Procurement of spore-free Bacillus anthracis for molecular typing outside of BSL3 environment

Authors

  • D.S. King,

    1.  Center for Biological Defense, College of Public Health, University of South Florida, Tampa, FL, USA
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    • *

      DSK and VAL contributed equally to this article.

  • V.A. Luna,

    1.  Center for Biological Defense, College of Public Health, University of South Florida, Tampa, FL, USA
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    • *

      DSK and VAL contributed equally to this article.

  • A.C. Cannons,

    1.  Center for Biological Defense, College of Public Health, University of South Florida, Tampa, FL, USA
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  • P.T. Amuso

    1.  Center for Biological Defense, College of Public Health, University of South Florida, Tampa, FL, USA
    2.  Florida Department of Health, Bureau of Laboratories, Tampa, FL, USA
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Vicki Ann Luna, Center for Biological Defense, College of Public Health, University of South Florida, 3602 Spectrum Blvd, Tampa, FL 33612, USA. E-mail: vluna@health.usf.edu

Abstract

Aims:  To (i) develop a protocol that would eliminate or greatly reduce sporulation within Bacillus anthracis vegetative cells, and (ii) harvest an adequate number of cells and sufficient DNA suitable for molecular methods including Riboprint® analysis and pulse field gel electrophoresis (PFGE).

Methods and Results:  Seven strains of B. anthracis (Ames, French B2, Heluky, Kruger, Pasteur, Sterne, and Vollum) were grown at 37, 42 and 45°C under normal air, enhanced CO2, microaerophilic, and anaerobic conditions on solid media and subcultured in two broths with and without supplements. The bacterial cells were centrifuged and washed. Slides made from the cell pellets were stained with Malachite Green and observed for the presence of spores. Cell preparations were subjected to 80°C for 30 min and processed for and analysed by either Riboprinter® or PFGE. Multiple pellets of each strain were processed, stained, placed onto solid culture media, incubated for 7 days and observed for growth. The cell preparations yielded clear and reproducible results with both molecular methods. None of the cell preparations yielded growth on the culture media.

Conclusions:  This method eliminated viable spores in cell preparations of B. anthracis, yet still allowed the growth of vegetative cells to provide sufficient DNA suitable for analysis by Riboprinter® and PFGE.

Significance and Impact of the Study:  This method will provide safe cell preparations, prevent instrument contamination, and may be useful for other aerobic and anaerobic spore-formers.

Introduction

Like other Bacillus species, Bacillus anthracis has two forms, the metabolically active vegetative cell and the dormant endospore. It is the spore that initiates infection of a susceptible host and causes cutaneous, inhalational and gastric forms of anthrax, the latter two normally being rapidly lethal (Dixon et al. 1999; Mock and Fouet 2001). Endospore formation is triggered in the cell by a variety of adverse environmental conditions, such as a change in temperature, increase in oxygen or decrease in carbon dioxide levels, change in pH, nutrient depletion, waste and toxin accumulation, or desiccation (Van Ness 1971; Yazdany and Lashkari 1975; Dragon and Rennie 1995; Baweja et al. 2008). This is easily demonstrated in the laboratory where sporulation occurs readily on any number of different culture media, with large numbers of spores being produced. Thus, the cultivation of virulent B. anthracis is limited to the biosafety level 3 (BSL3) laboratory where personnel can be protected from accidental exposure by aerosols.

Because of the importance of protecting personnel from accidental exposure, the ubiquitous presence of spores in B. anthracis cell and nucleic acid preparations prevents the removal of the preparations from the BSL3 environment. However, many molecular examinations cannot be performed in a BSL3 laboratory because of space limitations. In contrast BSL2 laboratories are usually larger and contain instruments used for Riboprinting, pulsed field gel electrophoresis (PFGE), and nucleic acid sequencing. In order to solve this dilemma, boiling, filtration and gamma irradiation have been useful in either removing or inactivating the spores in DNA preparations and retaining the usefulness of the nucleic acid for molecular applications such as PCR (Dauphin et al. 2008; Dauphin and Bowen 2009). Many laboratories then perform a sterility check by using a portion of the DNA preparation to inoculate growth plates and look for growth or no growth after a period of days. Yet, these methods (boiling, filtration and gamma irradiation) do not yield DNA that is suitable for either PFGE or Riboprinting. In addition, irradiated DNA, although suitable for PCR is not a likely choice for most laboratories because of the considerable personnel training needed, stringent national certification requirements, safety issues and cost when complying with Federal regulations DOE 10 CFR 835 (Department of Energy) and NRC 10 CFR 20 (U.S. Nuclear Regulatory Commission).

