Expression of icaA and icaD genes by quantitative real-time PCR in correlation to biofilm synthesis among methicillin-resistant coagulase-negative staphylococci

Abstract number: 1733_395

Foka A., Chini V., Petinaki E., Dimitracopoulos G., Spiliopoulou I.

Objectives: Among pathogens causing hospital infections methicillin-resistant coagulase–negative staphylococci (MR-CNS) have become predominant, especially in neonatal intensive care units (nICU). This results their ability to form biofilm, especially among patients with invasive procedures. The purpose of the present study was to investigate slime production in correlation to the presence and expression of ica operon among MR-CNS isolated from different patients hospitalised in a nICU.

Methods: During a two-year period 132 (MR-CNS) were isolated from different inpatients at the nICU. Biofilm production was detected by the qualitative and quantitative methods. PCR was performed for the detection of the four genes of the ica operon, (icaA, icaD, icaB, icaC) and of the insertion sequence element IS256. Fourteen representative strains, 11 slime-positive and three slime-negative, were selected for reverse transcription quantitative relative Real time PCR (qRT-PCR) for the detection of the expression levels of icaA and icaD genes. Total RNA was isolated by the Trizol method, and a part of the 23S rRNA was used as the reference gene in the relative quantification of ica genes using the SYBR® Green I chemistry; results were calculated by the Pfaffl method.

Results: Both methods for bioflilm formation revealed that 117 (89%) isolates were biofilm-positive. PCR showed a variation of possession of ica genes. The selected 14 strains were positive for icaA and icaD by PCR screening. Seven out of the 11 slime-positive strains carried also IS256. Among them, two showed high, two medium and three strains low expression levels of both ica genes by qRT-PCR. In the remaining four slime-positive strains that did not carry IS256, two showed medium and two low expression levels of both ica genes. Two out of the three slime-negative MR-CNS possessed IS256 and all three had low expression levels of ica genes.

Conclusions: The majority of MR-CNS associated with infections in the nICU produced biofilm. The insertion sequence element IS256 does not always inactivate the expression of ica operon. Other factors are involved in biofilm formation.

Session Details

Date: 31/03/2007
Time: 00:00-00:00
Session name: European Society of Clinical Microbiology and Infectious Diseases
Location: ICC, Munich, Germany
Presentation type:
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