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J Clin Microbiol. 2009 December; 47(12): 4168–4170.
Published online 2009 October 21. doi:  10.1128/JCM.01354-09
PMCID: PMC2786629

Incubation Alone Is Adequate as a Culturing Technique for Cardiac Rhythm Management Devices[down-pointing small open triangle]

Abstract

There exist no standardized methods for culturing cardiac rhythm management devices. To identify the most optimal culturing method, we compared various techniques that comprise vortex, sonication, and incubation or combinations thereof. Incubation alone yielded bacterial colony counts similar to those of other culturing combinations and is the least labor-intensive.

Permanent pacemakers (PPM) and implantable cardioverter-defibrillators (ICD) are being utilized increasingly because of an aging population (5, 9). About 180,284 PPM and 57,436 ICD were inserted into cardiac patients in the United States in 2003 (10), and worldwide there are 3 million functioning PPM and 180,000 indwelling ICD (2). Infection is the most common serious complication of PPM and ICD, which are collectively referred to as cardiac rhythm management devices (CRMD). The rates of infection of CRMD range from 0.13% to 19.9% (3, 7) with an average rate of 4% (4). Staphylococcus epidermidis and Staphylococcus aureus collectively account for 70 to 95% of CRMD infections (1, 6). A National Hospital Discharge Survey from 1996 to 2003 indicated that along with the rising use of cardiac medical devices, there has been a disproportional increase in the number of infections of such devices (10). The average cost of combined medical-surgical treatment of an infected CRMD was estimated at $35,000 (4).

There are no standardized methods for culturing explanted cardiac generators and leads of the CRMD. Although it has been demonstrated that sonication increases the yield of bacterial cultures from prosthetic joints (8), the utility of this diagnostic approach for CRMD has not been published. To identify the most optimal culturing method, we compared the bacterial yield of various techniques that comprise incubation, vortex, sonication and combinations thereof.

Cardiac devices.

We collected 20 similar cardiac pacemakers (St. Jude Medical, St. Paul, MN) that had been removed from patients because of battery failure. Cardiac generators were gently brushed to remove any debris and then sterilized by placing them for 20 min in a sterile container that contained 70% isopropyl alcohol. Individual devices were removed, washed with 100 ml of sterile normal saline, then placed in another sterile container that contained 100 ml of sterile normal saline, and placed in an autoclave at 125°C for 60 min. To maintain consistency, after finalizing each culturing method (methods A to D), each individual cardiac pacemaker was reused after repeating the above sterilization process. In addition, we also studied unused sterile silicone cardiac leads (St. Jude Medical, St. Paul, MN). These leads were sterilized for 20 min in an autoclave at 125°C.

Infection of devices.

Once the cardiac generators were individually sterilized, the normal saline was drained from the container and replaced with 100 ml of Trypticase soy broth (TSB). Ten cardiac generators were then individually incubated for 24 h at 37°C in a 105 CFU/ml suspension of a biofilm-producing clinical strain of methicillin (meticillin)-resistant S. aureus (MRSA). After formation of a biofilm, each cardiac generator was washed three consecutive times with 100 ml of normal saline to remove any planktonic bacteria and then placed in another sterile container with 100 ml of TSB for subsequent cultures as described below.

Cardiac leads were cut into 1-cm segments and were individually placed in sterile containers that contained 5 ml of TSB. Each 1-cm lead segment was then incubated with a 105 CFU/ml suspension of the same clinical isolate of MRSA for 24 h at 37°C. Thereafter, the lead segment was washed three consecutive times with 10 ml of sterile normal saline to remove any planktonic bacteria, placed in a new sterile container with 5 ml of TSB, and subsequently cultured by all four culture methods as described below.

Ten other cardiac generators and leads were infected with a biofilm-producing clinical strain of Pseudomonas aeruginosa that had caused CRMD infection, as P. aeruginosa is one of the most common gram-negative bacteria causing CRMD infections. Devices were infected with P. aeruginosa using the same methods used for MRSA.