The goal of this study was to define a protocol that would produce spore-free DNA in sufficient quantity for use with molecular methods like Riboprint® analysis and PFGE. Further examination of the bacterium’s life cycle shows that B. anthracis does not generate endospores when growing inside the mammalian host (Mock and Fouet 2001; Swartz 2001; Frazier et al. 2006). Even in the laboratory, sporulation is greatly reduced when cells are grown at higher temperatures such as 45°C (Baweja et al. 2008). Spores are not seen in direct examination of tissue or blood specimens in the veterinary or clinical microbiology laboratory (Dragon and Rennie 1995; Swartz 2001; Logan and Turnbull 2003). The spores are not observed until the bacteria are exposed to air, desiccation of tissue and fluids or to a nutrient-depleted microenvironment. In addition, spore production is stimulated during a temperature or pH change such as happens when the bacteria is grown in culture media or when the bacilli are released into the environment after an animal bleeds out and dies (Dragon and Rennie 1995; Frazier et al. 2006). Animals such as mice could be used as a reservoir for vegetative cells, but this is expensive and a waste of animals. Therefore, we tried to mimic the conditions found in a mammal in order to avoid or limit the production of spores using normal laboratory media. These conditions were high temperature mimicking those found in herbivores, low O2 and higher CO2 concentration, and use of enriched media and animal serum. The success of producing asporogenous B. anthracis cultures would allow the removal of nonviable vegetative cell preparations that could be safely used for the molecular methods of Riboprint® analysis and PFGE without the danger of spore contamination of instruments or human exposure.

A preliminary report on this work has been presented previously (D.S. King, V.A. Luna, A.C. Cannons and P.T. Amuso, Abstr. 109th General Meeting of the American Society for Microbiologists in Philadelphia, PA, abstr, Y-006, 2009).

Materials and methods

Bacterial strains

We utilized seven isolates of B. anthracis that were received from the Florida Department of Health, Bureau of Laboratories, Tampa, FL (FLDOH), the NIH Biodefense and Emerging Infections Research Resources Repository (BEI Resources, Bethesda, MD, USA) and isolated from an environmental sample (Luna et al. 2003). The different strains were Ames NR-411 (BB004)(pX01+, pX02+), French B2-NR-413 (BB0023)(pX01+, pX02+), Heluky (CBD 131)(pX01−, pX02+), Kruger B1-NR-412 (BB006)(pX01+, pX02+), Pasteur BC3132 (CBD 63)(pX01−, pX02+), Sterne BA3194 (BB001)(pX01+, pX02-), and Vollum NR-414 (BB0022)(pX01+, pX02+). Bacillus atrophaeus ATCC 49337 (CBD 994), Bacillus cereus ATCC 14579 (CBD 55), and Bacillus thuringiensis ssp. israelensis ATCC 35646 (CBD 61) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), while Bacillus subtilis ssp. subtilis NRRL NRS 744 (CBD 984) was procured from Agriculture Research Service Culture Collection (ARSCC, Peoria, IL, USA).

All manipulations of B. anthracis isolates were performed in a class 2 biological safety cabinet. Bacillus anthracis Pasteur and Heluky isolates were stored and handled in a BSL2 laboratory using BSL3 practices. All other B. anthracis isolates were handled in a BSL3 laboratory. This arrangement followed the Institutional Bio-safety Committee requirements at the University of South Florida (USF). All safety protocols and requirements required by US federal regulation DHHS 42 CFR 73 were strictly followed. Prior to performing the growth protocols, the bacteria were grown on tryptic soy agar supplemented with 5% sheep red blood cells (Blood Agar, BA) (Remel, Lenexa, KS, USA). The plates were incubated at either 30 or 35°C (±2) overnight (18–24 h) in ambient air and checked for adequate growth before proceeding.

Testing various growth conditions for minimal sporulation

We tested the bacteria under three different temperatures (37, 42 and 45°C) and under various O2 and CO2 conditions. Besides normal ambient conditions, the B. anthracis strains were also grown overnight on BA under anaerobic (20% CO2 residual O2 of <1%), microaerophilic (14% CO2 and <5%O2) and enriched CO2 (5% CO2) atmospheres using gas packs (Mitsubishi Gas Chemical Co., New York, NY, USA). Culture plates were placed into sealed containers with appropriate gas packs and incubated at 37, 42 and 45°C for 18 h ± 30 min. After incubation, the culture plates were examined for growth. Glass slides were prepared from the growth edges of colonies and stained using Malachite Green (Sigma–Aldrich, St Louis, MO, USA) for 5 min and counter stained 1 min with Safranin (Fisher Scientific, Suwannee, GA, USA) following standard directions (Hendrickson 1985). Between 50 and 100 microscopic fields were examined for spores at ×1000 magnification under oil using a light microscope (Carl Zeiss, Inc., Thornwood, NY, USA).

