Methods for Enhanced Culture Recovery of Francisella tularensis

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

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

Transport systems.Within 15 min of necropsy, spleen and liver tissues were frozen or transferred to Cary-Blair transport medium; ∼10 g was placed in cryovials and frozen on dry ice, and ∼2 g was placed in Cary-Blair transport medium and held at 4°C until transport to the laboratory (∼72 h). On arrival at the laboratory, frozen tissues were placed at −20°C and Cary-Blair samples were placed at 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 punctured 10 to 20 times with a sterile wooden stick. Tissue adhering to the stick was transferred to cysteine heart agar with chocolatized 9% sheep blood (CHAB), and then a 1-μl sterile loop was used to streak the plate for colony isolation. Plates were sealed immediately with parafilm and transported in ice coolers (∼15 to 20°C) until arrival at the Centers for Disease Control and 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 checked daily for characteristic F. tularensis growth. All recovered isolates 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 ampicillin per liter (CHAB-A) was also utilized. CHAB-A culture plates were inoculated in the laboratory and incubated at 37°C for 7 days.

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 animals and immediately cultured on the site of an investigation. In order to minimize airborne contamination, the mobile field station was moved into the animal facility and away from direct air-conditioning flow. Despite suboptimal growth temperatures (∼15 to 20°C) during transport (72 h), this approach yielded 5 cultures from group A animals and 13 cultures from group B animals on arrival at the laboratory. Upon 24 h incubation at 37°C, an additional three cultures from group A and three cultures from group B animals were obtained. After 4 more days of incubation, the last cultures were obtained: one culture from group A and two cultures from group B animals.

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

Antibiotic-supplemented media required for culture recovery from contaminated specimens.To determine if cultivable F. tularensis was present in culture-negative carcass tissues, CHAB medium was supplemented with a combination of antibiotics (CHAB-A) previously shown to preserve the viability of F. tularensis (8). With CHAB-A, 30 F. tularensis isolates were cultured from the 37 CHAB-negative carcasses, improving the recovery rate by 81.1% (Table 1). When side-by-side comparisons were performed with CHAB versus CHAB-A, we found that F. tularensis growth was dramatically inhibited by the presence of contaminating bacteria in carcass tissues (e.g., Fig. 1). Thus, isolation of F. tularensis from contaminated specimens requires the utilization of 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 utility in the recovery of live organisms from tissues that cannot be immediately cultured (Table 2). For recovery of F. tularensis from carcasses, a statistically significant difference between transport systems was observed. The recovery rate from frozen tissue was 88.8%, while the recovery rate from Cary-Blair medium was only 11.1% (P ≤ 0.05, McNemar's test) (Table 2). In the majority of cases, cultures initiated from tissues transported in Cary-Blair medium were highly contaminated with Pseudomonas, Staphylococcus, and Proteus species.

There was no statistically significant difference observed when transport systems were compared for recovery of F. tularensis from live animals. The recovery rate from frozen tissue was 94.4%, while the recovery rate from Cary-Blair medium was 83.3% (P ≥ 0.05, McNemar's test) (Table 2).