Methods for Enhanced Culture Recovery of Francisella tularensis

Francisella tularensis is found in a wide variety of hosts andextrahost environments, making culture recovery a diagnosticchallenge. Here we demonstrate improved recovery times and goodsensitivity (90%) when cultures were inoculated on the siteof an investigation using fresh tissues. For contaminated specimens,antibiotic supplementation of enriched cysteine heart agar bloodculture medium improved recovery of F. tularensis by 81.1%.For transport of tissues, immediate freezing yielded culturerecovery rates as high as 94%.

INTRODUCTION

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Introduction

Francisella tularensis, the etiologic agent of the zoonoticdisease tularemia, is found throughout the Northern Hemisphere(Holarctic region) (3, 6). Natural infections with F. tularensishave been found in a wide range of vertebrates ( $~$ 150 species)and invertebrates ( $~$ 100 species), as well as contaminated soil,water, and vegetation, showing that F. tularensis has the abilityto spread and survive in widely differing natural environments(7, 10). In nature, the true reservoirs of the bacterium areunknown, although hosts such as ticks, rodents, and protozoahave been suggested (1, 7, 10). The ecology of tularemia isalso poorly understood due in large part to the broad distributionof F. tularensis in nature.

The prevalence and distribution of F. tularensis in nature hasrecently become of more general interest due to heightened concernsthat F. tularensis might be used as an agent of bioterrorism(5). Identification of F. tularensis by culture presents problemsbecause the organism is highly infectious, fastidious, and growsslowly in vitro (4, 12). F. tularensis is also extremely difficultto recover from field specimens, with recovery rates from carcassesat only 30% (9). Nevertheless, the availability of such culturesis essential for detailed molecular epidemiology studies andfor our understanding of how biotype and strain variations canimpact the natural cycles of F. tularensis. Further, in thecase of an intentional release of F. tularensis, the earliestagent recovery will expedite its characterization and control.

Here, we evaluated several culture and transport systems fortheir use in enhancing recovery of F. tularensis cultures. Comparisonswere performed using samples from a tularemia outbreak in prairiedogs (Cynomys ludovicianus) (2).

MATERIALS AND METHODS

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Materials and Methods

Outbreak specimens.
For comparative evaluations, prairie dogs were divided intogroup A (46 dead animals) and group B (20 live animals, euthanized).All group A and B animals were previously tested and classifiedas F. tularensis subsp. holarctica (type B) positive (11).

On-site specimen processing.
All group A and B prairie dogs were necropsied on-site by usinga mobile field station. The field station, a 6- by 10-ft closedtrailer with fold-out workbenches, contained personal protectiveequipment, biohazard containment supplies, surface sterilizationsolvents, sterile necropsy equipment, tissue collection vials,and culture media. Prairie dogs were surface sterilized withethyl alcohol and necropsied, and tissues were surgically removed.Personal protection included single-use disposable closed-frontgowns, N95 masks, glasses, and double gloves.

Transport systems.
Within 15 min of necropsy, spleen and liver tissues were frozenor transferred to Cary-Blair transport medium; $~$ 10 g was placedin cryovials and frozen on dry ice, and $~$ 2 g was placed in Cary-Blairtransport medium and held at 4°C until transport to thelaboratory ( $~$ 72 h). On arrival at the laboratory, frozen tissueswere placed at –20°C and Cary-Blair samples were placedat 4°C. For evaluations, tissues were cultured 3 weeks later.

Culture recovery of F. tularensis.
Within 15 min of necropsy, spleen and liver tissues were punctured10 to 20 times with a sterile wooden stick. Tissue adheringto the stick was transferred to cysteine heart agar with chocolatized9% sheep blood (CHAB), and then a 1-µl sterile loop wasused to streak the plate for colony isolation. Plates were sealedimmediately with parafilm and transported in ice coolers ( $~$ 15to 20°C) until arrival at the Centers for Disease Controland Prevention laboratory, Fort Collins, Colo. ( $~$ 72 h later).All plates were then transferred to a biosafety level 3 laboratory,where they were incubated at 37°C for 5 more days and checkeddaily for characteristic F. tularensis growth. All recoveredisolates were confirmed as F. tularensis (11).

CHAB supplemented with 7.5 mg of colistin, 2.5 mg of amphotericin,0.5 mg of lincomycin, 4 mg of trimethoprim, and 10 mg of ampicillinper liter (CHAB-A) was also utilized. CHAB-A culture plateswere inoculated in the laboratory and incubated at 37°Cfor 7 days.

RESULTS

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Results

Rapid recovery of F. tularensis cultures.
To see if recovery times for F. tularensis could be improved,necropsy specimens were collected from all group A and B animalsand immediately cultured on the site of an investigation. Inorder to minimize airborne contamination, the mobile field stationwas moved into the animal facility and away from direct air-conditioningflow. Despite suboptimal growth temperatures ( $~$ 15 to 20°C)during transport (72 h), this approach yielded 5 cultures fromgroup A animals and 13 cultures from group B animals on arrivalat the laboratory. Upon 24 h incubation at 37°C, an additionalthree cultures from group A and three cultures from group Banimals were obtained. After 4 more days of incubation, thelast cultures were obtained: one culture from group A and twocultures from group B animals.

For live animals (group B), the sensitivity of on-site cultureinoculation was 90% (Table 1), demonstrating the utility ofthis technique when testing fresh tissues. In comparison, thesensitivity for carcass tissues (group A) was only 19.6% (Table1), with a surprisingly low recovery rate of 17% from animalsdead less than 24 h (data not shown). The majority of culturesinitiated from carcass tissues (n = 37) yielded only Pseudomonas,Staphylococcus, or Proteus species.

