Luc Bissonnette

On the evolution of Tn21-like multiresistance transposons: Sequence analysis of the gene (aacC1) for gentamicin acetyltransferase-3-I(AAC(3)-I), another member of the Tn21-based expression cassette

SummaryThe aminoglycoside-3-O-acetyltransferase-I gene (aacC1) from R plasmids of two incompatibility groups (R1033 [Tn1696], and R135) was cloned and sequenced. In the case of R1033, it was shown that theaacC gene is coded by a precise insertion of 833 bp between theaadA promoter and its structural gene in a Tn21 related transposon (Tn1696). This insertion occurs at the same target sequence as that of the OXA-1 β-lactamase gene insertion in Tn2603. Upstream of theaacC gene, we found an open reading frame (ORF) which is probably implicated in the site-specific recombinational events involved in the evolution of this family of genetic elements. These results provide additional confirmation of the role of Tn21 elements as naturally occurring interspecific transposition and expression casssettes.

Significant Linkage for Tourette Syndrome in a Large French Canadian Family

Family and twin studies provide strong evidence that genetic factors are involved in the transmission of Gilles de la Tourette syndrome (TS) and related psychiatric disorders. To detect the underlying susceptibility gene(s) for TS, we performed linkage analysis in one large French Canadian family (127 members) from the Charlevoix region, in which 20 family members were definitely affected by TS and 20 others showed related tic disorders. Using model-based linkage analysis, we observed a LOD score of 3.24 on chromosome 11 (11q23). This result was obtained in a multipoint approach involving marker D11S1377, the marker for which significant linkage disequilibrium with TS recently has been detected in an Afrikaner population. Altogether, 25 markers were studied, and, for level of significance, we derived a criterion that took into account the multiple testing arising from the use of three phenotype definitions and three modes of inheritance, a procedure that yielded a LOD score of 3.18. Hence, even after adjustment for multiple testing, the present study shows statistically significant evidence for genetic linkage with TS.

Diagnosing infections—current and anticipated technologies for point-of-care diagnostics and home-based testing

In recent years, we have witnessed many transitions in healthcare systems around the globe. For example, population expansion and ageing, and the human immunodeficiency virus (HIV)-AIDS epidemics, have exerted pressure to decentralize the practice of healthcare outside of traditional settings to bring care to those in need. Upstream of patient management, diagnosis is aimed at adequately orienting medical decisions, and considerable efforts have been made to make this process faster and more efficient. However, there are several diseases and medical conditions that may/will benefit from technologies and tests that can be performed closer to the patient, at the point of care or even in the home. In this review, and in light of the paradox that technology and assay developers and healthcare officials must take into consideration for advancing human health in developed and developing countries, we present an overview of rapid diagnosis of infectious diseases at the point of care and of technologies that may contribute to enhancement of the worldwide point-of-care testing market.

Analytical comparison of nine PCR primer sets designed to detect the presence of Escherichia coli/Shigella in water samples

The analytical performance of 9 different PCR primer sets designed to detect Escherichia coli and Shigella in water has been evaluated in terms of ubiquity, specificity, and analytical detection limit. Of the 9 PCR primer sets tested, only 3 of the 5 primer sets targeting uidA gene and the primer set targeting tuf gene amplified DNA from all E. coli strains tested. However, of those 4 primer sets, only the primer set targeting the tuf gene also amplified DNA from all Shigella strains tested. For the specificity, only the primer sets targeting the uidA gene were 100% specific although the primer sets targeting 16S rRNA, phoE, and tuf genes only amplified Escherichia fergusonii as non-specific target. Finally, the primer set targeting the 16S-ITS-23S gene region, was not specific as it amplified DNA from many other Enterobacteriaceae species. In summary, only the assay targeting the tuf gene detected all E. coli/Shigella strains tested in this study. However, if it becomes important to discriminate between E. coli and E. fergusonii, assays targeting the uidA gene would represent a good choice although none of them were totally ubiquitous to detect of the presence of Shigella strains.

Detection of target DNA using fluorescent cationic polymer and peptide nucleic acid probes on solid support

BackgroundNucleic acids detection using microarrays requires labelling of target nucleic acids with fluorophores or other reporter molecules prior to hybridization.ResultsUsing surface-bound peptide nucleic acids (PNA) probes and soluble fluorescent cationic polythiophenes, we show a simple and sensitive electrostatic approach to detect and identify unlabelled target nucleic acid on microarray.ConclusionThis simple methodology opens exciting possibilities for applied genetic analysis for the diagnosis of infections, identification of genetic mutations, and forensic inquiries. This electrostatic strategy could also be used with other nucleic acid detection methods such as electrochemistry, silver staining, metallization, quantum dots, or electrochemical dyes.

Next revolution in the molecular theranostics of infectious diseases: microfabricated systems for personalized medicine

The molecular diagnosis of infectious diseases is currently going through a revolution sustained by the regulatory approval of amplification tests that have been shown to be equivalent or superior to existing gold standard methods. The recent approval of a microarray system for the pharmacogenomic profiling of cytochrome P450-mediated drug metabolism is paving the way to novel, rapid, sensitive, robust and economical microfabricated systems for point-of-care diagnostics, which are utilized closer and closer to the patient’s bedside. These systems will enable the multiparametric genetic evaluation of several medical conditions, including infectious diseases. This forecoming revolution will position molecular theranostics in a broader integrated view of personalized medicine, which exploits genetic information from microbes and human hosts to optimize patient management and disease treatment.

