Investigation of biofilm formation in clinical isolates of Staphylococcus aureus

Abstract

As with many other bacterial species, the most commonly used method to assess staphylococcal biofilm formation in vitro is the microtiter plate assay. This assay is particularly useful for comparison of multiple strains including large-scale screens of mutant libraries. When such screens are applied to the coagulase-negative staphylococci in general, and Staphylococcus epidermidis in particular, they are relatively straightforward by comparison with microtiter plate assays used to assess biofilm formation in other bacterial species. However, in the case of clinical isolates of Staphylococcus aureus, including methicillin-resistant S. aureus, we have found it necessary to employ specific modifications including precoating of the wells of the microtiter plate with plasma proteins and supplementation of the medium with both salt and glucose. In this chapter, we describe the microtiter plate assay in the specific context of clinical isolates of S. aureus and the use of these modifications. A second in vitro method, which also is generally dependent on coating with plasma proteins and supplementation of the growth medium, is the use of flow cells. In this method, bacteria are allowed to attach to a surface and then monitored with respect to their ability to remain attached to the substrate and differentiate into mature biofilms under the constant pressure of fluid shear force. Although flow cells are not applicable to large-scale screens, we have found that they provide a more reproducible and accurate assessment of the capacity of S. aureus clinical isolates to form a biofilm. They also provide a means of analyzing structural differences in biofilm architecture and isolating bacteria and/or spent media for analysis of physiological and metabolic changes associated with the adaptive response to growth in a biofilm. While a primary focus of this chapter is on the use of in vitro assays to assess biofilm formation in clinical isolates of S. aureus, it is important to emphasize two additional considerations. First, it has become increasingly evident that biofilm formation in S. epiderimidis and S. aureus is not equivalent. Additionally, to date, most studies with S. aureus have been done with a very limited number of strains, almost all of which are derived from the NCTC strain designated 8325, and we have found that these strains are not representative of the most relevant clinical isolates. As with the specific elements of our flow cell system, we have written this chapter to reflect our focus on clinical isolates of S. aureus and the specific methods that we have found most reliable in that context. Second, as is often the case, in vitro methods do not necessarily reflect events that occur in vivo. Several in vivo methods to assess biofilm formation have been described, and these generally fall into one of two categories. The first focuses directly on staphylococcal diseases that are generally thought to include a biofilm component (e.g., endocarditis, osteomyelitis, septic arthritis). A discussion of these models is also beyond the scope of this chapter, but examples are easily found in the staphylococcal literature. The second approach uses some form of implanted device in an attempt to focus more directly on implant-associated biofilms. We use a model in which a small piece of Teflon catheter is implanted subcutaneously in mice and used as a substrate for colonization. We have the advantage of using bioluminescent derivatives of S. aureus clinical isolates and the IVIS(R) imaging system. However, because this system is not generally available, we restrict technical comments in this chapter to our use of an implanted catheter model evaluated by direct microbio-logical analysis of explanted catheters (2).

Fig. 1

Diagram of Stovall flow cell.

Fig. 2

A 20-mL sterile “male” Luer-Lok fitted syringe is connected to a small (~6 in.) section of sterile tubing by means of a “female” Luer-Lok adapter. The other end of the sterile tubing is subsequently attached to the flow cell output manifold. This apparatus is used to introduce plasma into the flow cell circuit.

Fig. 3

A small (~6 in.) section of sterile tubing containing a “female” Luer-Lok adapter at one end is connected to the flow cell input manifold. This “female” Luer-Lok-equipped end of the sterile tubing is subsequently placed in a 50-mL beaker containing 25 mL of resuspended 20% human plasma.

Fig. 4

Prior to sterilization of the media reservoir, a three-way stopcock is fitted to the external tubing and placed in the “off” position.

Fig. 5

The “female” Luer-Lok adapter on the section of tubing connected to the flow cell input manifold is removed and replaced with a “male” Luer-Lok adapter. The flow cell may now be aseptically connected to the sterilized media reservoir by means of the three-way stopcock.

Fig. 6

The flow cell is connected to a peristaltic pump by placing each of the three pieces of tubing between the flow cell input manifold and the bubble trap apparatus into adjacent pump channels.

Fig. 7

Bubble traps in the flow cell apparatus are sequentially filled by turning a bubble trap stopcock to the “on” position. Each bubble trap cylinder is filled approximately half full with medium, and then the bubble trap stopcock is returned to the “off” position. This process is repeated for each of three bubble traps per flow cell.

Fig. 8

Tubing just upstream of the flow cell chamber is prepared for inoculation by cleansing with a sterile alcohol pad followed by application of self-sealing tape (included in the Stovall Flow Cell Kit).

Fig. 9

The needle of an insulin syringe containing the flow cell inoculum is carefully inserted through the self-sealing tape and into the lumen of the tubing just upstream of the flow cell chamber. The bacterial inoculum (0.5 mL) is slowly introduced into the flow cell chamber while the upstream pinch clamp is closed and the downstream pinch clamp is open. After full injection of the inoculum, the injection site is cleaned with a sterile alcohol pad, and the downstream pinch clamp is closed.

Fig. 10

After inoculation, the flow cell chamber is placed upside down in a 37°C incubator. Growth at 37°C is maintained without medium flow for 1 h, after which the flow cell is returned to an upright position and medium flow is resumed.

Fig. 11

The flow cell tubing should not be impacted as it enters or exits the incubator. The flow cell chamber should be level with the incubator surface at all times throughout the experiment (a small weight may be used for this purpose).