Delayed Toxin Production Research Articles

Unique Clindamycin-Resistant Clostridioides difficile Strain Related to Fluoroquinolone-Resistant Epidemic BI/RT027 Strain.

During a surveillance study of patients in a long-term care facility and the affiliated acute care hospital in the United States, we identified a Clostridioides difficile strain related to the epidemic PCR ribotype (RT) 027 strain associated with hospital outbreaks of severe disease. Fifteen patients were infected with this strain, characterized as restriction endonuclease analysis group DQ and RT591. Like RT027, DQ/RT591 contained genes for toxin B and binary toxin CDT and a tcdC gene of identical sequence. Whole-genome sequencing and multilocus sequence typing showed that DQ/RT591 is a member of the same multilocus sequence typing clade 2 as RT027 but in a separate cluster. DQ/RT591 produced a similar cytopathic effect as RT027 but showed delayed toxin production in vitro. DQ/RT591 was susceptible to moxifloxacin but highly resistant to clindamycin. Continued surveillance is warranted for this clindamycin-resistant strain that is related to the fluoroquinolone-resistant epidemic RT027 strain.

Effect of Cultured Celery Juice, Temperature, and Product Composition on the Inhibition of Proteolytic Clostridium botulinum Toxin Production

Clostridium botulinum may be of concern in prepared refrigerated meals, for which strict cold chain management cannot be guaranteed. This study evaluated the effect of temperature, product composition, and cultured celery juice powder (CCJP) as a source of nitrite on the inhibition of botulinum toxin formation in two experimental (meat- and vegetable-based) prepared meals. Data obtained from the challenge study were compared with a published mathematical model to determine whether the model is fail-safe with regard to the tested meals. Treatments were inoculated with proteolytic C. botulinum, vacuum packaged, cooked at 90°C for 10 min, and assayed for botulinum toxin at appropriate intervals in samples stored at 10, 15, or 20°C for up to 8 weeks. None of the treatments stored at 10°C for 8 weeks supported toxin production by proteolytic C. botulinum. The addition of CCJP delayed toxin production by 1 and 3 weeks in cauliflower potatoes and in Dijon pork, respectively, stored at 15°C. Toxin production was delayed by 1 week at 20°C when CCJP was added to the cauliflower potatoes. This study found that the predictive model was fail-safe but was overly conservative for the experimental meals described. Finally, this study confirms that product composition, the addition of nitrite via CCJP, storage time, and temperature play important roles in the inhibition of toxin formation by proteolytic C. botulinum.

Factors That Contribute to the Botulinal Safety of Reduced-Fat and Fat-Free Process Cheese Products

The effects of fat, type of natural cheese, and adjunct process cheese ingredients were evaluated to determine factors that contribute to the botulinal safety of reduced-fat (RF) process cheese products stored at 30°C. In the first set of experiments, pasteurized process cheese products (PPCPs) were formulated using full-fat (FF) Cheddar, 30% RF Cheddar, or skim milk (SM) cheese as cheese-base types and were standardized to 59% moisture, pH 5.75, 2.8 or 3.2% total salts, and 15 to 19% fat. Subsequent trials evaluated the effect of fat levels and adjunct ingredients in PPCPs made with SM, RF, and FF cheese (final fat levels, less than 1, 13, and 24%, respectively). When fat levels of PPCPs were comparable (15.1, 19.1, and 16.2 for product manufactured with SC, RF, and FF cheese, respectively), botulinal toxin production was delayed for up to 2 days in PPCPs formulated with SM compared with RF or FF cheese; however, the effect was not statistically significant. When fat levels were reduced to less than 1% in SM PPCPs, toxin production was delayed 2 weeks in products made with SM compared with RF or FF cheese manufactured with 13 or 24% fat, respectively. The antibotulinal effect of adjunct ingredients varied among the products manufactured with different fat levels. Sodium lactate significantly delayed toxin production (P < 0.05) for all fat levels tested, whereas β-glucan fat replacer did not delay toxin production. An enzyme-modified cheese used as a flavor enhancer significantly delayed toxin production (P < 0.05) in SM (less than 1% fat) products but had little to no inhibitory effect in RF (13% fat) and FF (24% fat) cheese products. Similarly, monolaurin increased the time to detectable toxin in SM products but was ineffective in RF or FF cheese products. These results verify that RF PPCPs exhibit greater safety than FF products and that safety may be enhanced by using certain adjunct ingredients as antimicrobials.

