Vladimir E. Chizhikov

Sequencing of the intergenic 16S-23S rRNA spacer (ITS) region of Mollicutes species and their identification using microarray-based assay and DNA sequencing.

Abstract We have completed sequencing the 16S-23S rRNA intergenic transcribed spacer (ITS) region of most known Mycoplasma , Acholeplasma , Ureaplasma , Mesoplasma , and Spiroplasma species. Analysis of the sequence data revealed a significant interspecies variability and low intraspecies polymorphism of the ITS region among Mollicutes . This finding enabled the application of a combined polymerase chain reaction–microarray technology for identifying Mollicutes species. The microarray included individual species-specific oligonucleotide probes for characterizing human Mollicutes species and other species known to be common cell line contaminants. Evaluation of the microarray was conducted using multiple, previously characterized, Mollicutes species. The microarray analysis of the samples used demonstrated a highly specific assay, which is capable of rapid and accurate discrimination among Mollicutes species.

Biological Enrichment of Mycoplasma Agents by Cocultivation with Permissive Cell Cultures

ABSTRACT In this study, we describe our results on the evaluation of the ability of different permissive mammalian cell lines to support the biological enrichment of mycoplasma species known to be bacterial contaminants of cell substrates. The study showed that this approach is able to significantly improve the efficiency of mycoplasma detection based on nucleic acid testing or biochemical technologies (e.g., MycoAlert mycoplasma detection). Of 10 different cell lines (Vero, MDBK, HEK-293, Hep-G2, CV-1, EBTr, WI-38, R9ab, MDCK, and High Five) used in the study, only MDCK cell culture was found to support the efficient growth of all the tested mycoplasmas (Mycoplasma arginini, M. bovis, M. fermentans, M. gallinaceum, M. gallisepticum, M. synoviae, M. hominis, M. hyorhinis, M. orale, M. salivarium, and Acholeplasma laidlawii) known to be most frequently associated with contamination of cell substrates and cell lines in research laboratories or manufacturing facilities. The infection of MDCK cells with serial dilutions of each mycoplasma species demonstrated that these common cell line contaminants can be detected reliably after 7-day enrichment in MDCK cell culture at contamination levels of 0.05 to 0.25 CFU/ml. The High Five insect cell line was also found to be able to support the efficient growth of most mycoplasma species tested, except for M. hyorhinis strain DBS1050. However, mycoplasma growth in insect cell culture was demonstrated to be temperature dependent, and the most efficient growth was observed when the incubation temperature was increased from 28°C to between 35 and 37°C. We believe that this type of mycoplasma enrichment is one of the most promising approaches for improving the purity and safety testing of cell substrates and other cell-derived biologics and pharmaceuticals.

Genotyping of Measles Virus in Clinical Specimens on the Basis of Oligonucleotide Microarray Hybridization Patterns

ABSTRACT An oligonucleotide microarray hybridization method for identification of most known measles virus (MV) genotypes was developed. Like the conventional genotyping method, the microarray relied on detecting sequence differences in the 450-nucleotide region coding for the COOH-terminal 150 amino acids of the nucleoprotein (N). This region was amplified using PCR primers binding to all known MV genotypes. The microarray included 71 pairs of oligonucleotide probes (oligoprobes) immobilized on glass slides. Each pair consisted of a genotype-specific oligoprobe, which matched the sequence of only one target genotype, and a control oligoprobe, which contained mismatches at the nucleotide positions unique to this genotype. A pattern recognition algorithm based on cluster analysis of the ratios of hybridization signals from specific and control oligoprobes was used to identify the specific MV genotype. Following the initial validation, the method was used for rapid genotyping of two panels of coded samples. The results of this study showed good sensitivity (90.7%), specificity (100%), and genotype agreement (91.8%) for the new method compared to the results of genotyping conducted using phylogenetic analysis of viral sequences of the C terminus of the N gene. In addition, the microarray demonstrated the ability to identify potential new genotypes of MV based on the similarity of their hybridization patterns with those of known MV genotypes.

Oligonucleotide Microarrays for Identification of Microbial Pathogens and Detection of Their Virulence-Associated or Drug-Resistance Determinants

Microarrays are spatially ordered arrays with ligands chemically immobilized in discrete spots on a solid matrix, usually a microscope slide. Microarrays are a high-throughput large-scale screening system enabling simultaneous identification of a large number of labeled target molecules (up to several hundred thousand) that bind specifically to the immobilized ligands of the array. DNA microarrays represent a promising tool for clinical, environmental, and industrial microbiology since the technology allows relatively rapid identification of large number of genetic determinants simultaneously, providing detailed genomic level information regarding the pathogen species, including identification of their virulence-associated factors and the presence of antibiotic resistance genes. In this chapter, we describe key aspects and methodologies important for the development and use of DNA microarrays for microbial diagnostics.

