Clostridium perfringens: Comparative effects of heat and osmotic stress on non-enterotoxigenic and enterotoxigenic strains

Accepted Manuscript Clostridium perfringens: Comparative Effects of Heat and Osmotic stress on nonenterotoxigenic and enterotoxigenic strains Cinthia Carolina Abbona, Patricia Virginia Stagnitta PII:

S1075-9964(16)30021-X

DOI:

10.1016/j.anaerobe.2016.03.007

Reference:

YANAE 1549

To appear in:

Anaerobe

Received Date: 30 June 2015 Revised Date:

18 March 2016

Accepted Date: 18 March 2016

Please cite this article as: Abbona CC, Stagnitta PV, Clostridium perfringens: Comparative Effects of Heat and Osmotic stress on non-enterotoxigenic and enterotoxigenic strains, Anaerobe (2016), doi: 10.1016/j.anaerobe.2016.03.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Clostridium perfringens: Comparative Effects of Heat and Osmotic

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stress on non-enterotoxigenic and enterotoxigenic strains

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Cinthia Carolina Abbona1,*, Patricia Virginia Stagnitta2

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Cuyo, Mendoza, Argentina. E-mail address: [email protected] Tel +54

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0261 15686 9806. 2 Departamento de Química Biológica Facultad de Química

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Bioquímica y Farmacia, Universidad Nacional de San Luis, San Luis, Argentina.

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E-mail address: [email protected]

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*Corresponding author

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SUMMARY

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IBAM-CONICET and Facultad de Ciencias Agrarias, Universidad Nacional de

C. perfringens isolates associated with food poisoning carries a chromosomal

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cpe gene, while non-foodborne human gastrointestinal disease isolates carry a

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plasmid cpe gene. The enterotoxigenic strains tested produced vegetative cells

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and spores with significantly higher resistance than non-enterotoxigenic strains.

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These results suggest that the vegetative cells and spores have a competitive

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advantage over non-enterotoxigenic strains. However, no explanation has been

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provided for the significant associations between chromosomal cpe genotypes

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with the high resistance, which could explain the strong relationship between

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chromosomal cpe isolates and C. perfringens type A food poisoning. Here, we

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analyse the action of physical and chemical agent on non-enterotoxigenic and

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enterotoxigenic regional strains. And this study tested the relationship between

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the sensitivities of spores and their levels SASPs (small acid soluble proteins)

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production in the same strains examined.

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KEY WORDS: Clostridium perfringens; food safety; SASPs; C-cpe strains; heat

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resistance; osmotic stress; antibiotic resistance.

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1. INTRODUCTION

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Clostridium perfringens is a Gram-positive anaerobic spore-forming bacterium

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known to be the most widely distributed pathogen in nature (1-3). In many

ACCEPTED MANUSCRIPT countries more than half of all outbreaks of foodborne diseases are caused by

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meat and meat products, which are mostly associated with the toxoinfection

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produced by C. perfringens (4, 5). A few bacteria can survive after cooking food.

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They can be multiplied to infections doses if the environmental conditions are

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adequate (2, 6, 7). The presence of spores in some cooked food may be

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unavoidable. Even though the food cooking destroys the vegetative forms and

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spores of heat-sensitive strains (6-9), the heat-resistant spores can germinate

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and develop in unsuitable cooked and cooled meat (10). If already cooked meat

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is not cooled down, spores of C. perfringens can germinate and the number of

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vegetative cells will rapidly increase (11). C. perfringens isolates are classified

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into one of five types, types A through E (12, 13), based upon their abilities to

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produce the four major lethal toxins, the alpha, beta, epsilon and iota toxins.

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The major lethal toxins, however, are not the only medically important toxins;

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some C. perfringens isolates can produce C. perfringens enterotoxin (CPE).

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The CPE enterotoxin is produced by nearly all cases of C. perfringens type A

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food poisoning which involves enterotoxigenic type A C. perfringens isolates,

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expression of CPE by some type C and D C. perfringens isolates have also

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been described (14-16). This bacteria is generally considered to be a food

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pathogen since some bacteria isolated are able to produce and release CPE in

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the gastrointestinal tract, resulting in diarrhea (17-19).

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The gene encoding the enterotoxin (cpe) is only carried by 1 to 5% of all C.

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perfringens isolates in general (20), and by 11 to 15.6% in foodborne isolates

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(18, 19). The cpe gene is usually located in the bacterial chromosome (C-cpe),

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but in some cases in the plasmid (P-cpe) (21-23). C. perfringens food poisoning

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was mainly associated with strains carrying the C-cpe gene (20), but plasmid-

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borne (P-cpe) strains are now also recognized as causative factors of

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foodborne (24).