We tested several filter sterilized broths to determine the fastest and best growth of the strains. The first was a modified heart infusion broth (Remel) supplemented with sodium bicarbonate (NaHCO3) (Sigma–Aldrich) and heat-inactivated horse serum (SHIB) (Remel and/or Lonza, Walkersville, MD, USA) (De et al. 2002). The other broths were SHIB supplemented additionally with 0·25–3·0% (v/v) glucose (Sigma–Aldrich) (Dawes and Mandelstam 1970), SHIB supplemented only with 5% (v/v) glycerol (Liu et al. 2004) (Fisher Scientific), standard tryptic soy broth (TSB) (Becton Dickinson, Sparks, MD, USA), and finally TSB with horse serum and NaHCO3. The TSB with horse serum was made at a 2× concentration, and horse serum and sodium bicarbonate were added to a final concentration of 50% and 0·8% respectively. To start the protocol, one isolated colony of bacteria was first inoculated onto a BA plate and incubated overnight (18 h ± 30 min) at 37°C. The next day, 4 ml of broth was inoculated by using a small (10 μl) disposable loop that had been touched to the outer edge of 4–5 colonies. For the broth cultures, 5-ml microcentrifuge tubes (cat # T2076; Argos Technology, Elgin, IL, USA) were used to limit the air volume. All broths were incubated for 1–8 h at 37°C under ambient conditions with no shaking. If no growth was seen, the tubes were incubated longer, up to 72 h.

After it was determined which broths best supported the growth of the different strains, the bacterial growth in the broths were examined for spore formation. At different time points, the bacteria/broth mixture was centrifuged for 10 min at 11 627 g (10 000 rev min−1) in a Sorvall Legend centrifuge by Thermo Scientific (Fisher Scientific). The supernatant was decanted and the cell pellet was observed for size and stained for the presence of spores. When the optimal incubation time for an adequate pellet lacking spores was determined, the bacteria/broth mixture was centrifuged as above, and washed with sterile dH2O three times. Prior to the final centrifugation, the pellet was resuspended in 2 ml of sterile dH2O to an optical density at 600 nm adjusted to read between 0·2 and 0·37 using a Beckman Coulter DU640 spectrophotometer (Fullerton, CA, USA). About 1 μl of the pellet was removed, stained with malachite green/safranin and examined.

In order to verify that few or no spores were present in the sample, the cell pellet was resuspended in 100 μl sterile dH2O and heated to 80°C for 10 or 30 min. The entire mixture was used to inoculate a BA plate that was incubated at 37°C for up to 7 days. Finally, 500 μl of TSB was added to the now empty tube and incubated with the plates above. On day seven, the entire broth in the tube was used to inoculate another BA plate that was again incubated as above. Multiple samples (5–10) for each B. anthracis strain were processed in this manner to determine whether 10 or 30 min killed all of the bacterial cells.

Final cell preparation for Riboprint and PFGE

Bacteria were grown overnight on BA plates at 37°C in a microaerophilic increased CO2 environment (14% CO2 and <5%O2) using microaerophilic gas packs (Mitsubishi Gas Chemical Co.). Growth at the edges of 4–5 colonies were used to inoculate both SHIB and TSB broths and incubated at 37°C for 4 h. The bacterial cells were centrifuged, washed two times, resuspended, and adjusted to the optical density at 600 nm as mentioned earlier followed by a third and final centrifugation. Slides were made from the cell pellets and stained. The bacterial cells were resuspended in 40 μl of the Riboprint® sample buffer and were heated to 80°C for a full 30 min. Thirty micro-litres of the heated mixture were then transferred to the sample reservoir for the Riboprinter® (Dupont-Qualicon, Wilmington, DE, USA). The remaining 10 μl of cell/buffer mixture was plated onto a BA plate, and 500 μl TSB was added to the tube that had contained the cells. All plates and tubes were incubated for 7 days at 37°C as a second confirmation of spore absence or deactivation. After treatment with lysing agents as per the Riboprinter® protocol, samples were loaded onto the instrument. Molecular analysis of DNA was carried out on the Riboprinter®, using three different enzymes, AseI (New England Biolabs, Ipswich, MA, USA), EcoRI and PvuII (Dupont-Qualicon). The RiboPrinter® performed subsequent biochemical steps and gel image analysis. Normalized data from the RiboPrinter® was imported into the BioNumerics® (Applied-Maths, Austin, TX, USA) software package via program scripts that were provided by Dupont-Qualicon. A unweighted pair-group method with arithmetic mean (UPGMA) tree was produced using the Pearson Correlation with an optimization of 1·56% and a position tolerance of 1·0%. Uncertain bands were ignored following the recommendation by Dupont-Qualicon.

