The classification of bacterial survival strategies in the presence of antimicrobials.

The survival of bacteria under antibiotic therapy varies in nature and is based on the bacterial ability to employ a wide range of fundamentally different resistance mechanisms. This great diversity requires a disambiguation of the term 'resistance' and the development of a more precise classification of bacterial survival strategies during contact with antibiotics. The absence of a unified definition for the terms 'resistance', 'tolerance' and 'persistence' further aggravates the imperfections of the current classification system. This review suggests a number of original classification criteria that will take into account (1) the bacterial ability to replicate in the presence of antimicrobial agents, (2) existing evolutionary stability of a trait within a species, and (3) the presence or absence of specialized genes that determine the ability of a microorganism to decrease its own metabolism or switch it completely off. This review describes potential advantages of the suggested classification system, which include a better understanding of the relationship between bacterial survival in the presence of antibiotics and molecular mechanisms of cellular metabolism suppression, the opportunity to pinpoint targets to identify a true bacterial resistance profile. The true resistance profile in turn, could be used to develop effective diagnostic and antimicrobial therapy methods, while taking into consideration specific bacterial survival mechanisms.

Microarray-based analysis of the BRAF V600 mutations in circulating tumor DNA in melanoma patients.

BACKGROUND: Circulating tumor DNA (ctDNA) holds great potential for cancer therapy and can provide diagnostic and prognostic information before and during treatment. METHODS: Plasma DNA samples from 97 melanoma patients, 20 healthy donors and 3 patients with benign skin tumors were analyzed by microarray analysis and droplet digital PCR (ddPCR). RESULTS: A microarray for simultaneous detection of six BRAF V600 mutations in ctDNA has been developed. The method allows the detection of 0.05% mutated DNA from WT DNA background. For paired samples (pre-surgery plasma and tumor tissue) isolated from 74 patients, the concordance of genotypes between tumor DNA and ctDNA was 65% (48/74). BRAF mutations in ctDNA were detected in 27/50 patients with BRAF-positive tumors and in 3/24 patients with BRAF wild-type tumors. The presence of ctDNA BRAF mutations in 23 plasma samples from melanoma patients undergoing therapy correlated significantly with tumor progression (P=0.005). The increase in cell-free DNA levels measured by ddPCR also correlated with disease progression (P=0.02). The concordance of results obtained by microarray identification of BRAF mutations and those obtained by ddPCR was 91%. CONCLUSION: The novel microarray-based approach can be a useful non-invasive tool for accurate identification of ctDNA BRAF mutations to monitor disease progression.

Exogenous contaminating DNA in Taq polymerases: A method to avoid false-positive results when detecting the blaTEM gene.

In the course of developing an assay to identify genes responsible for antibiotic resistance in gram-negative bacteria, it has been found that standard (not DNA-free) Taq DNA polymerases were contaminated with blaTEM gene fragments that varied in length and quantities. The complete blaTEM gene sequence was either absent or was detected in infinitesimal amounts. We developed an approach to avoid false-positive findings caused by contaminating blaTEM gene sequences in conventional polymerases. The method is based on selection of a target sequence to be detected within the blaTEM gene in such a way that the chosen sequence is amplified with primers incapable of amplifying contaminating DNA sequences of the polymerase.

Detection of second-line drug resistance in Mycobacterium tuberculosis using oligonucleotide microarrays.

BACKGROUND: The steady rise in the spread of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) requires rapid and reliable methods to identify resistant strains. The current molecular methods to detect MTB resistance to second-line drugs either do not cover an extended spectrum of mutations to be identified or are not easily implemented in clinical laboratories. A rapid molecular technique for the detection of resistance to second-line drugs in M. tuberculosis has been developed using hybridisation analysis on microarrays. METHODS: The method allows the identification of mutations within the gyrA and gyrB genes responsible for fluoroquinolones resistance and mutations within the rrs gene and the eis promoter region associated with the resistance to injectable aminoglycosides and a cyclic peptide, capreomycin. The method was tested on 65 M. tuberculosis clinical isolates with different resistance spectra that were characterised by their resistance to ofloxacin, levofloxacin, moxifloxacin, kanamycin and capreomycin. Also, a total of 61 clinical specimens of various origin (e.g., sputum, bronchioalveolar lavage) were tested. RESULTS: The sensitivity and specificity of the method in the detection of resistance to fluoroquinolones were 98% and 100%, respectively, 97% and 94% for kanamycin, and 100% and 94% for capreomycin. The analytical sensitivity of the method was approximately 300 genome copies per assay. The diagnostic sensitivity of the assay ranging from 67% to 100%, depending on the smear grade, and the method is preferable for analysis of smear-positive specimens. CONCLUSIONS: The combined use of the developed microarray test and the previously described microarray-based test for the detection of rifampin and isoniazid resistance allows the simultaneous identification of the causative agents of MDR and XDR and the detection of their resistance profiles in a single day.