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C-cpe strains, both vegetative cells and spores, display enhanced resistance to

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the heat, osmotic and nitrites stress in comparison to P-cpe strains (6, 25). In

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addition, the vegetative cells and spores of C. perfringens type A with C-cpe

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gene survive in larger quantities than without C-cpe gene from isolated food

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poisoning outbreaks (6, 9, 26). Neither the deletion of the C-cpe gene nor the

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curing of plasmid in P-cpe strains alters its susceptibilities, suggesting that the

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ACCEPTED MANUSCRIPT resistance properties of C-cpe strains are linked with parameters other than

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CPE production (25, 27). Thus, the basis for resistance of C-cpe strains to heat

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and other environmental stress factors remains unknown.

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Clostridium species have the capacity to form metabolically inert spores, which

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are extremely resistant to heat, environmental stress, radiation and toxic

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chemicals (28-31). The spores of Clostridium species contain a group of

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proteins that protect DNA in the spores. The proteins are called small acid-

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soluble proteins type α/β (SASP), which surround the spore DNA. The SASP

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alter both physical structure and chemical reactivity in the spore protecting the

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DNA from the heat and some genotoxic chemicals (8, 30). Spores of

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Clostridium species contain multiple α/β-type, but not γ-type, SASPs and three

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genes (ssp1, 2 and 3) coding for α/β-type SASPs (32). However, the possible

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relationship between SASP and the high resistance of C-cpe strains remain

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unknown. In general, these kinds of proteins can be determined by

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polyacrylamide gel electrophoresis at low pH (8, 33, 34). We here analyse the

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level of SASP production in the same strains which were examined for

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resistance to different agents. Thereby, this study searches if the higher

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resistance of strains C-cpe is associated with a higher level of SASP

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production.

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On the other hand, the increased resistance to antimicrobial agents demands

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periodic reviews of the sensitivity profiles, which can detect the emergence of

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resistant strains and trends in hospitals and different geographical areas (35-

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39). Antimicrobial susceptibility testing is useful and important for bacterial

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species that have acquired resistance mechanisms. Tetracycline (37, 40-43)

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and metronidazole (44) resistance is a phenotype commonly found in anaerobic

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pathogen C. perfringens. For this reason, we tested the antimicrobial

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susceptibility.

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Even though there are differences across regions in terms of sensitivity to

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various factors including the spores produced by different strains (45), here we

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report the higher resistance of C-cpe strains with regard to P-cpe strains. In

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addition, we carried out an assay to test the level of SASP protein production

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and to compare between C-cpe and P-cpe strains.

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2. MATERIALS AND METHODS

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Five strains of enterotoxigenic C. perfringens with the enterotoxin gene in chromosomal location (C-cpe), which were isolated from meat (E1, E2, E3, E4,

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E5) (46); and five non-enterotoxigenic strains (NE1, NE2, NE3, NE4, NE5) were isolated from spices (47). All of them were included in this study. All isolates were maintained as stock cultures in cooked meat medium (Oxoid) and stored at -20°C. The starters vegetative cultures (6 ml) o f each strain were prepared by

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2.1 Bacteria and growth conditions.

inoculating 0.1 ml of cooked meat medium stock culture into 6 ml of fluid thioglycolate broth (FTG; Difco),all of which were overnight growth at 37°C. The

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sporulation cultures were prepared by inoculating 0.2 ml of starter FTG medium culture into 10 ml of Tortora modified medium (TMM) (46), which was then incubated at 37°C for 7 days. The presence of sporu lating cells in each culture was checked by microscopy.

2.2 Heat effect on vegetative cells.

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A 0.2 ml aliquot of an overnight FTG medium starter culture was inoculated into

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6 ml of FTG medium and grown for 2 h at 37°C. After mixing, a 0.1 ml aliquot of

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the 2 h FTG medium culture, it was aseptically withdrawn and serially diluted

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(dilutions range, 10-2 to 10-7) with sterile FTG medium. The diluted samples

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were plated onto brain heart infusion agar (BHIA; BD Biosciences) plates to

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determine the total number of vegetative cells present in each FTG medium

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culture at the start of heating (i.e., at the zero-time point of the experiment).

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The remainder of each 2 h FTG medium culture was heated at 55°C. After

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blending, a 0.1 ml aliquot of each heated culture was removed. At 55°C, the

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aliquots were removed every 5 min for up to 30 min. The aliquots were diluted

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(dilutions range, 10-2 to 10-7) with sterile FTG medium, and dilutions were

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immediately plated onto BHIA plates. After 16 to 20 h of anaerobic incubation,

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colonies on the BHIA plates were counted to determine the number of viable

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CFU, which were present per milliliter of heated culture at each time point. The

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CFU average values were graphed and the decimal reduction value (D value)

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was determined for vegetative cells of enterotoxigenic and non-enterotoxigenic

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strains.

