Overview report on the application of double digest selective label (DDSL) bacteria genotyping technique for identification of strains and certification of commercially used bacteria.

The objective of this report is to demonstrate the potential of the proposed simple typing technique, double digest selective label (DDSL), which was initially developed to identify clinical isolates of Pseudomonas aeruginosa, for other bacterial species including Salmonella enterica, Clostridium difficile, Staphylococcus aureus, and Bacillus subtilis. The technique is based on digestion of bacterial genomic DNA with two restriction enzymes and simultaneous labeling fragments with biotinylated deoxycytidine triphosphate in fill-in reaction by Taq polymerase. The number and distribution of generated DNA fragments can be optimized by selecting restriction enzymes. DDSL is fast, reproducible, cost effective and sufficiently discriminatory typing method applicable for identification of bacterial strains at laboratories having no access to expensive sequencing equipment and with limited funding and lack of skilled personnel. Data concerning the potential of the technique for short-term epidemiological surveillance and bacterial strain certification are presented and discussed. Multiple locus variable number tandem repeat analysis performed on our set of Clostridium difficile isolates did not demonstrate sufficient discriminatory power both with TR6 and TR10 loci on a set of 24 isolates. In contrast, the DDSL analysis resolved all isolates into individual strains.

[An Efficient Method for Genetic Certification of Bacillus subtilis strains, Prospective Producers of Biopreparations].

Genetic certification of commercial strains of bacteria antagonistic to phytopathogenic microorganisms guarantees their unequivocal identification and confirmation of safety. In Russia, unlike EU countries, genetic certification of Bacillus subtilis strains is not used. Based on the previously proposed double digestion selective label (DDSL) fingerprinting, a method for genetic identification and certification of B. subtilis strains was proposed. The method was tested on several strains differing in their physiological and biochemical properties and in the composition of secondary metabolites responsible for the spectrum of antibiotic activity. High resolving power of this approach was shown. Optimal restriction endonucleases (SgsI and Eco32I) were determined and validated. A detailed protocol for genetic certification of this bacterial species was developed. DDSL is a universal method, which may be adapted for genetic identification and certification of other bacterial species.

Molecular epidemiology of Pseudomonas aeruginosa in intensive care units over a 10-year period (1998-2007).

Pseudomonas aeruginosa is one of the leading nosocomial pathogens in intensive care units (ICUs). The source of this microorganism can be either endogenous or exogenous. The proportion of cases as a result of transmission is still debated, and its elucidation is important for implementing appropriate control measures. To understand the relative importance of exogenous vs. endogenous sources of P. aeruginosa, molecular typing was performed on all available P. aeruginosa isolated from ICU clinical and environmental specimens in 1998, 2000, 2003, 2004 and 2007. Patient samples were classified according to their P. aeruginosa genotypes into three categories: (A) identical to isolate from faucet; (B) identical to at least one other patient sample and not found in faucet; and (C) unique genotype. Cases in categories A and B were considered as possibly exogenous, and cases in category C as possibly endogenous. A mean of 34 cases per 1000 admissions per year were found to be colonized or infected by P. aeruginosa. Higher levels of faucet contamination were correlated with a higher number of cases in category A. The number of cases in category B varied from 1.9 to 20 cases per 1000 admissions. This number exceeded 10/1000 admissions on three occasions and was correlated with an outbreak on one occasion. The number of cases considered as endogenous (category C) was stable and independent of the number of cases in categories A and B. The present study shows that repeated molecular typing can help identify variations in the epidemiology of P. aeruginosa in ICU patients and guide infection control measures.

Application and evaluation of double digest selective label (DDSL) typing technique for Pseudomonas aeruginosa hospital isolates.

This study describes the application and evaluation of a recently developed fast bacterial typing technique (double digest selective label - DDSL) for hospital isolates of Pseudomonas aeruginosa. The protocol was based on a simultaneous double digestion/labelling reaction which was performed in a single reaction tube. After agarose gel separation selectively tagged restriction fragments were transferred using deonised water to a nylon membrane and visualized by a colour reaction. Starting from overnight culture, turn around time using this technique was only 8 h. The DDSL typing technique was applied for 77 hospital isolates. Among them 63 isolates were also typed by PFGE and the typing results were compared with those of DDSL. In conclusion, both techniques discriminated bacterial isolates into the same major clusters. DDSL proved to be as discriminatory as PFGE but much faster and easier to set up in a standard microbiological laboratory.