Microarray with LNA-probes for genotyping of polymorphic variants of Gilbert's syndrome gene UGT1A1(TA)n.

BACKGROUND: Gilbert's syndrome is a common metabolic dysfunction characterized by elevated levels of unconjugated bilirubin in the bloodstream. This condition is usually caused by additional (TA) insertions in a promoter region of the uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) gene, which instead of the sequence А(TА)6TАА contains А(TА)7TАА. While the condition itself is benign, it presents elevated risk for patients treated with irinotecan, a common chemotherapy drug. METHODS: The technique is based on hybridization analysis of a pre-amplified segment of the UGT1A1 gene promoter performed on a microarray. Specific probes containing locked nucleic acids (LNA) were designed and immobilized on the microarray to provide accurate identification. RESULTS: A microarray has been developed to identify both common and rare variants of UGT1A1(TA)n polymorphisms. In total, 108 individuals were genotyped. Out of these, 47 (43.5%) had homozygous wild-type genotypes (TA)6/(TA)6; 41(38%) were heterozygotes (TA)6/(TA)7; and 18 (16.7%)--homozygotes (TA)7/(TA)7. In two cases (1.8%), rare genotypes (TA)5/(TA)7 and (TA)5/(TA)6 were found. The results were in full agreement with the sequencing. In addition, synthetic fragments corresponding to all human allelic variants [(TA)5, (TA)6, (TA)7, (TA)8] were successfully tested. CONCLUSIONS: The developed microarray-based approach for identification of polymorphic variants of the UGT1A1 gene is a promising and reliable diagnostic tool that can be successfully implemented in clinical practice.

Separate production of single-stranded DNA is not necessary: circuit denaturation of double-stranded DNA followed by hybridization of single strands on oligonucleotide microchips.

An approach to circuit renaturation-hybridization of dsDNA on oligonucleotide microchips is described. A close circuit cycling device has been developed, and the feasibility of the proposed technique was demonstrated on two platforms. First, a commercial microchip for detection of rifampicin resistance in Mycobacterium tuberculosis was used. Hybridization of a 126 nt long single-stranded DNA (ssDNA) fragment of the rpoB gene according to manufacturer's protocol has been compared to hybridization of the same double-stranded DNA (dsDNA) fragment using the developed approach. Hybridization signals obtained by both methods were comparable in intensity and correlated closely. Second, a 22 nt long hairpin-forming oligonucleotide was designed and hybridized with a custom microchip containing probes complementary to both strands of the oligonucleotide. Conventional hybridization of this oligonucleotide did not yield any significant signals. Cleavage of the hairpin loop resulted in the formation of a 9 bp long intermolecular duplex. Hybridization of the duplex using the suggested technique yielded strong signals. The proposed approach allows analyzing target DNA in double-stranded form bypassing the preparation of single-stranded targets. Moreover, both complementary chains could be analyzed simultaneously, providing a reliable internal control. Being combined with fragmentation this method opens new possibilities in analyzing ssDNA with complex secondary structure.

An oligonucleotide microarray for multiplex real-time PCR identification of HIV-1, HBV, and HCV.

We describe a novel microarray-based approach for simultaneous identification and quantification of human immunodeficiency virus type 1 (HIV-1) and hepatitis B and C viruses (HBV and HCV) in donor plasma specimens. The method is based on multiplex real-time RT-PCR performed within the microarray hydrogel pads. Double-stranded amplification products are simultaneously detected using nonspecific SYBR Green I dye due to the reaction run in separate pads bearing 5'-immobilized specific primers. Both the sensitivity and specificity of the assay, based on 132 blood specimens analyzed, were 100% (56, 26, and 8 specimens were seropositive to HBV HCV and HIV-1, respectively; 22 were positive to both HIV-1 and HCV and 2 positive to all three viruses; 18 samples were pathogen-negative). The dynamic range of the quantitative analysis covered a six-order interval ranging from 100 to 106 genome equivalents per assay. The 95% detection limits were 14 gEq for HIV-1, 10 gEq (1.7 IU) for HBV, and 15 gEq (7.5 IU) for HCV per assay. The proposed approach is considered to be versatile and could be adapted for simultaneous identification and quantification of numerous genetic targets.

Discrimination of Bacillus anthracis and closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microarray.

