Heat resistance differences are common between both vegetative cells and spores of Clostridium perfringens type F isolates carrying a chromosomal vs plasmid-borne enterotoxin gene

ABSTRACT

Clostridium perfringens type F isolates utilize C. perfringens enterotoxin (CPE) to cause food poisoning (FP) and nonfoodborne gastrointestinal diseases. The enterotoxin gene (cpe) can be located on either the chromosome or plasmids, but most FP isolates carry a chromosomal cpe (c-cpe) gene. Our 2000 article in Applied and Environmental Microbiology (66:3234–3240, 2000, https://doi.org/10.1128/aem.66.8.3234-3240.2000https://doi.org/10.1128/AEM.66.8.3234-3240.2000) determined that vegetative cells and spores of c-cpe isolates are more heat resistant than those of plasmid cpe (p-cpe) isolates, which is favorable for their survival in improperly cooked or held food. However, that 2000 article was recently retracted (90:e00249-24, 2024, https://doi.org/10.1128/aem.00249-24). To our knowledge, the 2000 article remains the only study reporting that heat resistance differences are common between both vegetative cells and spores of type F c-cpe isolates vs type F p-cpe isolates. To confirm and preserve this information in the literature, the heat resistance portion of the 2000 study has been repeated. The 2024 results reproduced the 2000 results by indicating that, relative to the surveyed type F p-cpe isolates, the vegetative cells of surveyed type F c-cpe isolates are ~2-fold more heat resistant and the spores of most surveyed c-cpe isolates are ~30-fold more heat resistant. However, consistent with several reports since our 2000 paper, one surveyed type F c-cpe isolate (which did not appreciably sporulate in 2000 but sporulated in 2024) produced spores with intermediate heat sensitivity, confirming that spores of some type F c-cpe isolates lack exceptional heat resistance.

IMPORTANCE

Clostridium perfringens type F food poisoning (FP), which is the second most common bacterial cause of FP, involves the production of C. perfringens enterotoxin. While the enterotoxin gene (cpe) can be located on either the chromosome or plasmids in type F isolates, most FP cases are caused by chromosomal cpe isolates. The current results support the conclusion that the vegetative cells and spores of type F chromosomal cpe isolates are often more heat resistant than vegetative cells and spores of type F plasmid cpe isolates. Greater heat resistance should favor the survival of the spores and vegetative cells of those chromosomal cpe isolates in temperature-abused food, which may help explain the strong association of type F chromosomal cpe strains with FP.

MATTERS ARISING

To cause type F food poisoning (FP) or nonfoodborne human gastrointestinal diseases (NFD), Clostridium perfringens type F strains must produce enterotoxin (1). The enterotoxin gene (cpe) is either chromosomal or located on large plasmids (2–4). Type F strains carrying a chromosomal cpe gene (c-cpe strains) cause ~70% of type F FP cases, while type F plasmid cpe strains (p-cpe strains) cause NFD, as well as ~30% of type F FP cases (3–6).

In 2000, we reported in this journal (7) the seminal observation that both vegetative cells and spores of type F c-cpe FP isolates often exhibit greater heat resistance than those of type F p-cpe NFD isolates. This observation was informative for understanding type F FP since enhanced heat resistance should improve survival of type F c-cpe FP isolates in temperature-abused foods and would help explain the strong association of type F c-cpe isolates with FP. Recently that 2000 article was retracted (8) for reasons unrelated to the heat sensitivity findings. To our knowledge, no subsequent study has reported greater heat resistance is common for both vegetative cell and spores of type F c-cpe vs p-cpe isolates, so this point should be retained in the literature. Consequently, we recently re-assayed the vegetative cell and spore heat resistance properties for the same type F strains surveyed in our 2000 article.

