Comparison of two non-invasive methods of microbial analysis in surgery practice: incision swabbing and the indirect imprint technique.

BACKGROUND: A variety of methods exist to take samples from surgical site infections for cultivation; however, an unambiguous and suitable method has not yet been defined. The aim of our retrospective non-randomized study was to compare two non-invasive techniques of sampling material for microbiologic analysis in surgical practice. We compared bacteria cultured from samples obtained with the use of the swab technique, defined in our study as the gold standard, with the indirect imprint technique. METHODS: A cotton-tipped swab (Copan, Brescia, Italy) was used; the imprints were taken using Whatman no. 4 filter paper (Macherey-Nagal, Duren, Germany) cut into 5×5 cm pieces placed on blood agar in a Petri dish. To culture the microorganisms in the microbiology laboratory, we used blood agar, UriSelect 4 medium (Bio-Rad, Marnes-la-Coquette, France), and a medium with sodium chloride (blood agar with salt). After careful debridement, a sample was taken from the incision surface by swab and subsequently the same area of the surface was imprinted onto filter paper. The samples were analyzed in the microbiology laboratory under standard safety precautions. The cultivation results of the two techniques were processed statistically using contingency tables and the McNemar test. Those samples that were simultaneously cultivation-positive by imprint and -negative by swabbing were processed in greater detail. RESULTS: Over the period between October 2008 and March 2013, 177 samples from 70 patients were analyzed. Sampling was carried out from 42 males and 28 females. One hundred forty-six samples were from incisions after operations (21 samples from six patients after operation on the thoracic cavity, 73 samples from 35 patients after operation on the abdominal cavity combined with the gastrointestinal tract, 52 samples from 19 patients with other surgical site infections not included above) and 31 samples from 11 patients with no post-operative infection. One patient had a sample taken both from a post-operative and a non-post-operative site. Coincidently, the most frequent cultivation finding with both techniques was a sterile one (imprint, 62; swab, 50). The microorganism cultivated most frequently after swabbing was Pseudomonas aeruginosa (22 cases), compared with Escherichia coli when the filter paper (imprint) was used (31 cases). The imprint technique was evaluated as more sensitive compared with swabbing (p=0.0001). The κ statistic used to evaluate the concordance between the two techniques was 0.302. Of the 177 samples there were 53 samples simultaneously sterile using the swab and positive in the imprint. In three samples colony- forming units (CFU) were not counted; 22 samples were within the limit of 0-25×10(1) CFU/cm(2), 20 samples within the limit of 25×10(1)-25×10(2) CFU/cm(2), five within the limit of 25×10(2)-25×10(3) CFU/cm(2), and three of more than 25×10(4) CFU/cm(2). CONCLUSIONS: The hypothesis of swabbing as a more precise technique was not confirmed. In our study the imprint technique was more sensitive than swabbing; the strength of agreement was fair. We obtained information not only on the type of the microorganism cultured, but also on the number of viable colonies, expressed in CFU/cm(2).

The differences in the isoelectric points of biofilm-positive and biofilm-negative Candida parapsilosis strains.

The isoelectric points of 39 Candida parapsilosis strains were determined by means of capillary isoelectric focusing. The value of the isoelectric point corresponded well with cell surface hydrophobicity, as well as with the ability to form biofilm in these yeasts.

[The yeast biofilm in human medicine]. / Kvasinkový biofilm v humánní medicíne.

In recent years, the role of Candida yeasts as causative agents of nosocomial infections has increased. One of the important virulence factors contributing to the development of such infections is biofilm production. This virulence factor enables yeast to colonize both native surfaces and artificial implants. The most common sources of infection are patients themselves, in particular the gastrointestinal tract and skin. The vectors of exogenous yeast infections are predominantly the hands of the health personnel and contaminated medical instruments. The adhesion of yeasts to the implant surfaces is determined both by implant surface and yeast characteristics. This is followed by proliferation and production of microcolonies and extracellular matrix. The final biofilm structure is also influenced by the production of hyphae and pseudohyphae. The entire process of biofilm production is controlled by numerous regulatory systems, with the key role being played by the quorum sensing system. Like the adhered bacterial cultures, candidas growing in the form of a biofilm are highly resistant to antimicrobial therapy. Resistance of yeast biofilms to antifungals is a complex process with multiple contributing factors. These are especially increased gene expression (e.g. genes encoding the so called multidrug efflux pumps), limited penetration of substances through the extracellular matrix, inhibited cell growth and altered microenvironment in deeper biofilm layers. The concentrations of antifungals able to effectively affect the biofilm cells exceed, by several orders of magnitude, the values of conventionally determined MICs. High biofilm resistance results in ineffective antifungal therapy of biofilm infections. Therefore, if possible, the colonized implant should be removed. Conservative therapy should involve antifungals with a proven effect on the biofilm (e.g. caspofungin). The most effective measure in fighting biofilm infections is prevention, especially adhering to aseptic techniques when manipulating with implants and their correct maintenance.

Capillary isoelectric focusing--useful tool for detection of the biofilm formation in Staphylococcus epidermidis.

The biofilm formation is an important factor of S. epidermidis virulence. Biofilm-positive strains might be clinically more important than biofilm-negative ones. Unlike biofilm-negative staphylococci, biofilm-positive staphylococci are surrounded with an extracellular polysaccharide substance. The presence of this substance on the surface can affect physico-chemical properties of the bacterial cell, including surface charge. 73 S. epidermidis strains were examined for the presence of ica operon, for the ability to form biofilm by Christensen test tube method and for the production of slime by Congo red agar method. Isoelectric points (pI) of these strains were determined by means of Capillary Isoelectric Focusing. The biofilm negative strains focused near pI value 2.3, while the pI values of the biofilm positive strains were near 2.6. Isoelectric point is a useful criterion for the differentiation between biofilm-positive and biofilm-negative S. epidermidis strains.

[Determination of sensitivity of biofilm-positive forms of microorganisms to antibiotics]. / Stanovení citlivosti k antibiotikum u biofilmpozitivních forem mikrooganizmu.

Nosocomial infections caused by biofilm-positive microorganisms are a serious therapeutic problem. In the biofilm, microorganisms are protected against adverse effects of the external environment, including the action of antibiotics. It is well known that the values of minimum inhibitory concentrations (MIC) determined for planktonic forms do not correspond to the actual concentrations of antibiotics necessary for the eradication of bacteria in a biofilm. The purpose of the study was to propose a method of determining minimum biofilm inhibitory concentrations (MBIC) and minimum biofilm eradication concentrations (MBEC) and to compare these values with MIC values. Biofilm-positive strains of Staphylococcus epidermidis were cultured so as to form a biofilm layer on polystyrene pegs. The biofilm on the pegs was then exposed to the action of antibiotics and after 18 hours we determined the minimum biofilm inhibitory concentration (MBIC). The evaluation of minimum biofilm eradication concentrations was done colorimetrically from the metabolic activity of surviving cells. MBIC and MBEC values were many times higher than MIC values. We selected such a duration of the biofilms cultivation on the pegs of the plate, which ensured that the number of bacterial cells corresponded to standard MIC assessment. The MBEC values established in our study indicate that the currently used concentrations of tested antibiotics cannot be used in monotherapy for an efficacious eradication of a biofilm. The MBEC determination is a far more laborious and time-consuming method than the determination of MIC, but the use of plates with pegs facilitates the handling of biofilms. The advantage of our method is the possibility of standardization of the size of the inoculum and thus of the whole MBEC assessment.