| dc.description.abstract | Central venous catheters (CVC) are a known source of nosocomial blood
stream infections. However, standard cultivation methods to identify causal
pathogens of catheter-related blood stream infections (CRBSI) on catheters often
fail. In this study, I compare the traditional cultivation method to the cultivation-independent
PCR-DGGE method to examine bacterial colonization on central
venous catheters retrieved from cancer patients.
In order to study the biofilm communities that colonize the catheters, an
optimized sonication protocol was developed to remove biofilm bacteria from
their substratum. I showed that a sonicating power of 12 Watts for 5 minutes
could remove bacteria, such as Pseudomonas putida and Staphylococcus
epidermidis cells, from a glass surface without killing the bacteria.
Twenty-four catheters were retrieved from cancer patients and used in this
study. Five out of the 24 catheter samples (21%) showed growth in at least one
of the culture media used. The isolates recovered from the catheters belonged to
five bacterial species, including Staphylococcus aureus, S. epidermidis, S.
hominis, a Staphylococcus sp., and Streptococcus agalactica.
The PCR-DGGE method showed that 100% of the catheters were
colonized by bacteria. Furthermore, unlike the cultivation assay demonstrated
that contaminated catheters were colonized by only one or two bacterial species,
DGGE analysis showed that all the catheters in this study possessed a mixed-bacterial
community of at least 4 bacterial species. By analyzing the sequences of the DGGE bands, Enterococcus faecalis (100%), E. faecium (96%) and
Roseomonas sp. (88%) were the most predominant species detected on the
catheters. Staphylococcus epidermidis, S. hominis, S. aureus, Corynebacterium
sp. and Serratia sp. were detected on 30-54% of the catheter samples. Other
bacterial species, such as E. coli, Aeroccous sp.. Micrococcus sp. and alpha
proteobacterium were detected on less than 30% of the catheters. In addition,
scanning electron microscopy (SEM) confirmed the DGGE findings in that
biofilms were found on all the catheter samples. Biofilms were found in the
lumens of the catheters but rarely associate with the outer surface.
The catheters were rinsed prior to the sonication process and the rinsing
buffers were also analyzed by culturing and DGGE assays as described for the
catheter samples. None of the buffer samples showed any positive growth in the
growth media but all of them showed positive amplification by a pair of
eubacterial universal primers and showed similar DGGE profiles as their
respective catheter samples, showing that even gentle rinsing could remove
enough biofilm cells for PCR-DGGE analysis.
In conclusion, the PCR-DGGE method is a superior method in identifying
biofilm microbial communities on catheters. This method could detect and identify
“viable but non-culturable” bacteria that were missed by the conventional
cultivation methods. Furthermore, this study also revealed that some bacterial
species (such as Enterococcus, Roseomonas spp. and Serratia spp.), that have
yet been recognized as a major cause of catheter related infections, can
potentially be important catheter-associated pathogens. | |