To compare heat resistance properties between vegetative cells of these type F strains, 2 h FTG cultures were shifted to 55°C. Aliquots were removed every 2 min for up to 20 min, plated onto BHI agar, and, after overnight anaerobic incubation at 37°C, CFU were counted to determine D₅₅, i.e., the time necessary at 55°C to reduce vegetative cell viability by one log. The 2024 results recapitulated those from 2000 (Table 1). The D₅₅ for vegetative cells of the six surveyed type F c-cpe strains averaged 9.6 min (range 6.3–14.8 min) in the 2024 analyses, compared to a 13.1 min D₅₅ average (range 11.2–16.5 min) for the same strains in 2000. In contrast, the D₅₅ for vegetative cells of the seven surveyed type F p-cpe strains averaged 5.1 min (range 3.0–7.8 min) in 2024, compared to a mean of 6.7 min (range 5.0–9.1 min) in 2000. Therefore, in both our 2024 and 2000 analyses, the surveyed type F c-cpe vegetative cells averaged ~2-fold more heat resistance at 55°C than the surveyed type F p-cpe vegetative cells. This finding differs from those in another study (9) reporting no vegetative cell viability differences between c-cpe vs p-cpe type F strains at 60°C. However, the D₆₀ values in that study were very small, so this higher temperature may mask strain-related differences in vegetative cell heat resistance.

TABLE 1.

Vegetative cell heat resistance of cpe-positive C. perfringens strains

Isolate	Origin	D value (55°C) min-2024	D value (55°C) min-2000	Isolate references
Isolates carrying a chromosomal cpe gene
NCTC8239	Humans, Europe, 1950s	11.7	16.5	(10)
191–10	Humans, United States, 1990s	14.8	11.2	(10)
C1841	Humans, United States, 1980s	8.9	13.6	(10)
FD1041	Humans, United States, 1980s	6.3	12.5	(10)
E13	Humans, United States, 1960s	7.5	12.1	(10)
NCTC10239	Humans, Europe, 1950s	8.3	12.3	(10)
Average		9.6 ± 1.3	13.1 ± 0.9
Isolates carrying a plasmid cpe gene
F5603	Humans, Europe, 1980s	5.6	5.6	(10)
F4969	Humans, Europe, 1980s	6.5	9.1	(10)
NB16	Humans, Europe, 1980s	7.8	8.6	(10)
B40	Humans, Europe, 1980s	4.9	5.0	(11)
222	Veterinary, United States, 1990s	3.0	6.7	(10)
153	Veterinary, United States, 1990s	4.9	6.8	(10)
485	Veterinary, United States, 1990s	3.2	5.0	(10)
Average		5.1 ± 0.65	6.7 ± 1.5

To compare spore heat resistance among these same type F strains, Duncan-Strong (DS) sporulation medium cultures were grown for 24 h at 37°C. Aliquots of each culture were heat-shocked at 70°C to kill vegetative cells and induce spore germination. A 0.1 mL aliquot of each heat-shocked DS culture was serially diluted with PBS and plated onto BHI agar plates to establish the number of viable spores at time zero. The remainder of each non-heat-shocked DS culture was incubated at 100°C for 1–80 min. At each time-point, a 0.1 mL aliquot was removed, diluted with PBS, and plated onto BHI agar. BHI plates were anaerobically incubated overnight at 37°C. Colonies, which developed from germinated spores, were counted to determine viable spores/mL and calculate D₁₀₀, i.e., the time required at 100°C to cause a one log reduction in spore viability.

Again, results of the 2024 analyses resembled the 2000 results (Table 2). Spores of the five type F c-cpe strains surveyed in both studies averaged a D₁₀₀ of 53.3 min (range 43.3–64.3 min) in 2024 vs an average D₁₀₀ of 60 min (range 30–124 min) measured in 2000. In contrast, spores of the seven surveyed type F p-cpe strains averaged a D₁₀₀ of 1.8 min (range 1.3–3.4 min) in 2024 vs an average D₁₀₀ of 1.0 min (range 0.5–1.9 min) reported in 2000. Therefore, compared to the type F p-cpe spores, spores of the five type F c-cpe strains examined in both 2000 and 2024 exhibited exceptional spore heat resistance, i.e., ~30-fold or ~60-fold higher D₁₀₀ values.

TABLE 2.