Culturing techniques.

We compared four culturing methods. (i) Method A combined vortexing, sonication, and incubation. Each device was placed in an individual sterile container that had either 100 ml (for each cardiac generator) or 5 ml (for each 1-cm lead segment) of TSB. Each device was then individually vortexed for 30 s. Subsequently, 0.1-ml aliquots of the vortexed suspension were obtained, and aliquots of serial 10-fold dilutions were inoculated onto blood agar plates. The device was kept in the vortexed solution and then sonicated for 5 min, following which 0.1-ml aliquots were cultured as described above. Finally, the device was incubated in the same suspension that had been vortexed and then sonicated for 24 h at 37°C, and then 0.1-ml aliquots were cultured as described above. (ii) Method B combined vortexing and incubation. Method B comprised only the steps of vortexing followed by incubation as described for method A. (iii) Method C combined sonication and incubation. Method C included only the steps of sonication followed by incubation, as described for method A. (iv) Method D (incubation) consisted of only incubation for 24 h, as described for method A.

The mean bacterial colony counts determined by different culture methods were compared by analysis of variance (ANOVA) method and two-tailed pairwise Student t tests using STATA software (version 8.2; Stata Corp, College Station, TX). A P value of ≤0.05 indicated a significant difference.

Incubation alone (method D) of cardiac generators and leads with MRSA yielded bacterial colony counts similar to those found by methods A (vortexing-sonication-incubation), B (vortexing-incubation), and C (sonication-incubation) (P > 0.65, ANOVA). For P. aeruginosa, incubation alone (method D) of cardiac generators yielded bacterial colony counts similar to those found by method B (P = 0.063, t test) and colony counts higher than those found by methods A and C (P ≤ 0.004, t test). Additionally, incubation alone (method D) of cardiac leads with P. aeruginosa yielded bacterial colony counts similar to those found by methods A (P = 0.32, t test) and B (P = 0.98, t test) and colony counts higher than those found by method C (P = 0.002, t test). Furthermore, for vortexed and/or sonicated devices, subsequent incubation increased the bacterial colony counts by 2 to 3 log units for cardiac generators (P < 0.001, t test) and by 3 to 4 log units for leads (P < 0.001) for both MRSA and P. aeruginosa (Fig. (Fig.11).

FIG. 1.
Results for both MRSA and P. aeruginosa from 10 cardiac generators and 10 cardiac leads. Subsequent incubation of vortexed and/or sonicated generators increased bacterial colony counts by 2 to 3 log units (P < 0.001, t test) and by 3 to 4 log ...

Although sonication cultures of prosthetic joints reportedly yield a high bacterial count (8), the results of this in vitro study indicate that incubation alone of CRMD is more than adequate. Similar results were obtained when cardiac generators and leads were incubated with 103 CFU/ml of MRSA and P. aeruginosa. Since the final yields of all four studied culture methods were similar, incubation alone is as good as any studied combination of culturing methods. Not only does incubation alone yield adequate growth of gram-positive and -negative bacteria, it also can help eliminate false-positive results by decreasing the risk of laboratory contamination that could occur with more labor-intensive culture techniques, including vortexing and sonication. Although it takes longer to obtain the culture results of incubated devices, this technique requires less work time of the laboratory technician than any other culture technique studied and, therefore, may save costs. On the basis of the results of this in vitro study, we recommend that all potentially infected CRMD be incubated for at least 24 h to increase the yield, even if the devices are to be vortexed or sonicated. However, since our study was performed in vitro, it is not possible to state with certainty how well these culturing techniques would work in vivo or in a clinical setting with infected implantable cardiac devices.

Acknowledgments

This study was supported by funds from Baylor College of Medicine, Houston, TX.

Footnotes

[down-pointing small open triangle]Published ahead of print on 21 October 2009.

REFERENCES

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Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)