileged central nervous system. The secretion of a variety of inflammatory cytokines and chemokines from glial cells leads to loss of myelin, disruption of oligodendrocyte integrity, and axonal loss. These events, in large measure, affect progressive neural atrophy. How brain inflammatory activities affect transendothelial migration of leukocytes into the brain and alter the process of myelination are the focal points for MS research activities.

Animals↗