A New Twist to the Kirby-Bauer Antibiotic Susceptibility Test Activity-Increasing Antibiotic Sensitivity of Pseudomonas fluorescens through Thermal Stress.

INTRODUCTION

Antibiotic sensitivity and the effect of temperature on microbial growth are two standard laboratory activities found in most microbial laboratory manuals. We have found a novel way to combine the two activities to demonstrate how temperature can influence antibiotic sensitivity using a standard incubator in instructional laboratory settings. This activity reinforces the important concepts of microbial growth and temperature along with Kirby-Bauer antibiotic susceptibility testing. We found that Pseudomonas fluorescens can be manipulated to become more sensitive to several antibiotics by simply increasing growth temperature and exposing the organism to various antibiotics. No additional equipment is required beyond a standard incubator.

Pseudomonas fluorescens is an excellent choice for this activity since it is a safe alternative to Pseudomonas aeruginosa, a biosafety level 2 agent. Pseudomonads are important to explore in the microbiology laboratory since Pseudomonas aeruginosa poses a serious issue in health care settings, as this organism is known to be a multi-drug-resistant pathogen (6). More importantly, P. fluorescens is a good alternative in the laboratory to P. aeruginosa since it is also pigmented (5) and a possible reservoir of antibiotic resistance genes (4). In addition, it grows best at room temperatures and can easily be thermally stressed by placing in a standard 35ºC to 37ºC incubator.

PROCEDURE

We run the Kirby-Bauer susceptibility test similar to that described in most lab manuals and use an American Type Culture Collection strain of P. fluorescens (Strain 13525). Briefly, P. fluorescens cultures are grown overnight at room temperature (20°C–25ºC), then suspended in either nutrient broth or sterile saline to match the turbidity of a 0.5 McFarland Standard and swabbed onto Mueller-Hinton (MH) agar plates (3). Antibiotic disks are usually dispensed using a commercial multidisc dispenser and incubated for an additional 24 hours at room temperature. We have found that six antibiotics work well in this activity (chloramphenicol, streptomycin, penicillin, neomycin, tetracycline, and erythromycin). Zones of inhibition are measured and compared to standardized tables usually published in the laboratory manual or provided with the antibiotic disks.

To thermally stress the organism, the above procedure is slightly modified. After swabbing the MH agar plates, these are placed at 35ºC to 37ºC for 24 hrs. P. fluorescens are inhibited at these temperatures and will not grow. After 24 hours the instructor or students can dispense antibiotic disks and continue incubation at room temperature overnight, where P. fluorescens will grow. The next lab period, control plates (dispensed with antibiotic after swabbing the bacterium) are compared with the thermally stressed plates. These comparisons, using the Kirby-Bauer antibiotic sensitivity method, show measurable differences in zones of inhibition when using several antibiotics.

Another variation that we found to work well is to perform the experiment setting the incubators to 30ºC or 32ºC. We have found that P. fluorescens will tolerate these temperatures better than 35ºC or 37ºC. With this variation, both control plates (P. fluorescens grown with antibiotic disks at 25ºC) and thermal stress plates can have antibiotic disks dispensed at the same time. The plates are placed at their appropriate temperature and the results can be read the next day or placed at 4ºC until the next laboratory period. Typical antibiotic zones of inhibition with the various stress temperatures and their interpretations are found in Table 1.

TABLE 1.

Zones of inhibition of P. fluorescens (ATCC 13525 strain) grown at various temperatures.

	Temperatures
Antibiotic discs	25˚C	30˚C	32˚C	37˚C
Tetracycline (30μg)	20 mm (S)	22 mm (S)	25 mm (S)	28 mm (S)
Chloramphenicol (30 μg)	9 mm (R)	13 mm (I)	17 mm (I)	12 mm (I)
Penicillin (10 Units)	0 mm (R)	0 mm (R)	0 mm (R)	0 mm (R)
Neomycin (5 μg)	14 mm (I)	15 mm (I)	18 mm (S)	18 mm (S)
Erythromycin (15 μg)	0 mm (R)	0 mm (R)	0 mm (R)	0 mm (R)
Streptomycin (10 μg)	10 mm (R)	16 mm (S)	22 mm (S)	14 mm (I)

S, sensitive; I, intermediate; R, resistant.

Safety issues

The P. fluorescens cultures (ATCC Strain 13525) used are considered biosafety level 1 agents (www.attc.org) and should be handled accordingly.

CONCLUSION

By simply thermally stressing a P. fluorescens culture plate prepared for the Kirby-Bauer test and comparing it to a similar plate placed at optimal temperature, students can easily see how temperature affects microbial metabolism and antibiotic sensitivity. We typically present this laboratory as an open ended activity, informing students that we are investigating the effect of elevated temperature on antibiotic sensitivity. We use the ATCC strain 13525 to avoid antibiotic sensitivity variations observed in laboratory strains of P. fluorescens. This strain is hypersensitive to tetracycline, neomycin, and streptomycin upon thermal stress, as indicated in Table 1. In addition to the increase in antibiotic sensitivity, we noted that P. fluorescens grown at elevated temperatures of 30ºC and 32ºC do not have the bright green pigment when grown at room temperature, thus adding another dimension of inquiry as to what could be happening at the elevated temperatures. Undoubtedly, a search of the literature will uncover a number of papers that explain what effect elevated temperature can have on membrane, efflux pumps, and gene regulation in this bacterium (1, 2). This is an excellent activity to demonstrate how environmental factors such as thermal stress can affect bacterial metabolic processes and lead to phenotypic changes. The results are easily discernable, as noted by the differences in pigment coloration and antibiotic sensitivity. Students are excited to see and measure changes in P. fluorescens that can be easily manipulated in the laboratory by varying temperature.