Analytical limits of four β-glucuronidase and β-galactosidase-based commercial culture methods used to detect Escherichia coli and total coliforms

Colilert (Colilert), Readycult Coliforms 100 (Readycult), Chromocult Coliform agar ES (Chromocult), and MI agar (MI) are beta-galactosidase and beta-glucuronidase-based commercial culture methods used to assess water quality. Their analytical performance, in terms of their respective ability to detect different strains of Escherichia coli and total coliforms, had never been systematically compared with pure cultures. Here, their ability to detect beta-glucuronidase production from E. coli isolates was evaluated by using 74 E. coli strains of different geographic origins and serotypes encountered in fecal and environmental settings. Their ability to detect beta-galactosidase production was studied by testing the 74 E. coli strains as well as 33 reference and environmental non-E. coli total coliform strains. Chromocult, MI, Readycult, and Colilert detected beta-glucuronidase production from respectively 79.9, 79.9, 81.1, and 51.4% of the 74 E. coli strains tested. These 4 methods detected beta-galactosidase production from respectively 85.1, 73.8, 84.1, and 84.1% of the total coliform strains tested. The results of the present study suggest that Colilert is the weakest method tested to detect beta-glucuronidase production and MI the weakest to detect beta-galactosidase production. Furthermore, the high level of false-negative results for E. coli recognition obtained by all four methods suggests that they may not be appropriate for identification of presumptive E. coli strains.

Rapid Concentration and Molecular Enrichment Approach for Sensitive Detection of Escherichia coli and Shigella Species in Potable Water Samples

ABSTRACT In this work, we used a rapid, simple, and efficient concentration-and-recovery procedure combined with a DNA enrichment method (dubbed CRENAME [concentration and recovery of microbial particles, extraction of nucleic acids, and molecular enrichment]), that we coupled to an Escherichia coli/Shigella-specific real-time PCR (rtPCR) assay targeting the tuf gene, to sensitively detect E. coli/Shigella in water. This integrated method was compared to U.S. Environmental Protection Agency (EPA) culture-based Method 1604 on MI agar in terms of analytical specificity, ubiquity, detection limit, and rapidity. None of the 179 non-E. coli/Shigella strains tested was detected by both methods, with the exception of Escherichia fergusonii, which was detected by the CRENAME procedure combined with the E. coli/Shigella-specific rtPCR assay (CRENAME + E. coli rtPCR). DNA from all 90 E. coli/Shigella strains tested was amplified by the CRENAME + E. coli rtPCR, whereas the MI agar method had limited ubiquity and detected only 65 (72.2%) of the 90 strains tested. In less than 5 h, the CRENAME + E. coli rtPCR method detected 1.8 E. coli/Shigella CFU whereas the MI agar method detected 1.2 CFU/100 ml of water in 24 h (95% confidence). Consequently, the CRENAME method provides an easy and efficient approach to detect as little as one Gram-negative E. coli/Shigella cell present in a 100-ml potable water sample. Coupled with an E. coli/Shigella-specific rtPCR assay, the entire molecular procedure is comparable to U.S. EPA Method 1604 on MI agar in terms of analytical specificity and detection limit but provides significant advantages in terms of speed and ubiquity.

Infectious Disease Management through Point-of-Care Personalized Medicine Molecular Diagnostic Technologies

Infectious disease management essentially consists in identifying the microbial cause(s) of an infection, initiating if necessary antimicrobial therapy against microbes, and controlling host reactions to infection. In clinical microbiology, the turnaround time of the diagnostic cycle (>24 hours) often leads to unnecessary suffering and deaths; approaches to relieve this burden include rapid diagnostic procedures and more efficient transmission or interpretation of molecular microbiology results. Although rapid nucleic acid-based diagnostic testing has demonstrated that it can impact on the transmission of hospital-acquired infections, we believe that such life-saving procedures should be performed closer to the patient, in dedicated 24/7 laboratories of healthcare institutions, or ideally at point of care. While personalized medicine generally aims at interrogating the genomic information of a patient, drug metabolism polymorphisms, for example, to guide drug choice and dosage, personalized medicine concepts are applicable in infectious diseases for the (rapid) identification of a disease-causing microbe and determination of its antimicrobial resistance profile, to guide an appropriate antimicrobial treatment for the proper management of the patient. The implementation of point-of-care testing for infectious diseases will require acceptance by medical authorities, new technological and communication platforms, as well as reimbursement practices such that time- and life-saving procedures become available to the largest number of patients.

Method for rapid and sensitive detection of Enterococcus sp. and Enterococcus faecalis/faecium cells in potable water samples

We have developed a rapid and robust technological solution including a membrane filtration and dissolution method followed by a molecular enrichment and a real-time PCR assay, for detecting the presence of Enterococcus sp. or Enterococcus faecalis/faecium per 100 mL of water in less than 5 h and we compared it to Method 1600 on mEI agar in terms of specificity, sensitivity, and limit of detection. The mEI and the Enterococcus sp.-specific assay detected respectively 73 (64.0%) and 114 (100%) of the 114 enterococcal strains tested. None of the 150 non-enterococcal strains tested was detected by both methods with the exception of Tetragenococcus solitarius for the Enterococcus sp. assay. The multiplexed E. faecalis/faecium assay efficiently amplified DNA from 47 of 47 (100%) E. faecalis and 27 of 27 (100%) E. faecium strains tested respectively, whereas none of the 191 non-E. faecalis/faecium strains tested was detected. By simultaneously detecting the predominant fecal enterococcal species, the E. faecalis/faecium-specific assay allows a better distinction between enterococcal strains of fecal origin and those provided by the environment than Method 1600. Our procedure allows the detection of 4.5 enterococcal colony forming units (CFU) per 100 mL in less than 5 h, whereas the mEI method detected 2.3 CFU/100 mL in 24 h (95% confidence). Thus, our innovative and highly effective method provides a rapid and easy approach to concentrate very low numbers of enterococcal cells present in a 100 mL water sample and allows a better distinction between fecal and environmental enterococcal cells than Method 1600.