The cell preparations used for PFGE were grown the same way as for the Riboprint® analysis except that glucose and threonine (0·3% and 0·36% v/v final concentration respectively) were added to TSB (Rudolph et al. 1998; Luna et al. 2000). For CBD 63 and CBD 131 that grew slowly in SHIB supplemented with glucose, TSB with and without glucose were made. The cell preparations were embedded in 2% low melting point agarose (Incert agarose; Lonza, Rockland, ME, USA) to make 1% blocks, processed and the DNA digested with the restriction enzymes AscI and NotI (double digest), SgfI, SmaI (Promega, Madison, WI), or SbfI (New England Biolabs). These enzymes were chosen after performing a virtual digestion on the LaserGene MapDraw program (DNAStar, Madison, WI, USA). The blocks along with a standard PFGE lambda DNA ladder (0·05–1 Mb) (Bio-Rad, Hercules, CA, USA) were melted at 80°C for 30 min and approximately half of the melted plugs were loaded into the wells of a 1% agarose gel (SeaKem Gold; Lonza). The remaining portion (c. 50%) of each plug was used to inoculate a BA that was then incubated at 37°C for up to 7 days. The plugs were electrophoresed in 0·5× Tris-borate–EDTA (pH 8·0) running buffer using a CHEF-Mapper XA (contour-clamped horizontal electrophoresis) instrument (Bio-Rad) at 6 V cm−2 for 20 h at 14°C with initial switch time of 2 s and final switch time of 30 s. Gels were stained as previously described (Rudolph et al. 1998; Luna et al. 2000). DNA bands were visualized under UV light and photographed using the Bio-Rad GelDoc with Quantity One (Bio-Rad) computer software.

Reproducibility and sterility testing

In order to ensure that the Riboprint® and PFGE patterns were reproducible and corresponded to known patterns of B. anthracis Pasteur and Sterne strains, multiple cell preparations were made and processed completely through Riboprinter® and PFGE analysis. The resulting Riboprint® and PFGE patterns were examined and compared as described earlier. The non-anthracis Bacillus species were grown overnight on BA under ambient atmospheric conditions at 35°C (±2). These plates were used to inoculate SHIB as described earlier for the Riboprint® instrument or colonies were used directly from plates as per the Riboprinter® protocol. The resulting patterns were examined for any changes.

Although all cell preparations of the two Pasteur and one Sterne strain never yielded growth and appeared microscopically to be consistently spore-free, sterility testing was expanded to include more B. anthracis strains known to make spores more abundantly. Cell preparations of each B. anthracis strain were prepared as described earlier. A slide was made from each preparation and stained, and the entire cell preparation was then placed onto BA that was incubated at 37°C (±2) for 7 days and examined for growth.

Results

Testing various growth conditions for asporogenous cultures

After 18 h incubation, all of the isolates grew at 42°C with normal air (ambient), enhanced CO2 and microaerophilic atmospheres. The numbers of colonies were profuse (>300 CFU), but the colony sizes were usually smaller (<4 mm) than colonies seen on the original BA incubated at 35°C with ambient air (5–9 mm) (Table 1). When stressed under both the anaerobic atmosphere and 42°C, only BB001 and BB006 grew with colonies <1 mm in only the first quadrant of the plate (10–20 colonies), while the other five strains did not grow at all. At 45°C, none of the strains grew in the anaerobic atmosphere (Table 1). CBD 131 failed to grow at this temperature no matter the variation of O2 or CO2. The other strains grew at 45°C, producing 50 to >300 colonies that were generally small (<1–2 mm). Microscopic examination revealed profuse spores (2–10 spores per field) present in the slides made from colonies grown in ambient air and enhanced CO2 and rare spores (1–2 spores per slide) in the slides made from the microaerophilic cultures.

Table 1.   Growth characteristics* of seven Bacillus anthracis strains on Blood Agar under different temperatures and atmospheric conditions
Identification numberStrainIncubation temperature (°C)Ambient airEnhanced CO2MicroaerophilicAnaerobic
  1. G, growth on the solid media; SG, slight growth, only on the primary streak area; NG, no growth; MC, medium sized colonies (diameter range of 3–4 mm); SC, small colonies (diameter range 1–2 mm); VSC, very small colonies (diameter < 1 mm). No notation for colony size implies diameter range of 5–8 mm.