Nonproteolytic Clostridium botulinum Toxigenesis in Cooked Turkey Stored under Modified Atmospheres

The ability of nonproteolytic Clostridium botulinum type B spores to grow and produce toxin in cooked, uncured turkey packaged under modified atmospheres was investigated at refrigeration and mild to moderate abuse temperatures. Cook-in-bag turkey breast was carved into small chunks, surface-inoculated with a mixture of nonproteolytic C. botulinum type B spores, packaged in O2-impermeable bags under two modified atmospheres (100% N2 and 30% CO2:70% N2), and stored at 4, 10, and 15°C. Samples were analyzed for botulinal toxin and indigenous microorganisms, as well as subjected to sensory evaluation, on days 0, 7, 14, 28, 42, and 60. Given sufficient incubation time, nonproteolytic C. botulinum type B grew and produced toxin in all temperature and modified atmosphere treatment combinations. At moderate temperature abuse (15°C), toxin was detected by day 7, independent of packaging atmosphere. At mild temperature abuse (10°C), toxin was detected by day 14, also independent of packaging atmosphere. At refrigeration temperature (4°C), toxin was detected by day 14 in product packaged under 100% N2 and by day 28 in product packaged under 30% CO2:70% N2. Reduced storage temperature significantly delayed toxin production and extended the period of sensory acceptability of cooked turkey, but even strict refrigeration did not prevent growth and toxigenesis by nonproteolytic C. botulinum. At all three storage temperatures, toxin detection preceded or coincided with development of sensory characteristics of spoilage, demonstrating the potential for consumption of toxic product when spoilage-signaling sensory cues are absent.

Effect of Headspace CO2 Concentration on Toxin Production by Clostridium botulinum in MAP, Irradiated Fresh Pork

The effects of five initial levels of CO2 (15, 30, 45, 60, and 75%) and three irradiation doses (0, 0.5, and 1.0 kGy) on toxin production by Clostridium botulinum in inoculated fresh pork were studied using factorial design experiments. Headspace CO2 levels increased in all samples during storage at 15°C. In most treatments, spoilage preceded toxigenesis. Toxin production occurred faster in samples initially packaged with 15 to 30% of CO2 while higher levels of CO2 (45–75%) delayed toxin production. Low-dose irradiation delayed toxin production at all levels of CO2 in the package headspace. Contrary to expectations, including a CO2 absorbent in the package enhanced toxin production by C. botulinum. This was attributed to production of H2 by the CO2 absorbent, possibly resulting in a decrease in the oxido-reduction potential of the meat.

Effect of chlorine injury on heat-labile enterotoxin production in enterotoxigenic Escherichia coli.

Heat-labile enterotoxin (LT) production was examined in chlorine-injured and noninjured populations of enterotoxigenic Escherichia coli (ETEC) by passive immune hemolysis and Y-1 mouse adrenal tumor cell assays. Sublethally injured populations showed reduced LT production after 1, 2.5, and 4 h incubation in trypticase soy broth plus 0.25% glucose, pH 8.0. Reduction was observed during injury, resuscitation, and for at least 1.5 h following repair. LT levels comparable with that present in noninjured cells were found after 24 h incubation in the same medium, indicating delayed toxigenesis rather than permanent damage. Chlorinated populations failed to incorporate [14C]glucose until repair was completed suggesting a possible explanation for delayed toxin production. The results indicate a temporary loss of virulence among sublethally injured ETEC in chlorinated waters.

