Bacterial infections of central venous catheters (CVCs) cause much morbidity and mortality, and are usually diagnosed by concordant culture of blood and catheter tip. However, studies suggest that culture often fails to detect biofilm bacteria. This study optimizes X-ray micro-focus computed tomography (X-ray µCT) for the quantification and determination of distribution and heterogeneity of biofilms in in vitro CVC model systems.
Bacterial culture and scanning electron microscopy (SEM) were used to detect Staphylococcus epidermidis ATCC 35984 biofilms grown on catheters in vitro in both flow and static biofilm models. Alongside this, X-ray µCT techniques were developed in order to detect biofilms inside CVCs. Various contrast agent stains were evaluated using energy-dispersive X-ray spectroscopy (EDS) to further optimize these methods. Catheter material and biofilm were segmented using a semi-automated matlab script and quantified using the Avizo Fire software package. X-ray µCT was capable of distinguishing between the degree of biofilm formation across different segments of a CVC flow model. EDS screening of single- and dual-compound contrast stains identified 10 nm gold and silver nitrate as the optimum contrast agent for X-ray µCT. This optimized method was then demonstrated to be capable of quantifying biofilms in an in vitro static biofilm formation model, with a strong correlation between biofilm detection via SEM and culture. X-ray µCT has good potential as a direct, non-invasive, non-destructive technology to image biofilms in CVCs, as well as other in vivo medical components in which biofilms accumulate in concealed areas.
Citrobacter rodentium is a Gram-negative, murine-specific enteric pathogen that infects epithelial cells in the colon. It is closely related to the clinically relevant human pathogen, enterohemorrhagic Escherichia coli (EHEC), a leading cause of haemorrhagic colitis and haemolytic uremic syndrome. We have previously reported that a novel antimicrobial peptide, wrwycr, compromises bacterial DNA repair and significantly reduces the survival of acid-stressed EHEC, suggesting an antimicrobial strategy for targeting the survival of ingested EHEC. This study examines the impact of peptide pretreatment on survival of the closely related murine pathogen, C. rodentium, before and after acid stress, using both in vitro and in vivo investigations. Peptide pretreatment of C. rodentium significantly and dramatically increases acid-stress-induced killing in a peptide-dose-dependent and time-dependent manner. Reduction in survival rates after brief pretreatment with peptide (25–65 µM) followed by 1 h at pH 3.5 ranges from 6 to 8 log fold relative to untreated C. rodentium, with no detectable bacteria after 65 µM peptide-acid treatment. Using a C57BL/6 mouse model of infection, peptide pretreatment of C. rodentium with wrwycr prior to orogastric gavage eliminates evidence of infection based on C. rodentium colonization levels, faecal scores, colonic histology, faecal microbiome and visual observation of overall animal health. These findings provide compelling evidence for the role of the peptide wrwycr as a potential strategy to control the growth and colonization of enteric pathogens.