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TABLE 1. Comparison of F. tularensis recovery rates for on-site culture and antibiotic-supplemented CHAB (CHAB-A)

Antibiotic-supplemented media required for culture recovery from contaminated specimens.
To determine if cultivable F. tularensis was present in culture-negativecarcass tissues, CHAB medium was supplemented with a combinationof antibiotics (CHAB-A) previously shown to preserve the viabilityof F. tularensis (8). With CHAB-A, 30 F. tularensis isolateswere cultured from the 37 CHAB-negative carcasses, improvingthe recovery rate by 81.1% (Table 1). When side-by-side comparisonswere performed with CHAB versus CHAB-A, we found that F. tularensisgrowth was dramatically inhibited by the presence of contaminatingbacteria in carcass tissues (e.g., Fig. 1). Thus, isolationof F. tularensis from contaminated specimens requires the utilizationof antibiotics to suppress growth of inhibitory bacterial species.

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FIG. 1. Comparison of carcass tissues plated on CHAB (left panel) and on CHAB-A (right panel).

Comparison of transport systems.
Two transport systems were also evaluated for their utilityin the recovery of live organisms from tissues that cannot beimmediately cultured (Table 2). For recovery of F. tularensisfrom carcasses, a statistically significant difference betweentransport systems was observed. The recovery rate from frozentissue was 88.8%, while the recovery rate from Cary-Blair mediumwas only 11.1% (P $<=$ 0.05, McNemar's test) (Table 2). In the majorityof cases, cultures initiated from tissues transported in Cary-Blairmedium were highly contaminated with Pseudomonas, Staphylococcus,and Proteus species.

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TABLE 2. Comparison of transport methods for culture recovery of F. tularensis from live and dead animals^a

There was no statistically significant difference observed whentransport systems were compared for recovery of F. tularensisfrom live animals. The recovery rate from frozen tissue was94.4%, while the recovery rate from Cary-Blair medium was 83.3%(P $>=$ 0.05, McNemar's test) (Table 2).

DISCUSSION

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Discussion

Due in large part to the hazards associated with culture ofF. tularensis and the low funding priority of tularemia researchin recent decades, little effort has focused on the evaluationof different systems for the efficient recovery of live organismsfrom field specimens. In the present study, we identified andevaluated culture and transport systems that demonstrate goodsensitivity, improved culture recovery times, and recovery ratesas high as 84.8% from infected carcasses, specimens that areusually highly contaminated and pose a significant challengefor isolating F. tularensis. Although these methods were evaluatedon specimens infected with F. tularensis subsp. holarctica (typeB), we expect the methods to be fully applicable to F. tularensissubsp. tularensis (type A), as these two subspecies have highlysimilar culture characteristics and growth requirements.

Culture is often not the preferred diagnostic method for F.tularensis when a rapid result is required, as it is a fastidious,slow-growing organism. However, by inoculating CHAB plates onthe site of an investigation, we were able to shorten culturerecovery times that would have otherwise been delayed by shipping(e.g., 3 to 6 days saved by eliminating normal transport times).This technique also showed very good sensitivity (90% recoveryrate) when cultures were initiated from tissues of live F. tularensis-infectedanimals. Most likely, this success was due to the freshnessof the specimens used for culture; as soon as the animals wereeuthanized, tissues were excised and immediately cultured, thusminimizing environmental contamination.

Recovery of live F. tularensis from field specimens has historicallyproven to be a significant diagnostic challenge. Here, we showedin the case of carcasses that recovery of F. tularensis is negativelyaffected by the growth dynamics of competing bacteria and thatthis effect occurs within 24 h of death. Antibiotic supplementationof CHAB media controlled the growth of contaminating bacteriaand significantly improved the ability to recover F. tularensis(P $<=$ 0.05), allowing for an increase in sensitivity of 81.1%.

Whether other bacteria deplete nutrients required for growthof F. tularensis and/or produce bacteriocins affecting F. tularensisgrowth requires further study. Nonetheless, these findings haveimportant implications when attempting to isolate F. tularensisfrom a variety of samples that contain other bacterial species.To date, CHAB-A media has proved useful for culture of F. tularensisfrom carcasses and urine. Future evaluations of this media willalso be important using water, soil, and grass or hay.

In this study, we also compared culture recovery from infectedtissues after use of different transport systems. Freezing oftissues from carcasses yielded isolates in 75% of cases andafforded significantly greater transport recovery than did Carey-Blairmedium (P $<=$ 0.05). This difference is likely due to postmortemgrowth dynamics of competing bacteria that continue to evolvein Carey-Blair medium. We conclude that freezing of tissuesis more likely to preserve predeath bacterial distributionsand enable improved recovery of F. tularensis. Alternatively,Cary-Blair medium supplemented with the antibiotics describedhere may also be used for transport but will require futureevaluation.

In summary, utilization of the culture and transport systemsdescribed and evaluated in this study should prove useful fortesting a wide variety of samples, thus providing importantnew insights into the ecology, epidemiology, and transmissioncycles of F. tularensis and improving our bioterrorism responsecapabilities.

ACKNOWLEDGMENTS

We thank members of the Diagnostic and Reference Section includingSandra Urich, Brook Yockey, and Scott Bearden for technicalassistance.

FOOTNOTES

* Corresponding author. Mailing address: Diagnostic and Reference Section, Bacterial Zoonoses Branch, Centers for Disease Control and Prevention, P.O. Box 2087, Rampart Rd., Foothills Campus, Fort Collins, CO 80522. Phone: (970) 266-3524. Fax: (970) 221-6476. E-mail: nzp0{at}cdc.gov.

REFERENCES

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