  2. *All plates were incubated for 18 h ± 30 min.

CBD 63Pasteur37GGGG, VSC
42GG, MCG, MCNG
45NGG, SCG, SCNG
CBD 131Heluky37GGGG, VSC
42GG, MCG, MCNG
45NGNGNGNG
BB001Sterne37GGGSG, VSC
42GG, MCG, MCSG, VSC
45G, SCG, SCG, VSCNG
BB004Ames37GGGSG, VSC
42G, MCG, MCGNG
45G, MCSG, SCG, VSCNG
BB006Kruger37G, MCG, MCG, MCSG, VSC
42G, SCG, SCG, SCSG, VSC
45G, SCSG, SCG, VSCNG
BB022French B237GGGSG, VSC
42GGGNG
45G, MCSG, SCG, SCNG
BB023Vollum37GGGG, SC
42GGGNG
45G, SCNGG, VSCNG

After the same incubation time at 37°C, the culture plates grown under anaerobic conditions contained very few (10–20) and small (<1 mm) colonies, while the plates grown in enriched CO2 produced numerous (>300) larger colonies (5–8 mm) (Table 1). The media plates grown under microaerophilic conditions yielded as many colonies in the same size range as those grown in the enhanced CO2. This growth (amount and colony size) was also comparable to that found on the plates incubated in ambient air. Upon microscopic examination, spores were not observed in the colonies grown in either anaerobic or microaerophilic environments. In contrast, the colonies grown in enhanced CO2 and in ambient air produced numerous spores.

All of the strains grew at 37°C with most producing large colonies (diameter range 5–10 mm) in 18 h. BB006 consistently yielded colonies smaller than the other strains regardless of the temperature or the culture media. This is a characteristic of BB006. However, at 37°C the colonies for BB006 were large enough to facilitate touching the edges of colonies without touching closer to the colony’s centre where spores may reside with the vegetative cells. The difference in colony size from one temperature to the other may explain why no spores were seen in the slides made from the colonies incubated in the microaerophilic atmosphere at 37°C, yet there were rare spores seen on the slides made from colonies incubated in the same atmosphere at the higher temperatures. It was very difficult to touch only the edge of the small colonies incubated at 42 and 45°C and not also get some of the colony closer to the centre. In addition, 37 ± 2°C is close to or overlaps the body temperature of human and many herbivores (i.e. bovine = 38·3 ± 0·5°C, caprine = 39·1 ± 0·5°C; equine = 37·6 ± 0·5°C; ovine = 39·1°C) (Dr Alberto Van Olphen, CBD, Personal Communication).

Because SHIB is used by the Laboratory Response Network reference laboratory at FDOH, it was the first broth tested. While five of the strains grew well in the broth, two strains (CBD 131 and BB004) did not grow even after an extended incubation time of 72 h (Table 2). Adding glycerol or glucose to the SHIB did not support sufficient growth in all of the tested strains for Riboprint® analysis. BB0023 grew in the SHIB with glycerol, but not at all in the SHIB with glucose and surprisingly, CBD 63 (Pasteur) grew much slower in both supplemented broth preparations than in the original SHIB. In addition, neither CBD 131 nor BB001 (Sterne) grew at all in the SHIB with glycerol. Thus, because all of the strains grew in TSB, we then tested TSB supplemented with horse serum and NaHCO3. All of the strains except CBD 131 grew in this broth. The other Bacillus species (B. atrophaeus, B. cereus, B. subtilis, and B. thuringiensis) grew in both the SHIB and TSB.

Table 2.   Growth of seven Bacillus anthracis strains in two growth media broths with and without additives, incubated at 37°C
Identification numberStrainMedia tested
SHIB*SHIB + glucoseSHIB + glycerolTSBTSB + glucoseTSB + Horse serum + NaHCO3
  1. *SHIB, heart infusion broth with added horse serum and NaHCO3; TSB, trypic soy broth.

  2. †G, growth in tube that produces sufficient cell pellet when centrifuged for further work; NG, no growth; SG, slight growth, or light haze of growth in broth so that the cell pellet was not large enough for further work.

