David Drake and Dr. Richard Robison, Molecular and Microbiology
Introduction
Our initial goal was to develop and evaluate the effectiveness of a new selective medium for isolating Francisella species from environmental samples, including those from soil, feces, and ponds. We successfully developed the medium, CHAB-PACCVI, and used it to isolate a novel Francisella species from hot springs samples obtained in Salt Lake.
F.tularensis, subspecies tularensis (type A) and holarctica (type B) are the causative agents of tularemia, a bacterial zoonosis with several routes of transmission and a broad range of clinical forms. F. tularensis is one of the most infectious pathogenic bacteria known, requiring as few as 10 organisms to cause disease (Ingelsby 2001). Due to its extreme infectivity, ease of dissemination, and substantial capacity to cause illness and death, F. tularensis is classified as a Class A Select Agent by the CDC. Francisella species can survive and grow in one of the widest environmental ranges of any studied pathogen. Indeed, the organism has been isolated from soil, feces, water, and aerosol environmental samples. Despite this broad environmental range, a method for direct isolation of F. tularensis environmental soil, lake, and fecal samples did not exist until 2009. Isolation from these sources required passage through mice or guinea pigs, wasting precious time and resources and impeding our ability to investigate natural outbreaks of tularemia. However, in 2009, a selective media was developed to grow and isolate Francisella spp. from seawater and seaweed samples. This media, called CHAB-PACCV (cysteine heart agar with 9% chocolatized sheep blood, containing polymyxin B, amphotericin B, cyclohexamide, cefepime, and vancomycin), included a mixture of antimicrobials with activity against Gram-negative and Gram-positive bacteria as well as fungi, but not Francisella. However, the media was not evaluated with other environmental samples.
Methods
CHAB-PACCV was made as previously described. Briefly, the media base consisted of cysteine heart agar with 9% chocolatized sheep blood (CHAB). The following concentrations of antibiotic supplements were added: 8 x 10^4 U/L polymyxin B, 2.5 mg/L amphotericin B, 4.0 mg/L cefepime, 100mg/L cyclohexamide, and 4.0 mg/L vancomycin. Selectivity tests showed that the medium effectively inhibited the growth of several common soil contaminants: Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 11229, Bacillus subtilis ATCC 19659, and Candida albicans. However, Pseudomonas aeruginosa ATTC 15442 grew up uninhibited. Following this observation, we added imipenem at a concentration of 8 mg/L to the media formulation and subsequently called it CHAB-PACCVI. Imipenem has been shown to inhibit the growth of P. aeruginosa without affecting Francisella spp.
After the addition of Imipenem, the sensitivity of CHAB-PACCVI was confirmed with six Francisella strains: F. tularensis subsp. Tularensis (type A)(strain Schu S4 and 837), F. tularensis subsp. holarctica (type B)(strain 80501131), F. tularensis subsp. novicida (strain U112:CG69), F. philomiragia, and Live Vaccine Strain (LVS) derived from Francisella tularensis subsp. holarctica. 10 uL of each isolate was cultured from frozen stocks onto CHAB-PACCVI and incubated at 37 C for 5 days in 5% CO2.
To evaluate the effectiveness of CHAB-PACCVI on environmental samples, mud, water, pond scum and sediment samples were obtained from Wasatch Hot Springs in Salt Lake City, Utah on January 6, 2013. Approximately 5g of each environmental sample was placed into a new 15mL centrifuge tube and centrifuged at 8000 g for 15 min. The supernatant was decanted and the pellet was resuspended in 2mL of sterile .85% saline solution.
100uL of the suspensions was inoculated by spread-plating onto CHAB-PACCVI. Plates were incubated at 37 C for 72 h in 5% CO2. For screening, colonies with the smooth, greenish-white appearance of Francisella spp. grown on CHAB-PACCVI were picked and subcultured twice on CHAB plates. Purity of small, gram-negative cocci was confirmed by visualization of gram-stained preparations using brightfield microscopy. Freezer stocks of presumptive Francisella spp. isolates were made using Mueller- Hinton broth with 12.5% glycerol. Isolates were stored at -80 C.
The isolates were identified through several biochemical and genetic tests. Briefly, isolates were identified basted on (1) their production of oxidase and hydrogen sulfide, (2) their cellular fatty acid content (MIDI), (3) a Francisella-specific PCR test, and (4) a BLAST search based on sequences from the 16s rRNA and sdhA genes.
Results
Selectivity and sensitivity tests of CHAB-PACCVI showed that the modified medium grows each of the Francisella subspecies tested while inhibiting the growth of common environmental contaminants.
After imaging colonies isolated from the hot spring samples, 8 isolates remained as potential Francisella spp. Identification results for these isolates are illustrated in the following table:
Discussion
Three different Francisella spp. were isolated from the hot springs: F. philomiragia, F. novicida, and a novel species. These results confirmed that CHAB-PACCVI may be used for direct isolation of Francisella species from the environment. Future utilization of CHAB-PACCVI may prove valuable for investigating outbreaks of tularemia and understanding the ecology, epidemiology, and potential environmental sources of exposure for Francisella infections.
In the process of developing CHAB-PACCVI and testing its effectiveness, we inadvertently isolated a novel species of Francisella. This new species lacks a name and was only first described in 2008 when it was isolated from acutely ill patients from two states in the eastern United States. BLAST comparisons showed 100% similarity in the 16s rRNA and sdhA regions between the novel species and four of our isolates. Clinicians and microbiologists may need to become more aware of this new potential pathogen, as its geographical range and infectivity may be more common than recognized.
Further analysis of this novel organism will be required to understand its prevalence and pathogenicity. Prior to publishing this research, we hope to test the media on a second batch of samples from the hot springs as well as on homogenized ticks carrying Francisella spp. Following these tests, a paper will be submitted to Letters in Applied Microbiology for publication.