Spore cell heat resistance of cpe-positive C. perfringens strains

Isolate	Origin	D value (100°C) min-2024	D value (100°C) min-2000	Isolate references
Isolates carrying a chromosomal cpe gene
NCTC8239	Humans, Europe, 1950s	43.3	124	(10)
191–10	Humans, United States, 1990s	64.3	67	(10)
FD1041	Humans, United States, 1980s	49.7	32	(10)
E13	Humans, United States, 1960s	45.5	30	(10)
NCTC10239	Humans, Europe, 1950s	63.7	45	(10)
Average		53.3 ± 4.5	60.0 ± 18
C1841	Humans, United States, 1980s	5.6		(10)
Isolates carrying a plasmid cpe gene
F5603	Humans, Europe, 1980s	1.4	0.6	(10)
F4969	Humans, Europe, 1980s	1.7	0.5	(10)
NB16	Humans, Europe, 1980s	3.4	1.9	(10)
B40	Humans, Europe, 1980s	1.4	1.6	(11)
222	Veterinary, United States, 1990s	1.3	0.9	(10)
153	Veterinary, United States, 1990s	1.7	1.3	(10)
485	Veterinary, United States, 1990s	1.5	0.5	(10)
Average		1.8 ± 0.3	1.0 ± 0.3

Spore heat resistance for type F c-cpe strain C1841 was not determined in 2000 because it did not sporulate sufficiently. However, during the 2024 study, enough sporulation was obtained to calculate a D₁₀₀ for this strain. Interestingly, C1841 spores had an intermediate D₁₀₀ value of 5.6 min, i.e., more than surveyed type F p-cpe strains but less than the other surveyed type F c-cpe strains. Since our 2000 study, other type F c-cpe isolates producing spores that lack exceptional heat resistance have also been identified (6, 9, 12), but those strains are apparently less common than c-cpe isolates producing highly resistant spores. For example, using a different methodology from the current study, Grant et al. (6) reported ~80% vs ~20% of their surveyed c-cpe isolates make spores with, respectively, exceptional vs intermediate heat resistance. Notably, spores of c-cpe isolates surveyed by Grant et al. were consistently more heat resistant than spores of p-cpe strains, with the single exception of an unusual p-cpe strain producing spores with exceptional heat resistance.

Recently, Jaakkola et al. (9) distinguished two c-cpe type F groups, i.e., c-cpe group 1, which form very heat-resistant spores similar to most c-cpe isolates surveyed in our studies, vs c-cpe group 2, which usually make less heat-resistant spores and carry certain genes (e.g., the nanJ sialidase gene) absent from c-cpe group 1. Consistent with that report, C1841 formed spores with intermediate heat sensitivity and carries nanJ, while NCTC8239, NCTC10239, FD1041, or E13 form very heat-resistant spores and lack nanJ [this study and reference (13)]. However, exceptions to these patterns are evident, with Jaakkola et al. identifying (9) a c-cpe group 2 isolate forming spores with a 32 min D₉₉ and our reporting (12) that type F c-cpe strain 01E809, which is nanJ-positive, forms spores with a 50 min D₁₀₀; the 01E809 result is interesting since 01E803, another nanJ-positive type F c-cpe strain from the same type F FP outbreak, makes spores with a 0.7 min D₁₀₀ (12, 14).

Production of a small acid-soluble protein-4 (Ssp4) variant (12) with Asp at residue 36 is often important for type F c-cpe strains to make highly resistant spores. This Ssp4 variant is carried by 01E809, NCTC8239, NCTC10239, and 191-10 (12). In contrast, most type F p-cpe strains [including NB16, F4969, F5603, and 222 (12)] forming sensitive spores produce a Ssp4 with Gly at residue 36. The current study sequenced a portion of the C1841 ssp4 gene (Genbank accession number: PP726198), which like 01E803, encodes an Ssp4 with Gly at residue 36, offering an explanation for the less heat-resistant spores produced by C1841 vs the other surveyed c-cpe strains in this study.

Summarizing, the 2024 study confirmed 2000 findings that type F c-cpe FP strains often produce more heat-resistant vegetative cells and spores than most type F p-cpe strains and supports the emerging view that a minority of c-cpe FP isolates, like C1841, produce spores with intermediate heat sensitivity.