CBD 63PasteurG†SGSGGGG
CBD 131HelukyNGSGNGGGNG
BB001SterneGGNGGGG
BB004AmesNGSGSGGGG
BB006KrugerGGGGGG
BB022French B2GGGGGG
BB023VollumGNGGGGG

Other researchers have reported that depletion of glucose rapidly leads to sporulation (Dawes and Mandelstam 1970). SHIB does not contain glucose in the original recipe. Yet adding glucose to either SHIB or TSB did not delay or prevent sporulation. Interestingly, some of the strains (CBD 63 and CBD 131) grew less well or not at all (BB0023) with the added glucose in the SHIB. Although our normal PFGE protocol uses glucose in the growth media prior to cell preparation and lysing of cells, not all PFGE protocols require that glucose or threonine be included. This may be important for some isolates such as BB0023, but both CBD 63 and CBD 131 grew well in the TSB supplemented with glucose so that the PFGE blocks contained sufficient DNA to produce easily discernible DNA bands in the agarose gel and photographs. Growing an unknown isolate in TSB with and without glucose prior to PFGE block preparation would allow one to choose the best cell pellet to use.

Likewise, the optimal time frame of incubation in either broth for the bacteria to produce a spore-free pellet large enough for Riboprint® and PFGE use and still not produce spores was 4 h. The stained slides made from the cell pellets that were incubated 4 h or less presented solely vegetative cells. At 5 h incubation, slides revealed spores with the vegetative cells. We also noted that cell pellets made from the SHIB cultures were consistently much smaller than those produced from the TSB cultures. While there is enough growth to test for spores, the pellet from the SHIB cultures were not always sufficient in size from a single broth culture for use in the Riboprint® or PFGE experiments unless growth from two broths were combined. The pellets from BB0022 were consistently smaller than pellets from the other strains so two broths were always inoculated and combined for each Riboprint® or PFGE cell preparation. The pellets could be very compact as was seen with CBD 63 or extremely loose and fragile as observed with BB001. Most of the strains yielded cell pellets that fell between these two extremes. By using the Riboprint® buffer instead of H2O for the last rinse and a longer centrifugation step, the cell loss could be minimized, but we were never able to produce a compact pellet with the Sterne strain.

Riboprint® and PFGE studies

Previous testing in our lab demonstrated that a short 2-min heating encouraged germination of spores and did not kill vegetative cells and so this short time was not considered for use (Luna et al. 2003). Also, when we previously attempted higher temperatures such as 90 and 100°C, the DNA proved to be unsuitable for PFGE and Riboprint® analysis. Initially, the cell preparations were processed on the heat treatment station provided with the Riboprinter® as per the instrument protocol. This instrument takes 10 min to heat samples up to the target temperature of 80°C, remains at this temperature for 10 min and then takes 10 min to cool down to room temperature. Although no spores were seen microscopically, the Riboprinter® heat treatment station did not sufficiently kill the vegetative cells, and the cell preparations produced numerous colonies (>300). Therefore, we used a hot water bath to test the heating times. Turnbull’s laboratory reported that heating spores at 80°C for 10 and 30 min would adversely affect spores and yet might not be completely sporocidal (Turnbull et al. 2007). The 80°C temperature did prevent the growth of our cell preparations indicating that the vegetative cells were killed and that either there were no spores present or the temperature had damaged the rare spores so they could not germinate. The cell preparations from the TSB averaged 1·27 × 109 ± 0·56 CFU per sample (n = 50). Thus, the total log kill of cells was determined to be 9 logs. Even incubating the cells in the hot water bath for the shorter 10 min time frame prevented growth most of the time (9 log reduction). However, two pellets that were heated for solely 10 min grew rare colonies (1–3) on BA after overnight incubation indicating that either not all of the vegetative cells were killed or in complete prevention of germination. Although we could not find spores on any of the slides that were made from these samples, it does not mean that there might not be one or two spores in a cell pellet. It is possible that such rare spores or vegetative cells have been protected from the heat in the shorter time frame, multiplying when returned to normal temperature. In contrast, the cell preparations that were heated to 80°C for 30 min before incubation did not produce any bacterial growth. Additionally, the 30 min time of heating at 80°C did not affect the Riboprint® patterns obtained. Thus, 30 min was utilized for all procedures.

There was an optimal absorbance reading at OD600 for the pellets resuspended in the Riboprinter® buffer. When the cell preparation optical density at 600 nm ranged between 0·2 and 0·37, the bands were discernible and easily recognized by the Riboprinter®. When the optical density readings were <0·2, the Riboprint® patterns produced bands that were too light and easily missed. Suspensions with larger pellets gave higher readings displaying very dark bands and many times there were extra DNA bands in the Riboprint® patterns indicating that the digestions were probably not complete (data not shown).