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2.3 Heat effect on spores.

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The TMM cultures prepared and grown with each strains were heat shocked at

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75°C for 20 min, which killed the remaining vegetat ive cells and facilitated spore germination (48). A 0.1 ml aliquot of each heat-shocked TMM culture was then serially diluted with FTG medium to obtain dilutions ranging from 10-2 to 10-7. Each dilution was plated onto BHIA plates to establish the number of viable

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spores per milliliter of TMM culture at the zero-time point of the experiment. The remainder of each heat shocked TMM culture was heated at 100°C from 1

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to 60 minutes. At each time point, the heated TMM culture was mixed, and a 0.1

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ml aliquot was withdrawn and diluted from 10-2 to 10-7 with sterile FTG medium.

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The dilutions were then plated onto BHIA plates, which were incubated

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anaerobically at 37°C for 16 to 20 h. Colonies whic h developed from germinated

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spores that survived heating were counted to determine the number of viable

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spores present per milliliter of each heated TMM culture. The average data of

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enterotoxigenic and non-enterotoxigenic were graphed, and the D values were

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determined for spores.

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2.4 Effects of supplemental NaCl or NaNO2 on vegetative cell.

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A 0.2 ml aliquot of a starter FTG culture was inoculated into 6 ml FTG medium

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and grown for 2 h at 37°C. After mixing, a 0.1 ml a liquot of each 2 h FTG culture was serially diluted (dilutions from 10-2 to 10-7) with sterile water. The diluted samples were then plated onto BHIA, which were incubated anaerobically at 37°C for 16 to 20 h to determine the total number o f vegetative cells present in each FTG culture prior to the introduction of osmotic and nitrite stress. In order to evaluate the effects of those stresses, aliquots of the same diluted samples were plated onto BHIA supplemented with the following: a) for osmotic stress experiments, BHI (which contains 0.5% NaCl) was supplemented with 0, 1, 2, 3, or 4% NaCl; b) for nitrite stress experiments, BHIA was supplemented

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ACCEPTED MANUSCRIPT with 0, 0.1, 0.2, 0.3, or 0.4% NaNO2. After overnight incubation, CFU values were determined for the above plates, and those results were graphed to determine the effects of each stress condition on growth. To compare vegetative cell survival, a 2 h FTG culture was prepared and the

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total number of vegetative bacteria present at day zero was determined, as

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described above. The remainder of each 2 h culture was mixed and divided into

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1 ml. These aliquots were centrifuged and the pellets were washed with one of

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the following solutions: a) standard FTG (pH 7.6); b) FTG supplemented with an

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additional 3% NaCl; c) FTG supplemented with 0.35% NaNO2. Each washed

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pellet was suspended in the same modified FTG used for washing. Those

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samples were incubated at room temperature for up to 4 days. At defined time

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intervals, a 0.1 ml aliquot was removed from one tube of each sample, diluted

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(dilution range from 10-2 to 10-7) with sterile water, and then immediately plated

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onto BHIA. After an overnight anaerobic incubation, colonies on each BHIA

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plate were counted and the CFU values obtained were then graphed to

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determine the effects on survival of each type of stress.

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The addition of NaCl or NaNO2 (dissolved separately and filter sterilized with a

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0.45-µm Millipore filter) did not affect the pH of BHIA. 2.5 Spore survival or outgrowth in the presence of osmotic, nitrite-

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induced stress.

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A 0.2 ml aliquot of a starter FTG culture was inoculated into 10 ml of TMM. After

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overnight incubation at 37°C, the presence of sporu lating cells in each TMM

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culture was confirmed by phase-contrast microscopy. Following mixing, each

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sporulating TMM culture was heat shocked at 75°C fo r 20 min to kill any

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remaining vegetative cells and to promote subsequent spore germination and

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outgrowth. A 0.1 ml aliquot of each heat-shocked TMM culture was then serially

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diluted (dilution range from 10-2 to 10-7) with sterile water. Those diluted

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samples were plated onto BHIA plates, which were then incubated

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anaerobically overnight at 37°C to determine the to tal number of spores in each

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TMM culture that were capable of outgrowth in the absence of osmotic or

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NaNO2-induced stress.

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ACCEPTED MANUSCRIPT To determine the sensitivity of spore outgrowth to potentially stressful NaNO2 or

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osmotic conditions, we used BHIA plates containing supplemental NaCl or

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NaNO2, as described above for vegetative cell growth in the presence of these

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two stresses. After overnight anaerobic incubation, CFU values for each plate

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were graphed to determine the effects of NaNO2 or supplemental NaCl on

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spore outgrowth.