Effects of Sodium Chloride Reduction and Polyphosphate Addition on Clostridium Botulinum Toxin Production in Turkey Frankfurters

ABSTRACTMechanically deboned turkey meat emulsions were made with 1.0, 1.5, 2.0, 2.5, or 3.0% salt (NaCl), or with combinations of 1.5% or 2.0% salt with 0.4% sodium tripolyphosphate (TPP), sodium hexametaphosphate (HMP), or sodium acid pyrophosphate (SAPP). Sodium nitrite levels were constant at 150 ppm. Emulsions were inoculated with a mixture of 10 strains of C. botulinum (103/g) and incubated at 27°C. Increasing NaCl content from 1.0% to 3.0% delayed toxin production by 3 days on the average. Toxin production was detected earlier when TPP was added, HMP had no effect, and SAPP delayed toxin production.

Effects of Varying Levels of Chloride Salts on Clostridium Botulinum Toxin Production in Turkey Frankfurters

ABSTRACTInhibitory effects of sodium chloride (NaCl), potassium chloride (KCl), and magnesium chloride (MgCl2) on C. botulinum inoculated turkey frankfurter emulsions were determined. Ionic strengths (I.S.) of 0.42, 0.55, and 0.68 were compared (equivalent to 2.5%, 3.25%, and 4.0% NaCl). Sodium nitrite levels were constant at 150 ppm. Inoculated emulsions (103 spores/g) were incubated and maintained at 27°C. In‐creasing NaCl from I.S. 0.42 to 0.68 delayed toxin production from 4 days to at least 40 days. KCl was almost as effective as NaCl at I.S. 0.42, but inferior at higher levels. MgCl2 did not demonstrate any inhibitory effect. Fifty percent substitution of 2.5% NaCl with KCl or MgCl2 generally reduced time for toxin production to occur.

Inhibition of Clostridium botulinum Okra B by N-Acyl Amino Acid Ester (Nα-Cocoil Arginine Ethylester•DL-Pyrrolidone Carbonate)

An amino acid ester, Nα-cocoil-L-arginine ethylester·DL-pyrrolidone carbonate (CAE), was inhibitory to growth and toxin production of Clostridium botulinum okra in peptone-yeast extract-glucose (PYG) medium, pH 7.0, at 30°C. Addition of 10 mg of CAE/L to PYG medium delayed toxin production and 25 mg of CAE/L inhibited growth and toxin production, whereas 5 mg of CAE/L had no effect on both growth and toxin production.

Effect of carbon dioxide on toxin production by Clostridium botulinum

Ten strains of Clostridium botulinum (5 type A and 5 type B) were combined and tested for their ability to produce toxin in modified reinforced clostridial medium, pH 5.7, under different amounts of pressure of carbon dioxide at 30° C. At atmospheric pressure (101 kPa), 100% CO2 delayed toxin production when compared with an atmosphere of 100% N2. Increasing the pressure level of CO2 further delayed the onset of toxin production; however, CO2 at 891 kPa absolute did not totally inhibit production of toxin, as two of 10 samples became toxic between 4 and 8 weeks of incubation. Pressurized CO2 was lethal to C. botulinum, with the rate of decrease of recoverable colony-forming units being dependent on the amount of pressure of CO2 and the length of exposure. However, CO2 at 816 kPa absolute did not serve as a fully protective antibotulinal agent as some of the surviving spores were capable of germination and producing toxin within 8 weeks at 30° C.

Effect of the Parabens With and Without Nitrite on Clostridium botulinum and Toxin Production in Canned Pork Slurry

