Ultrastructure of a novel bacterial form located in Staphylococcus aureus in vitro and in vivo catheter-associated biofilms

Abstract

Bacterial biofilms are ubiquitous in nature, industry, and medicine, and understanding their development and cellular structure is critical in controlling the unwanted consequences of biofilm growth. Here, we report the ultrastructure of a novel bacterial form observed by scanning electron microscopy in the luminal vegetations of catheters from patients with active Staphylococcus aureus bacteremia. This novel structure had the general appearance of a normal staphylococcal cell but up to 10 to 15 times as large. Transmission electron microscopy indicated that these structures appeared as sacs enclosing multiple normal-sized (~0.6 µm) staphylococcal forms. Using in vitro cultivated biofilms, cytochemical studies using fluorescent reagents revealed that these structures were rich in lipids and appeared within 15 min after S. aureus inoculation onto clinically relevant abiotic surfaces. Because they appeared early in biofilm development, these novel bacterial forms may represent an unappreciated mechanism for biofilm surface adherence, and their prominent lipid expression levels could explain the perplexing increased antimicrobial resistance of biofilm-associated bacteria.

Figure 1.

Scanning electron microscopy (SEM) images of Staphylococcus aureus RN6390 (A–G, I, J) and ATCC 25923 (H, K, L) after inoculation onto glass or silicone coupons, showing large bacterial forms, some covered with short, fuzzy fibrillar material (H–L), after incubation for 15 min (A, C, E), 2 hr (H), 8 hr (G, I, J), 16 hr (D), or 48 hr (B, F, K, L) in tryptic soy broth with glucose (B–D, F–K), Hank’s balanced salt solution (A), or phosphate-buffered saline (E). (J, L) Higher magnifications of a portion of the images in I and K, respectively. Scale bar = 3 µm.

Figure 2.

Staphylococcus aureus RN6390 (A–D, F) and ATCC 25923 (E) incubated 15 min in Hank’s balanced salt solution on glass slides. Each row presents a differential interference contrast microscopy image followed by corresponding maximum intensity projections from deconvoluted z-stacks localizing cell surface material (red), DNA (blue), and the neutral lipid (green), followed by an overlay to emphasize the lipid as a hallmark of the large bacteria-like forms. Row A is a typical early biofilm containing cells in a matrix with scant lipids. Rows B–E contain large bacterial forms that stain prominently with the neutral lipid. Row D contains a circular form leaking internal contents; note the inset in the image highlighting the neutral lipid alone where a portion of the adjacent overlay has been placed to more clearly show staining for cell surface binding of wheat germ agglutinin and DNA in the isolated diplococcus on the left of the inset, while two coccal forms remain unstained within the periphery of the circular lipid structure on the left of the inset. Row F contains a sample stained for cell surface carbohydrates and DNA before staining for lipids, with the initial red and blue staining not evident where bacteria are covered with lipids, supporting the concept that the red and blue staining cannot penetrate into cocci covered with the neutral lipid. With the exception of the inset, all images are the same magnification. Scale bar = 5 µm.

Figure 3.

Phase contrast images of Staphylococcus aureus ATCC 25923 (A, B) and RN6390 (C, D) incubated for 15 min in Hank’s balanced salt solution on glass slides and then stained with Oil Red O (for neutral lipids), showing red staining only on the large bacterial structures. (A, C) Diplococcal-appearing forms. (D) A chain of cocci with arrows highlighting what appear to be cross-walls (similar topography to scanning electron microscopy images). These data confirm that the lipid is a unique hallmark of the large bacterial forms. Scale bars = 2 µm.

Figure 4.

Ultrastructure of vegetation in a silicone catheter removed (after 2 weeks in vivo) from a patient with active Staphylococcus aureus bacteremia. (A) Low-magnification scanning electron microscopy (SEM) image of catheter vegetation. (B–E) SEM images of vegetation showing large coccal forms, similar to the forms in Figure 1, with B containing coccal forms of varying sizes amid copious amorphous material, and C–E showing areas containing an almost confluent carpet of large coccal forms, with the arrow in E pointing to structures consistent with typically sized S. aureus coccal cells residing under the surface (and seen at higher magnification in the inset). (F) Transmission electron microscopy image of this vegetation showing saccular structures with coccal elements at the internal periphery, consistent with the image in E. Scale bars: A and E (inset) = 1 µm; B = 2 µm; C = 10 µm; D, E, F = 5 µm.

Figure 5.

Proposed development of novel bacterial forms. Scanning electron microscopy images of Staphylococcus aureus RN6390 suspended in Hank’s balanced salt solution and imaged 15 min (A–C) or 2 hr (D) after inoculation onto silicone coupons. (A) Low-magnification images showing tendency of cocci to arrange in clusters, seen at a higher magnification in B. (C, D) Clusters of bacteria become covered with an extracellular matrix, presumably containing the neutral lipid that stains with Oil Red O as well as LipidTOX. (E) Transmission electron microscopy image of catheter vegetation from a patient with active S. aureus bacteremia, showing mature forms that appear as sacs with peripheral distribution of coccal elements; note the comparatively clear appearance of the central portion of the sacs. (F) Diagram of the proposed development of the large bacterial structures where the solid black circle represents the lipid. Scale bar = 2 µm.