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To evaluate spore survival under osmotic or nitrite stress, a TMM culture was

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divided into several small tubes, which were washed with one of the following:

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standard FTG: a) FTG supplemented with 6% NaCl, pH 7.6; b) FTG

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supplemented with 0.5% NaNO2, pH 7.6. After washing, the bacteria were

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suspended in the same modified FTG used for washing and the suspended

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bacteria were then aliquoted into 1 ml tubes, which they were incubated at room

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temperature. To assess the effects of osmotic stress on spore survival, 0.1 ml

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aliquots were removed at 2-month intervals (for up to 6 months) from the tubes

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containing bacteria suspended in FTG supplemented with 6% NaCl, pH 7.6. To

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assess the effects of spore incubation in FTG containing 0.5% NaNO2 on spore

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survival, 0.1 ml aliquots were removed from the tube every month, for up to 3

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months. After an aliquot was withdrawn, the tube was sterilized and discarded.

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Removed aliquots were heat shocked at 75°C for 20 m in and then diluted (from

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10-2 to 10-7) with sterile water. Each dilution was immediately plated onto

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standard BHIA plates, which were anaerobically incubated overnight at 37°C.

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Colonies on each BHIA plate were counted the next morning to determine the

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number of viable spores that had been present per milliliter of culture at each

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measured time point for each stress treatment. The values were graphed to

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determine the log reduction in viable spores over 3 or 6 months.

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2.6 Statistical analyses.

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Statistical analyses were performed using the Student t test.

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2.7 Acid electrophoresis.

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The spores used were free of growing or sporulating cells and germinated spores, as determined by phase-contrast microscopy. Spores (~6 mg, dry weight) were dry ruptured, SASP were extracted with 1 ml and then 0.5 ml ice-

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ACCEPTED MANUSCRIPT cold 0.1 mM HCl, and both supernatant fluids were pooled, dialyzed in 3,500molecular-weight-cutoff dialysis tubing against 1% acetic acid, and lyophilized (49). The dry residue was dissolved in 30 µl 8 M urea, 15 µl acid gel diluent was added, aliquots from ~1.3 mg (dry weight) of spores were subjected to polyacrylamide gel electrophoresis at low pH, and the gel was stained with Coomassie blue as described elsewhere (49).

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2.8 Antimicrobial susceptibility testing.

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An inoculum concentration of 5 x 105 CFU/ml were incubated in FTG for 24 h at

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37°C under anaerobic conditions. All the strains we re tested twice for

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metronidazole and tetracycline, both were applied in eight concentrations 0.5, 1,

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2, 4, 8, 16, 32 and 64 mg/L. A suspension without antibiotic was used as

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control. The MIC (Minimum inhibitory concentration) was defined as the lowest

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antibiotic concentration that showed no turbidity after 18 h of incubation.

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3. RESULTS

3.1 The effects of heating vegetative cells.

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Enterotoxigenic

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enterotoxigenic strains were compared with respect to their heat sensitivities.

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The D values were determined for vegetative cells heated at 55° C. The Figure

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1 shows thermal death curve results obtained at 55° C. It is the average value

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from five enterotoxigenic and five non-enterotoxigenic strains. The D value for

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enterotoxigenic and non-enterotoxigenic strains was 11 and 4 min, respectively.

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Since microscopic inspection did not reveal the presence of any spores, these

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heat sensitivity differences were not attributed to the presence of heat-resistant

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spores in medium cultures.

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The D values measured for five enterotoxigenic strains surveyed were twice the

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D values of the five non-enterotoxigenic (the differences are statistically

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significant at P < 0.05). There was no significant difference (P > 0.05) between

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enterotoxigenic strains. Microscopic examination confirmed that only vegetative

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cells were present in FTG medium cultures of each isolate shown in Table 1.

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carrying a

chromosomal cpe gene and non-

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strains

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Consistent with this observation, no colonies were obtained when FTG medium

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cultures of each isolate were heat-shocked at 75°C for 20 min before plating on

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BHIA and overnight anaerobic incubated at 37°C.

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Figure 1: Thermal death curves for vegetative cells of enterotoxigenic (E) and nonenterotoxigenic (NE) strains. Vegetative cultures of E and NE were heated at 55°C for specified times, and the number of viable bacteria per milliliter of each heated culture was then determined. The CFU average value of five E and five NE were graphed.

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Table 1: Vegetative cells heat resistance of enterotoxigenic (E) and non-enterotoxigenic (NE) strains at 55°C. D value

E1 11.27

E2 11.27

E3 11.27

E4 11.26

E5 11.28

Average 11.27

D value

NE1 3.97

NE2 3.98

NE3 3.97

NE4 3.97

NE5 3.97

Average 3.97

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ACCEPTED MANUSCRIPT 3.2 The effects of heating on spores.

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Then the assays were performed to evaluate the heat sensitivity of spores.