ABSTRACTAntimiciobials were evaluated in thioglycollate broth at pH 6.5 for the ability to inhibit growth and toxin production by C. botulinum 12885A and ATCC 7949 (Type B). Methyl, ethyl, propyl, and butyl parabens (0.1%) and sorbic acid (0.2%) were effective in inhibiting growth of C. botulinum 12885A and ATCC 7949 in broth. Ethyl, propyl, and butyl parabens (0.1%) and sorbic acid (0.2%) inhibited toxin production by both strains in culture medium. Ethyl, propyl, butyl parabens (0.1%) and sorbic acid (0.2%) were individually added to a comminuted pork slurry having salt and sugar, with or without 40 ppm sodium nitrite. Cans were inoculated with a mixture of C. botulinum 12885A and ATCC 7949 spores. The canned product was abused by holding at 27°C and was observed over a 3‐month period for swollen cans. Swollen cans were examined for botulinal toxin by the mouse bioassay. Propyl and butyl paraben did not inhibit or delay toxin production. Ethyl paraben with or without nitrite delayed toxin production for 4 wk. Sorbic acid inhibited toxin for 3 wk; when 40 ppm nitrite was added to the sorbic acid treatment, toxin production was delayed for 4 wk.

Clostridium botulinum CONTROL BY SODIUM NITRITE AND SORBIC ACID IN VARIOUS MEAT AND SOY PROTEIN FORMULATIONS

ABSTRACTClostridium botulinurn inoculated frankfurter‐type mixtures formulated with different meats (mechanically deboned chicken. meat, beef, and pork) and/or soy proteins (textured, concentrate, and isolate) were incubated at 27°C. The effects of sodium nitrite and/ or sorbic acid on botulinal spore germination, outgrowth, and toxin production were determined at specified time intervals during the incubation period. Also, residual nitrite depletion and in some instances total microbial growth were measured. Sorbic acid alone (0.2%) or in combination with nitrite (80 pg/g) retarded spore germination and outgrowth, and delayed toxin production. These effects were of larger magnitude in mixtures formulated with both nitrite and sorbic acid and were greater in beef, pork, and soy proteins compared to mechanically deboned chicken meat. Nitrite (80 μg/g) alone was only slightly effective in delaying toxin production and of significance only in pork. The highest nitrite level (156 μg/g)) tested in a meat‐soy mixture was significantly inhibitory to toxin development. Botulinal germination, outgrowth, and toxin production were slower in all‐soy, control (nitrite/sorbic acid‐free) formulations than in all‐meat, control samples. Residual nitrite depletion was slower in soy formulations, especially of the textured form, compared to all‐meat treatments. Inclusion of nitrite and/or sorbic acid in the formulation did not significantly change the rate and extent of total microbial growth in meat‐soy formulations.

The Production of Phytotoxins from Surface Crop Residues

AbstractExperiments were conducted to determine if crop residues, common to eastern Washington, were likely to cause phytotoxicity problems with no‐till planting of winter wheat (Triticum aestivum L.) The possible role of microorganisms was assessed also. Mats (5 to 8 cm thick) of lentil (Lens culinaris Medic.), pea (Pisum sativum L.), wheat, barley (Hordeum vulgare L.), and bluegrass (Poa pratensis L.) residues were spread over bare soil in Sept. 1975. Water extracts of the residues and soil beneath them were bioassayed weekly for wheat‐seedling phytotoxins, and the residues were plated biweekly to determine the numbers of fungi, bacteria, and pseudomonads from Sept. 1975 through May 1976. All residues produced wheat‐seedling root inhibitors, but only after conditions became favorable for microbial growth. Toxin production from these straws was irregular and was generally preceded by wet weather with air temperatures above freezing but below 15°C. Lentil‐ and pea‐straw extracts were most toxic to wheat seedlings in the fall and early winter, causing up to 90% root growth inhibition but little or no inhibition later. Wheat‐ and barley‐straw extracts were less inhibitory. However, these residues intermittently produced seedling inhibitors in the fall, late winter, and until late April when the straw dried. Bluegrass straw extracts showed toxic substances primarily in late winter and early spring. The delayed toxin production could explain why fall‐killed bluegrass sod was detrimental to no‐till seeded spring wheat, while spring‐killed no‐till seeded sod produced excellent wheat yields. The toxin(s) extracted were apparently different from those described by other researchers, since they (i) inhibited root growth only, (ii) were produced at lower temperatures, (iii) were produced cyclically over longtime periods, (iv) were stable when autoclaved, and (v) were not soluble in the organic solvents tested.