A novel in situ method for characterizing catheter-associated biofilm formation Elliott W. Welker, Tanner J. Barnharst, Bridget E. Eklund, and Nathan A. Fisher North Dakota State University, Department of Veterinary and Microbiological Sciences, Fargo, ND Isolate Information Isolate ATCC Source Acinetobacter baumannii 19606 Urine Acinetobacter baumannii BAA-­‐747 Ear Pus Acinetobacter species 49139 Clinical Isolate Enterobacter cloacae 35030 Quality Control Strain Enterobacter cloacae 13047 Spinal Fluid Enterobacter cloacae 23355 None Speciﬁed Klebsiella pneumoniae 33495 Human Urinary Tract Klebsiella pneumoniae 700603 Urine Klebsiella pneumoniae 35657 Qualty Control Strain Pseudomonas aeruginosa 19429 Urine Pseudomonas aeruginosa 35554 Qualty Control Strain Pseudomonas aeruginosa 27853 Blood Culture Stenotrophomonas maltophilia K279a Blood Stenotrophomonas maltophilia D457R Branchial aspirate Stenotrophomonas maltophilia 2004-­‐24-­‐121 PaNent Table 1 The isolates were obtained from the American Type Culture Collection (ATCC). Isolates were selected based on their ability to form varying strengths of biofilms. Methods A panel of gram negative nosocomial bacteria were streaked for isolation onto Luria Bertani (LB) agar plates. Broth cultures were created in tryptic soy broth (TSB). 24G x ¾“ intravenous catheters were submerged in overnight broth cultures for two hours to facilitate attachment. Catheters were implanted inside the abdomen of the Blaptica dubia cockroach and incubated for 24 hours at 37°C for the in situ assay . Catheters were incubated in TSB broth at 37°C for 24 hours for the in vitro assay. The catheters were then removed or extracted from their respective incubation locations then washed with a saline solution. The catheters were placed in a solution of crystal violet for 15 minutes, washed with saline, then solubilized in ethanol. Aliquots were transferred to microplate and read at 600 nm. Fig. 4 Site of catheter insertion Fig. 3 Teflon intravenous catheter components Our research goal is to compare biofilm growth of several nosocomial isolates on intravenous catheters using a Blaptica dubia cockroach as a host. This novel method will also be compared to biofilm growth on the catheter using an in vitro model. Crystal violet stain will be used as a method to quantitatively asses biofilm growth. We hypothesize that the physiology of biofilm formation varies in an in vivo environment compared to in vitro due to immunological pressures. Fig. 2 Representative MicroCT images of a i n t r a v e n o u s c a t h e t e r i m p l a n t e d i n t o t h e abdominal cavity of Blaptica d u b i a . I m a g e s w e r e captured using the GE v (tome) x 240 kV microfocus X-ray computer topography system at the NDSU electron microscopy center. Panel A and B show lateral and longitudinal sections respectively. Panel C shows a computer 3-D model. Together these images show that the implanted catheters (the arrow) are positioned directly beneath the roach epidermis and that internal organs are not punctured during the implantation process. Fig. 1 Representative SEM images of intravenous catheters colonized by Stenotrophomonas maltophilia in the roach in situ model. Images were captured using a JOEL JSM-7600F scanning electron microscope at the NDSU electron microscopy center. Panel A shows biofilm accumulation (arrowhead) on the internal and external walls of the catheter. Panels B and C show the structure of biofilms including bacterial cells embedded within the extracellular polymeric substance (EPS), complex columnar architecture (COL), and association with insect immune cells, termed hemocytes (HE). Results & Discussion Fig. 5 results of Stenotrophomonas maltophilia in vitro and in vivo catheter biofilm formation The Stenotrophomonas maltophilia strains (Fig. 5) form statistically different amounts of biofilm in the in vitro and in vivo environments and in one strain (Sm 2004-24-121), the in vivo growth was markedly increased.