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Representative thermal death curves obtained at 100°C for heat-shocked TMM

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cultures of enterotoxigenic and non-enterotoxigenic strains are shown in Fig. 2.

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The D value for enterotoxigenic spores was approximately 20-fold-higher than

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non-enterotoxigenic spores. The D value for spores at 100°C was 30 min to

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enterotoxigenic strains. However, the D value was only 1.4 min to non-

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enterotoxigenic strains Table 2. A highly significant difference was obtained

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between enterotoxigenic and non-enterotoxigenic strains. Phase-contrast

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microscopy analysis confirmed that high levels of spores were present in heat-

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shocked TMM cultures of both enterotoxigenic and non-enterotoxigenic strains.

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Figure 2: Thermal death curves for sporulation cultures of strains enterotoxigenic (E) and nonenterotoxigenic (NE). Heat-shocked TMM cultures of E and NE were heated at 100°C for specified times, and the number of viable spores per milliliter of each culture was determined. The average data of five enterotoxigenic and five non-enterotoxigenic were graphed.

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Table 2: Heat resistance of spores produced by enterotoxigenic (E) and non-enterotoxigenic (NE) strains E1 E2 E3 E4 E5 Average D value 29.94 30.03 30.40 29.71 29.78 29.97

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NE1 1.32

NE2 1.35

NE3 1.36

NE4 1.35

NE5 1.36

Average 1.35

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The effect of NaCl was assayed on vegetative cell growth. The medium BHIA

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supplemented with <2% of additional NaCl was found to cause, on average, a

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1-log reduction in growth of non-enterotoxigenic strains (Fig. 3A). However, the

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supplementation of BHIA should be with approximately 3% of additional NaCl to

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cause an average 1-log drop in growth of enterotoxigenic strains. These

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differences of osmotic sensitivity were statistically significant at P < 0.01.

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Moreover, the addition of <0.25% NaNO2 to BHIA was sufficient to cause, on

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average, a 1-log reduction in growth of non-enterotoxigenic strains (Fig. 3B).

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Nevertheless, near to 0.35% NaNO2 had to be added to BHIA to decrease the

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average growth of enterotoxigenic strains by 1 log. In addition, these differences

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were statistically significant at P < 0.01. Besides, vegetative cells of the most

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sensitive enterotoxigenic strains were able to grow at higher concentrations of

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NaCl or NaNO2 than the most resistant non-enterotoxigenic strains.

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Figure 3: Effects of supplemental NaCl or NaNO2 on vegetative cell growth or spore outgrowth. The average concentration of supplemental NaCl (A) or NaNO2 (B) needed to cause a 1-log reduction in vegetative growth (left) or spore outgrowth (right) is shown for five enterotoxigenic and five non-enterotoxigenic strains. For each treatment, the concentration of supplemental NaCl or NaNO2 needed to reduce vegetative growth or spore outgrowth by 1 log was determined in three independent experiments. Error bars indicate standard deviations. Double asterisks indicate differences that were statistically significant at P < 0.01.

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ACCEPTED MANUSCRIPT 3.4 Effects of supplemental NaCl or NaNO2 on spore outgrowth.

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The effects of NaCl on spore outgrowth between enterotoxigenic and non-

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enterotoxigenic strains were compared. A 1-log reduction in spore outgrowth for

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non-enterotoxigenic strains (Fig. 3A) was caused with ~2.5% of additional NaCl

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to medium BHIA. Nevertheless, BHIA had to be supplemented with nearly 3.5%

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of additional NaCl to produce an average 1-log drop in spore germination and

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outgrowth enterotoxigenic strains. A significant difference (P < 0.01) in spore

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outgrowth sensitivity was obtained to osmotic stress between enterotoxigenic

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and non-enterotoxigenic strains

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Moreover, a 1-log reduction in spore outgrowth for non-enterotoxigenic strains,

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on average, was caused for the addition of ~0.3% NaNO2 to BHIA (Fig 3B). In

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addition, 1-log reduction in spore outgrowth for enterotoxigenic strains, on

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average, the medium BHIA had to be supplemented with nearly 0.5% NaNO2.

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Besides, these differences were significant (P < 0.01) between enterotoxigenic

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and non-enterotoxigenic strains. Therefore, no overlap in the sensitivity of spore

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outgrowth to NaCl or NaNO2 was seen between enterotoxigenic versus non-

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enterotoxigenic strains.

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3.5 Effects of supplemental NaCl or NaNO2 on vegetative cell survival.

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Vegetative cell survival was tested in FTG supplemented with 3% additional

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NaCl. The incubation of 1.5-day was sufficient to cause 1-log reduction in

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vegetative cell viability for non-enterotoxigenic strains (Fig. 4A). In contrast, the

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enterotoxigenic strains had to be incubated in this medium for nearly 3 days to

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get a similar 1-log decrease in vegetative cell viability. A significant difference

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(P < 0.01) was obtained between enterotoxigenic and non-enterotoxigenic

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strains in osmotic stress survival.