When digested with EcoRI or other enzymes and processed on the Riboprinter®, the DNA from pellets made after 4 h in SHIB gave reliable results for four different non-anthracis Bacillus species. These Riboprint® patterns matched those obtained from bacteria obtained directly off BA plates as per the manufacturer’s protocol. These also matched the patterns contained in the Riboprinter® computer database and were correctly identified. A UPGMA phylogenetic tree created using the BioNumerics program from Applied-Maths grouped the species together regardless of the method used (Fig. 1). When either SHIB or TSB was used for the B. anthracis isolates, the Riboprint® patterns produced after digestion with two different enzymes (AseI and PvuII) were reproducible and identified as belonging to B. anthracis, matching known patterns contained in the database. However, when TSB was used and the bacterial DNA was digested with EcoRI, the Riboprint® patterns contained extra bands (Fig. 2a). These extra bands did not occur with the DNA obtained from the SHIB grown bacteria. This problem was resolved by extending the digestion time in the Riboprinter® program when EcoRI was used (Fig. 2b).

Figure 1.

 Using Dice coefficients and UPGMA phylogenetic tree, a comparison of Riboprint® patterns from four Bacillus spp. cell preparations made from bacterial colonies grown on Blood Agar (BA) alone or also grown in SHIB. DNA is digested with EcoRI following standard manufacturer’s digestion protocol. The patterns created were not affected by the media type. Each species is clearly distinguished and separated from the other species no matter the media from which the bacteria were taken. For the lanes marked ‘SHIB’, the bacterial isolates were grown on BA overnight, followed by 4-h incubation in SHIB, centrifuged and then processed following manufacturer’s protocol. For the lanes marked ‘Blood plates’, the bacteria were grown on Blood Agar overnight and processed directly off the media plates as directed by manufacturer. SHIB, heart infusion broth with added horse serum and NaHCO3.

Figure 2.

 Riboprint® patterns ofBacillus anthracis Pasteur (CBD 63) after DNA is digested with EcoRI following manufacturer’s standard digestion time (a) and after extended time (b). Bacteria were grown first on Blood Agar overnight in microaerophilic atmosphere and subsequently grown in either TSB or SHIB for 4 h before processing. The patterns resulting from the extended digestion time gave results consistently recognized by the Riboprinter® as B. anthracis. SHIB, heart infusion broth with added horse serum and NaHCO3; TSB, tryptic soy broth.

For PFGE, we subjected the cells to the 80°C heat treatment after the cells were added to the 2% agarose, lysed, treated with restriction enzymes, and just prior to embedding the cell/agarose plug into the agarose gel. This meant that until the heating step, the samples would contain potentially viable cells that needed to be kept in the BSL3 laboratory. Previous studies in our laboratory revealed that the plugs always produced colonies on BA even after undergoing the lysing and restriction digestion steps. However, heating the plugs to 80°C for 30 min prevented all growth. We decided to perform the heating step after the plugs were lysed and the DNA digested with restriction enzymes because the BSL3 laboratory had room to hold the plugs. However, the bacterial cells could be heated for 30 min just prior to mixing the cells with the low melting point agarose when making the PFGE blocks.

In preparing cells for PFGE, it was observed that CBD 63 and CBD 131 grew in TSB supplemented with glucose and provided enough bacterial cells for the agarose blocks. The DNA bands from the two B. anthracis strains produced easily distinguishable clean distinct bands (Fig. 3). The number of bands produced by each enzyme digest matched with the expected number of bands. Electrophoresis of the SgfI digests produced 18 DNA bands. The SmaI digest produced 12 bands, while both the digest with SbfI and the double digest with AscI and NotI gave eight bands. Heating the plugs to 80°C for 30 min did not appear to harm the embedded DNA in the plugs. None of the stained microscopic slides made from the cell preparations revealed any spores. Finally, none of the agarose plugs yielded any bacterial colonies when they were placed onto BA plates and incubated indicating the absence of or deactivation of spores in the preparation.

Figure 3.

 Pulse field gel electrophoresis (PFGE) of Bacillus anthracis DNA digested with SgfI. Lane 1, Lambda ladder PFGE standard; lanes 2 and 3, B. anthracis Pasteur (CBD 63); lanes 4 and 5, B. anthracis Heluky (CBD 131).

Sterility studies

None of the numerous Riboprinter®– ready cell preparations or PFGE gel blocks made from the seven different B. anthracis strains yielded growth (Table 3). Multiple repeats of the process for each strain was performed to be certain that the results from strains that produce generous amounts of spores (Ames, Kruger, etc.) were as valid as the results from strains that are known to have more meagre spore production (Pasteur strains).

Table 3.   Results for sterility tests of cell preparations of seven Bacillus anthracis strains
Identification numberStrainBrothTests with zero growth
N (n%)
  1. SHIB, heart infusion broth with added horse serum and NaHCO3; TSB, trypic soy broth.