Molecular Detection of Drug-Resistant Mycobacterium tuberculosis with a Scanning-Frame Oligonucleotide Microarray

The increasing emergence of drug-resistant Mycobacterium tuberculosis poses significant threat to the treatment of tuberculosis (TB). Conventional drug susceptibility testing is time-consuming and takes several weeks because of the slow growth rate of M. tuberculosis and the requirement for the drugs to show antimycobacterial activity. Resistance to TB drugs in M. tuberculosis is caused by mutations in the corresponding drug resistance genes (e.g., katG, inhA, rpoB, pncA, embB, rrs, gyrA, gyrB), and detection of these mutations can be a molecular indicator of drug resistance. In this chapter, we describe the utility of a microarray-based approach exploiting short overlapping oligonucleotides (sliding-frame array) to rapidly detect drug resistance-associated mutations (substitutions, deletions, and insertions) in the pncA gene responsible for resistance ofM. tuberculosis to pyrazinamide (PZA) as an example for this approach. Hybridization of pncA-derived RNA or DNA with the microarray enables easy and simple screening of nucleotide changes in the pncA gene. Sliding-frame microarrays can be used to identify other drug-resistant TB strains that have mutations in relevant drug resistance genes.

Prevalence, Genotype Richness, and Coinfection Patterns of Hemotropic Mycoplasmas in Raccoons (Procyon lotor) on Environmentally Protected and Urbanized Barrier Islands

ABSTRACT Raccoons (Procyon lotor) are successful urban adapters and hosts to a number of zoonotic and nonzoonotic pathogens, yet little is known about their hemoplasma infections and how prevalence varies across habitat types. This study identifies hemotropic Mycoplasma species infection in raccoons from urban and undisturbed habitats and compares hemoplasma infection in sympatric urban cats (Felis catus) from the same geographic region. We collected blood from raccoons (n = 95) on an urban coastal island (n = 37) and an undisturbed coastal island (n = 58) and from sympatric urban cats (n = 39) in Georgia, USA. Based on 16S rRNA gene amplification, 62.1% (59/95) of raccoons and 17.9% (7/39) of feral cats were positive for hemoplasma. There was a greater percentage of hemoplasma-infected raccoons on the undisturbed island (79.3% [46/58]) than on the urban island (35.1% [13/37]; χ2 = 16.9, df = 1, P = 0.00004). Sequencing of the full-length 16S rRNA gene amplicons revealed six hemoplasma genotypes in raccoons, including five novel genotypes that were distinct from three known hemoplasma species identified in the sympatric cats. In addition, the hemoplasma genotypes detected in raccoons were not identified in sympatric cats or vice versa. Although all six hemoplasma genotypes were found in raccoons from urban and undisturbed islands, coinfection patterns differed between sites and among individuals, with the proportion of coinfected raccoons being greater in the undisturbed site. This study shows that raccoons are hosts for several novel hemoplasmas and that habitat type influences infection patterns. IMPORTANCE This study provides information about novel hemoplasmas identified in raccoons (Procyon lotor), which can be used for assessments of the prevalence of these hemoplasmas in raccoon populations and for future studies on the potential pathogenic impacts of these hemoplasmas on raccoon health. Raccoons from the undisturbed habitat had a higher prevalence of hemoplasma infection than urban raccoons. There does not appear to be cross-species transmission of hemotropic mycoplasmas between urban raccoons and feral cats. Raccoons appear to be hosts for several novel hemoplasmas, and habitat type influences infection patterns.

Enhanced Mid-Infrared Chemical Imaging (IRCI) Detection of DNA Microarrays

We report on the optimization of a recently proposed mid-infrared chemical imaging (IRCI) detection method for the analysis of DNA microarrays. The improved protocol allowed for a ten-fold reduction in the time needed to generate a mosaic image of an entire microarray and the production of IR images with high contrast that would facilitate data analysis and interpretation. Advantages of using this protocol were evaluated by applying it to the analysis of four virulence genes in the genomes of 19 strains of the food bacterial pathogen Yersinia enterocolitica.