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Moreover, the enterotoxigenic strains survived more than non-enterotoxigenic

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strains in FTG containing 0.35% NaNO2 (Fig. 4B). In fact, these differences

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were also statistically significant at P < 0.01. In addition, no overlap was

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obtained between enterotoxigenic versus non-enterotoxigenic strains in the

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presence of supplemental NaCl or NaNO2.

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Figure 4: Effects of supplemental NaCl or NaNO2 on vegetative cell (A) or spore (B) survival for enterotoxigenic and non-enterotoxigenic strains. (A) Average number of days of incubation in FTG supplemented with either 3% additional NaCl (left panel) or FTG containing 0.35% NaNO2 (right panel) needed to a cause a 1-log reduction in viability of vegetative cell cultures. (B) Average log reduction in viable spores. Upon incubation for 6 months in FTG containing 6% supplemental NaCl (left panel) or for 3 months in FTG containing 0.5% NaNO2 (right panel). For both panels, results shown are from three independent experiments for each strain.

ACCEPTED MANUSCRIPT Error bars indicate standard deviations, while double asterisks indicate differences that were statistically significant at P values of <0.01.

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3.6 Effects of supplemental NaCl or NaNO2 on spore survival.

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The spores were incubated for 6 months in FTG supplemented with 6% NaCl. It

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caused (on average) nearly a fourfold-higher log reduction in viability of spores

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produced by non-enterotoxigenic strains compared to spores of enterotoxigenic

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strains (Fig. 4B). Likewise, spores incubated for 3 months in FTG containing

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0.5% NaNO2 (Fig. 4B) of non-enterotoxigenic strains showed, on average,

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nearly a threefold-higher log reduction in viability compared to spores from

394

enterotoxigenic strains. These spore survivals showed a significant difference

395

(P < 0.01) between enterotoxigenic and non-enterotoxigenic strains in the

396

presence of supplemental NaCl or NaNO2.

397

3.7 Acid-gel electrophoresis.

398

The expression levels of proteins SASP were the same for the different strains

399

(Figure 5).

E2

E4

E5

NE2

NE3

NE4

NE5

AC C

EP

E1

TE D

400 401 402

M AN U

SC

RI PT

384 385 386

403 404 405 406 407 408

Figure 5: Acid-gel electrophoresis of SASP extracts from spores of various strains. Spores were prepared, purified, and extracted; the extracts were processed; aliquots were subjected to acidgel electrophoresis; and the gels were stained. The extracts were run from spores of the fourenterotoxigenic strains (lane 1 to 4) and four non-enterotoxigenic strains (lane 5 to 8).

409

3.8 Antimicrobial susceptibility testing.

410

C. perfringens C-cpe enterotoxigenic (E) and non-enterotoxigenic (NE) strains

411

were

412

metronidazole is shown in Table 3. The NE5 non-enterotoxigenic strain showed

evaluated

regarding

turbidity.

The

activity

of

tetracycline

and

ACCEPTED MANUSCRIPT 413

a high MIC 32 mg/L to tetracycline. Similarly, the enterotoxigenic E4, E5 and

414

non-enterotoxigenic NE3 strains showed a MIC 16 mg/L. Metronidazole was the

415

most active agent tested. Only the non-enterotoxigenic NE5 strain was the least

416

susceptible to metronidazole with a MIC 16 mg/L.

417 Table 3: Antimicrobial susceptibility: C. perfringens C-cpe enterotoxigenic (E) and not enterotoxigenic (NE) strains were evaluated regard of turbidity (+) or no turbidity (-). NE5 strain showed high resistance to tetracycline with a MIC 64 mg/L. In addition, the E4, E5 and NE3 strains were resistance with a MIC 16 mg/L. The NE5 strain was the only resistance to metronidazole with a MIC 16 mg/L.