  2. *BB004 and CBD 131 did not grow or grew too slowly in the broth to produce a cell pellet of sufficient size for Riboprint® or pulse field gel electrophoresis analysis.

CBD 63PasteurTSB105 (100)
SHIB90 (100)
CBD 131HelukyTSB110 (100)
SHIBND*
BB001SterneTSB100 (100)
SHIB98 (100)
BB004AmesTSB108 (100)
SHIBND*
BB006KrugerTSB102 (100)
SHIB96 (100)
BB022French B2TSB53 (100)
SHIB32 (100)
BB023VollumTSB76 (100)

Discussion

We were successful in generating cell preparations of B. anthracis vegetative cells that can be killed and used for Riboprint® analysis and PFGE with minimum or no spores being present. This uncomplicated method uses increased temperature, an atmosphere consisting of a low O2 and a high concentration of CO2, and shortened incubation times to avoid the development of spores. Although we have not perfectly mimicked the mammalian host environment, it appears that these few simple steps have achieved the goal of obtaining a cell pellet that does not contain viable spores. We have also been successful in using the method on seven different B. anthracis strains known to have various proficiencies in sporulation (from adequate to copious). Additionally, we have successfully produced spore-free or nonviable spore cell preparations for a limited number of other Bacillus species (e.g. B. cereus, B. atrophaeus, B. subtilis and B. thuringiensis).

The 37°C temperature that we used not only allows optimal colony size for the different strains, it also does not rid the strains of the pXO1 plasmid as the higher temperature (42·5°C) can do. Plus, most research and reference laboratories can easily accommodate this temperature and are familiar with B. anthracis characteristics at temperatures between 25 and 37°C.

Asporogenous mutants have been reported to naturally arise in continuous cultures of B. atrophaeus (Idachaba and Rogers 2001). This may well occur with B. anthracis. However, our method employs a short incubation time on BA plates (18 h) and even shorter incubation time in broths (4 h). Thus, mutations are not expected to occur readily and the low number of mutants that might arise in 4 h should not affect the results of either the Riboprint® or PFGE analyses.

We prefer TSB for cell preparation for the Riboprinter® because all of the strains grew in TSB (with or without glucose), TSB was easier to make and less expensive than SHIB, and TSB was the broth used in our protocol for PFGE. The fact that we needed to program the Riboprinter® to extend digestion times as is needed for the cells grown in TSB is moot because the instrument’s programmes are easy to modify.

Because the various strains exhibited different phenotypic characteristics on the solid culture media, it became obvious that personnel needed to be familiar with each strain. The size of colony diameter, adherence to the media and stickiness were factors that varied among the different strains. Extreme care had to be taken to touch only the edge of the colonies especially the small colonies of BB006 (Kruger). Twice, one or two colonies appeared on a plate indicating spore survival. However, both times this occurred at the initial handling of the particular strain. After personnel better understood the growth characteristics of that strain, the efficient processing of bacteria was then able to be performed and tested. Because of this we would recommend three points. Firstly, personnel processing the cell sample need to be completely familiar with a B. anthracis isolate’s phenotypic traits on the media before attempting to barely touch an edge of a colony in order to successfully avoid the thicker part of the colony edge (not to mention the colony centre) where spores might be located. Secondly, microscopic slides should be made of the cell preparations before using for any application to ensure that no spores are present. We used the Malachite Green/Safranin spore stain method so that we could have a permanent record of each cell preparation. This allowed the slides to be reviewed later by other personnel and the results confirmed. Thirdly, no samples should be removed from the BSL3 laboratory until the growth plates show that it is safe to do so.

In conclusion, we have developed a method to prepare B. anthracis cell preparations for large molecular typing procedures that are free of viable spores. This makes the cell preparations safer for handling and safer for manipulation in a BSL2 environment that are usually more spacious and able to accommodate large instruments such as the Riboprinter® and PFGE equipment. This method would preclude the contamination of the expensive equipment and could be expanded for use in other instruments. This method also appears to successfully avoid spores in cell preparations from other Bacillus species, and further investigation in this area will be pursued. Potentially, this method could be applied to other environmental aerobic and anaerobic spore-formers.

Acknowledgements

This work was supported by the United States Department of Defense, DOD Contract Number W911SR-07-C-0084. The B. anthracis isolates from NIH were graciously contributed by the NIH Biodefense and Emerging Infections Research Resources Repository, NIAID, NIH. Isolates obtained by the Florida Department of Health were generously donated by Lea Heberlein-Larson and Frank Reeves. We wish to thank Dr Alberto Van Olphen, DVM, PhD at CBD for his information on animal husbandry.

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