Analysis of 16S rRNA sequences is a commonly used method for the identification and discrimination of microorganisms. However, the high similarity of 16S and 23S rRNA sequences of Bacillus cereus group organisms (up to 99-100%) and repeatedly failed attempts to develop molecular typing systems that would use DNA sequences to discriminate between species within this group have resulted in several suggestions to consider B. cereus and B. thuringiensis, or these two species together with B. anthracis, as one species. Recently, we divided the B. cereus group into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, based on 16S rRNA, 23S rRNA and gyrB gene sequences and identified subgroup-specific makers in each of these three genes. Here we for the first time demonstrated discrimination of these seven subgroups, including subgroup Anthracis, with a 3D gel element microarray of oligonucleotide probes targeting 16S and 23S rRNA markers. This is the first microarray enabled identification of B. anthracis and discrimination of these seven subgroups in pure cell cultures and in environmental samples using rRNA sequences. The microarray bearing perfect match/mismatch (p/mm) probe pairs was specific enough to discriminate single nucleotide polymorphisms (SNPs) and was able to identify targeted organisms in 5min. We also demonstrated the ability of the microarray to determine subgroup affiliations for B. cereus group isolates without rRNA sequencing. Correlation of these seven subgroups with groupings based on multilocus sequence typing (MLST), fluorescent amplified fragment length polymorphism analysis (AFLP) and multilocus enzyme electrophoresis (MME) analysis of a wide spectrum of different genes, and the demonstration of subgroup-specific differences in toxin profiles, psychrotolerance, and the ability to harbor some plasmids, suggest that these seven subgroups are not based solely on neutral genomic polymorphisms, but instead reflect differences in both the genotypes and phenotypes of the B. cereus group organisms.

Oligonucleotide microchip for subtyping of influenza A virus.

BACKGROUND: Influenza A viruses are classified into subtypes depending on the antigenic properties of their two outer glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Sixteen subtypes of HA and nine of NA are known. Lately, the circulation of some subtypes (H7N7, H5N1) has been closely watched because of the epidemiological threat they present. OBJECTIVES: This study assesses the potential of using gel-based microchip technology for fast and sensitive molecular subtyping of the influenza A virus. METHODS: The method employs a microchip of 3D gel-based elements containing immobilized probes. Segments of the HA and NA genes are amplified using multiplex RT-PCR and then hybridized with the microchip. RESULTS: The developed microchip was validated using a panel of 21 known reference strains of influenza virus. Selected strains represented different HA and NA subtypes derived from avian, swine and human hosts. The whole procedure takes 10 hours and enables one to identify 15 subtypes of HA and two subtypes of NA. Forty-one clinical samples isolated during the poultry fall in Novosibirsk (Russia, 2005) were successfully identified using the proposed technique. The sensitivity and specificity of the method were 76% and 100%, respectively, compared with the 'gold standard' techniques (virus isolation with following characterization by immunoassay). CONCLUSIONS: We conclude that the method of subtyping using gel-based microchips is a promising approach for fast detection and identification of influenza A, which may greatly improve its monitoring.

Diversity of human immunodeficiency virus type 1 subtype A and CRF03_AB protease in Eastern Europe: selection of the V77I variant and its rapid spread in injecting drug user populations.

To characterize polymorphisms of the subtype A protease in the former Soviet Union, proviral DNA samples were obtained, with informed consent, from 119 human immunodeficiency virus type 1 (HIV-1)-positive untreated injecting drug users (IDUs) from 16 regions. All individuals studied have never been treated with antiretroviral drugs. The isolates were defined as IDU-A (n = 115) and CRF03_AB (n = 4) by using gag/env HMA/sequencing. The pro region was analyzed by using sequencing and original HIV-ProteaseChip hybridization technology. The mean of pairwise nucleotide distance between 27 pro sequences (23 IDU-A and 4 CRF03_AB) was low (1.38 +/- 0.79; range, 0.00 to 3.23). All sequences contained no primary resistance mutations. However, 13 of 23 (56.5%) subtype A isolates bore the V77I substitution known as the secondary protease mutation. V77I was associated with two synonymous substitutions in triplets 31 and 78, suggesting that all V77I-bearing viruses evolved from a single source in 1997. Hybridization analysis showed that 55 of 115 (47.8%) HIV-1 isolates contained V77I, but this variant was not found in any of 31 DNA samples taken from regions, where the HIV-1 epidemic among IDUs started earlier 1997, as well as in any of four CRF03_AB isolates. The results of analysis of 12 additional samples derived from epidemiologically linked subjects showed that in all four epidemiological clusters the genotype of the donor and the recipients was the same irrespective of the route of transmission. This finding demonstrates the transmission of the V77I mutant variant, which is spreading rapidly within the circulating viral pool in Russia and Kazakhstan. The continued molecular epidemiological and virological monitoring of HIV-1 worldwide thus remains of great importance.