RI PT

418 419 420 421 422 423

0,5

1

2

4

SC

Antibiotic (mg/L) 8

16

32

64

+

+ +

NE2

+

-

+

NE3

+

-

+

NE4

+

-

+

NE5

+

+ +

E1

+

+ +

E2

+

+ +

E3 E4 E5

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

+

-

+

-

+

-

-

-

-

-

-

-

-

+

-

-

-

-

-

-

-

-

-

-

-

+ + + +

+ +

+ +

-

+

-

-

-

-

+

-

+

-

-

-

-

-

-

-

-

-

-

+

-

-

-

-

-

-

-

-

-

-

-

+

+ +

-

-

-

-

-

-

-

-

-

-

-

-

-

+

-

+

-

+

-

+

-

+

-

-

-

-

-

-

-

+

-

+

-

+

-

+

-

+

-

-

-

-

-

-

-

4. DISCUSSION

AC C

426

-

EP

424 425

+ + +

M AN U

NE1

TE D

Strains

T M T M T M T M T M T M T M T M

427

4.1 Heat

428

This study provides more evidence on the fact that vegetative cells and spores

429

of enterotoxigenic strains are significantly more heat-resistant than non-

430

enterotoxigenic strains of C. perfringens. These differences agree with previous

431

studies (6, 9, 48, 50), which have revealed differences in heat sensitivity

432

between spores produced by different C. perfringens isolates. Besides, the

433

vegetative cells of enterotoxigenic strains carrying a chromosomal cpe showed

ACCEPTED MANUSCRIPT more tolerance to the heat. This resistance benefits the bacteria, which due to

435

their multiplication in improperly cooked food, cause food poisoning.

436

Even though this is one of the main factors that contributes to the food

437

poisoning (51, 52), C. perfringens only causes this effect if the CPE is present

438

(6, 53). For this reason, the resistance to heat seems to be an important

439

characteristic of virulence (6), which was present in the studied enterotoxigenic

440

C-cpe strains.

441

4.2 NaCl, NaNO2

442

The NaCl is a good antimicrobial agent due to the fact that the external water

443

activity is reduced (54). So much so that the incidences of C. perfringens type A

444

food poisoning outbreaks that involve cured meats is very low (55). Therefore,

445

our current results indicate that the spores and vegetative cells of

446

enterotoxigenic strains are particularly resistant to osmotic and nitrite-induced

447

stress compared to the non- enterotoxigenic ones. This higher resistance offers

448

further explanation for the connection between the enterotoxigenic strains and

449

food poisoning.

450

The spores are essentially free of internal water, through which it is not

451

surprising that NaCl does not have a significant effect on spore viability (25).

452

Notably for food safety, the spores of enterotoxigenic strains are particularly

453

tolerant to nitrite in relation to non-enterotoxigenic strains. The concentration of

454

nitrite to inhibit spore outgrowth was similar to another study (56). Interestingly,

455

vegetative cells of C-cpe strains are particularly nitrite resistant to both their

456

growth and survival. Previous studies (57) have shown that nitrites can inhibit a

457

number of clostridia enzymes and factors such as ferredoxin. The resistance of

458

vegetative cells and spores of enterotoxigenic strains to osmotic and nitrite-

459

induced stress are identified in this study. This could be important for the

460

survival and growth of C-cpe strains in food prior to human transmission.

461

However, food preservation factors can act synergistically (57, 58). More

462

studies should be carried out to characterize the specific mechanisms used to

463

resist temperature, osmotic, and nitrite stresses.

AC C

EP

TE D

M AN U

SC

RI PT

434

ACCEPTED MANUSCRIPT

467 468 469 470 471 472 473 474 475 476 477 478

The electrophoresis at low pH enabled us to define the bands that correspond tothe SASPs proteins. The C-cpe strains as well as the non-enterotoxigenic ones showed the same level of SASPs. Hence the high resistance in the enterotoxigenic strains would not be connected to the unsuitable expression of the ssp genes or to the reduced production of SASPs. These results are unlike

RI PT

466

what was exposed by other authors (34) and they agree with Raju and col in 2007 (30). In later investigations the presence of a new protein SSP4 will be determined in these regional strains. It is known that a substitution of Asp by Gly in remainder 36 is present in strains with sensitive spores (32).

SC

465

4.3 Acid-gel electrophoresis

Interestingly, recent molecular epidemiological studies showed that the C-cpe strains belong to a different MLST cluster (18, 59). The highest robustness of

M AN U

464

the latter strains might allow a competitive advantage of this lineage. The distinct resistance properties might be the result of horizontal gene transfer and vertical evolutionary adaption in a specific niche or environmental selection. 4.4 Antibiotics

480

The strains used in this study were obtained from food (46, 47). One of them

481

showed high tetracycline MIC in agreement with other publications (60-67).

482

Tetracycline-resistance has been the most common antibiotic resistance found

483

in C. perfringens (68, 69). The high percentage of C. perfringens isolates with an

484

MIC of ≥16 µg/ml for tetracycline is the resistance breakpoint for human

485

anaerobes and veterinary isolates (70, 71). The discovery of resistant C.

486

perfringens strains emphasizes the importance of surveying antimicrobial

487

susceptibility profiles of common bacterial pathogens. Based on the results,

488

tetracycline is a poor antimicrobial choice for the treatment of C. perfringens, due

489

to the reduced susceptibility of C. perfringens isolates. Furthermore, it is known

490

that exposure to tetracycline concentrations below the MIC may induce

491

conjugative transfer of tetracycline resistance plasmids from Bacteroides

492

species. The gene transfer can occur not only within the Bacteroides genus but

493

also between Gram-positive bacteria (72). Administration of tetracycline,

494

especially at a low dose for treatment of C. perfringens, could select resistant

AC C

EP

TE D

479

ACCEPTED MANUSCRIPT strains and might potentially stimulate the transfer of tetracycline resistance. The

496

strain that offered greater resistance to metronidazole also offered resistance to

497

tetracycline. The antibiotic indiscriminate use and overuse against anaerobic

498

bacteria impose a selective pressure (73, 74). This information is particularly

499

important in the face of increasing reports of resistance among anaerobic

500

bacteria isolated from humans, pigs, and poultry (75). The antibiotic exhibitions

501

near MIC impose a selective pressure. Therefore, the bacteria become resistant

502

under this condition (76). This current study confirms the low susceptibility to

503

tetracycline and metronidazole. Because of this, antibiotics should be used

504

judiciously in antibiotics for humans and animals (73). Even so, in some cases,

505

they can develop antibiotic resistance (63, 77, 78). The most common antibiotic

506

treatments for the C. perfringens associated with diarrhea includes beta-

507

lactamics

508

tetracycline (79, 80). This study provides a better understanding of the fact that

509

the resistance exists in the beginning of a treatment.

510

Although the antibiotics are of no value in treating a patient suffering from food

511

poisoning, but a possible threat could be the uptake of antibiotic-resistant

512

bacteria in high numbers into the human microflora and exchange of resistance

513

genes with components of the indigenous flora. C. perfringens clearly reveals an

514

antibiotic resistance development similar to other bacteria found in antibiotic

515

abundant habitats.

SC

amoxicillin),

erythromycin,

M AN U

and

metronidazole

and

TE D

(ampicillin

EP

516

RI PT

495

We thank C. C. Abbona for laboratory assistant and J. P. Mower for critical

518

reading of the manuscript. This work was supported by NIH (FIRCA-BB-R03-

519

TW008353-01 to J.D.P. and M.V.SP.), by ANPCyT (PICT-193 to M.V.S-P.), by

520

CONICET/NSF (RD#138/13 to M.V.S-P), and by NSF (#1062432 to Indiana

521

University), which supports the computer cluster on which many of the analyses

522

were performed.

AC C

517

ACCEPTED MANUSCRIPT

5. CONCLUSIONS

523

Enterotoxigenic cells and spores develop a high degree of resistance to heat. It

525

could increase their survival in unsuitably cooked or heated food. The greater

526

resistance to extreme temperatures and osmotic stress from C-cpe strains,

527

provide more suitable environment for food poisoning. The presence of acid

528

soluble proteins at similar levels could infer that the difference in expression is

529

not responsible for the increased resistance of the enterotoxigenic strains.

530

Thus, the results pf this study may contribute to the searching of an explanation

531

about higher resistance of C-cpe strains. Further research to identify factors

532

responsible for C-cpe strains resistance, as well as to elucidate the mechanism

533

of action of α/β-type SASP, will help in understanding the mechanism of

534

resistance of C-cpe strains spores to stress factors.

535

There are no significant differences in the antibiotic resistance among

536

enterotoxigenic and non-enterotoxigenic strains. However, metronidazole

537

resistance of regional strains should be considered when making antibiotic

538

treatment. This study contributes to the emerging view which indicates that the

539

C-cpe strains responsible for most food poisoning outbreaks are highly adapted

540

to their role as a foodborne pathogen. In conclusion, the broad-spectrum

541

resistance phenotype of enterotoxigenic strains highlights the need to use these

542

specific strains for food microbiology studies of C. perfringens and for

543

establishing food safety control parameters. This knowledge may well have

544

applied implications in the areas of food safety and food preservation.

AC C

EP

TE D

M AN U

SC

RI PT

524

ACKNOWLEDGEMENTS

545 546

We thank Dra. Ana María Stefanini de Guzmán for critical reading of the

547

manuscript. This work was supported by Secretaría de Ciencia y Técnica of

548

Universidad Nacional de San Luis. Project 8803.

6. BIBLIOGRAPHY

549 550 551 552

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651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700

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39.

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ACCEPTED MANUSCRIPT Highlights

The enterotoxigenic Clostridium perfringens strains with chromosomal cpe gene showed more tolerance to heat than non-enterotoxigenic strains.

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The spores and vegetative cells of enterotoxigenic strains are particularly resistant to osmotic and nitrite-induced stress.

The high resistance in the enterotoxigenic strains would not be connected to the unsuitable

expression of the ssp genes or to the reduced production of acid-soluble spore proteins (SASPs).

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The more common antibiotic treatment for C. perfringens includes metronidazole and

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tetracycline. The resistance exists in the beginning of a treatment.