Comparison of methods for recovery of Clostridium difficile from an environmental surface

Survival of Clostridium difficile in an aerobic environment is possible because of spore formation. When sodium taurocholate is substituted for the egg yolk of a selective medium, cycloserine-cefoxitin-fructose-agar (CCFA), enhanced recovery of C. difficile spores is shown. This selective medium (TCCFA) does not improve recovery of vegetative forms. In this study, dry and saline-moistened swabs, adhesive paddles, and Rodac plates containing CCFA and TCCFA were compared in their ability to recover C. difficile spores from an inoculated surface. Rodac plates grew 20 to 25 times as many spores on TCCFA as on CCFA. Saline-moistened swabs recovered fewer organisms than Rodac plates. Dry swabs and adhesive paddles rarely recovered spores. Prereduction of agar in an anaerobic chamber was not necessary for optimal spore recovery. Optimal growth of vegetative C. difficile required prereduced media. Agar prereduced for 2 h supported the growth of 12 C. difficile isolates as well as agar prereduced for 18 h. Vegetative cells of C. difficile survived for only 15 min in room air. Use of Rodac plates containing TCCFA is preferred for detection of C. difficile spores in the hospital environment.     

Efficiency of various bile salt preparations for stimulation of Clostridium difficile spore germination.

Taurocholate, desoxycholate, and cholate stimulated germination of Clostridium difficile spores in broth medium and enhanced recovery of C. difficile spores on a selective agar medium. Desoxycholate and some crude taurocholate preparations also inhibited multiplication of vegetative cells. At a concentration of 1.2 X 10(-2) M, sodium cholate inhibited multiplication of vegetative cells, but at concentrations of 1.2 X 10(-3) to 2.4 X 10(-3) M, it stimulated germination without inhibiting cell multiplication. Thus, pure sodium taurocholate and sodium cholate may effectively be incorporated in cefoxitin-cycloserine-fructose agar, whereas some crude preparations of sodium taurocholate decrease recovery on this medium.     

Use of sodium taurocholate to enhance spore recovery on a medium selective for Clostridium difficile.

Isolation of Clostridium difficile from fecal specimens has been facilitated by the development of a selective and differential medium, cefoxitin-cycloserinefructose agar (CCFA). We substituted 0.1% sodium taurocholate for the 2.5% egg yolk in CCFA and compared the growth of 15 isolates of C. difficile on the resulting medium with growth on conventional CCFA. The taurocholate-containing medium (TCCFA) quantitatively recovered vegetative forms of C. difficile in the same numbers as CCFA medium. Recovery of spores was a mean 1.7 log(10) higher on TCCFA than on CCFA. Thirty-six of 60 patient stool specimens growing C. difficile gave a heavier growth on TCCFA than on CCFA, and 9 failed to yield C. difficile on CCFA. TCCFA detected spores of 75 colony-forming units per ml from artificially inoculated fecal specimens when conventional stool culturing techniques were used. Fluorescence of colonies of C. difficile was more intense on TCCFA than on CCFA. TCCFA was simpler to prepare and, overall, was more sensitive than CCFA.     

Effect of adding sodium taurocholate to selective media on the recovery of Clostridium difficile from environmental surfaces.

The recovery of Clostridium difficile on a medium containing cefoxitin, cycloserine, fructose, and egg yolk was compared with that on media containing one of three preparations of sodium taurocholate. In aerobic environments contaminated with C. difficile, media containing either crude taurocholate from Mann Research Laboratories, New York, N.Y., or pure taurocholate from Sigma Chemical Co., St. Louis, Mo., recovered organisms significantly more often than did cefoxitin-cycloserine-fructose-egg yolk agar.     

The effects of storage conditions on viability of Clostridium difficile vegetative cells and spores and toxin activity in human faeces

AIMS:Clostridium difficile is a common nosocomial pathogen and as such diagnostic and research methods may necessitate storage of faecal specimens for long periods, followed by subsequent re-examination. This study investigated the effects of storage conditions upon the viability of this organism and its toxin. METHODS: Three genotypically distinct strains of C difficile (two clinical isolates including the UK epidemic strain, and an environmental isolate) were grown anaerobically at 37 degrees C for 72 hours in a pool of five faecal emulsions. Aliquots of each emulsion were stored at either -20 degrees C (frozen) or 4 degrees C (refrigerated). Emulsions were assayed for viable cells, spores, and cytotoxin titre before storage and at days 1, 3, 5, 7, 14, 28, and 56. An aliquot of each emulsion was also removed, assayed, and replaced in storage at each time point to investigate the effects of multiple freezing/refrigeration/thawing. RESULTS: Neither storage temperature nor multiple cycles of refrigeration/freezing and thawing adversely affected the viability of C difficile vegetative cells or spores. Single and multiple exposures of samples to 4 degrees C had little effect upon the C difficile toxin titre. Toxin titres of multiply frozen and thawed faeces became significantly lower than for refrigerated faeces (p < 0.01) by day 5 of the experiment in two of the three strains, and in all strains by day 28. Toxin titres of singly frozen faeces became significantly lower than for refrigerated faeces (p < 0.01) by day 56 of the experiment in two of the three strains. CONCLUSION: Storage temperature and multiple cycles of freezing (refrigeration)/thawing had minimal effects upon the viability of C difficile or its spores. Storage at 4 degrees C has no discernible effect on C difficile cytotoxin. However, storage at -20 degrees C has a detrimental effect upon C difficile cytotoxin, and multiple cycles of freezing and thawing may further adversely effect toxin titres.     

Atmospheric entry simulations of Mars lander bioload--experiments in support of Beagle 2

Simulations of the temperature and vacuum effects of Martian atmospheric entry upon Bacillus atrophaeus (formerly Bacillus subtilis var niger; 8058; NCIMB) endospores were carried out inside a purpose-built vacuum chamber. The work formed part of the study in support of planetary protection for the Beagle 2 Mars lander and investigated to what extent the outer surface of the lander's back heat shield would be sterilised during Mars atmospheric entry. The spores were heated to peak temperatures up to 300 degrees C over 30 s under vacuum conditions (10(-3) mbar). There was no effect on spore viability until peak temperatures reached 180-200 degrees C (12-15 s of heat exposure). Spore viability then fell rapidly with increasing temperature. Once peak temperatures exceeded 300 degrees C, no further spore viability was detected. The average heating rate was rapid (10 degrees C s(-1)); thus spores were exposed to peak temperatures for less than a second. These data inform on the process of determining bioburden reduction and control steps necessary for external surfaces of spacecraft which are non-sterile at launch, as well as providing new information about the ability of a model resistant organism to survive rapid, short-duration heating.     

Analysis of latex agglutination test for Clostridium difficile toxin A (D-1) and differentiation between C difficile toxins A and B and latex reactive protein.

Virulent toxigenic and avirulent non-toxigenic strains of Clostridium difficile gave a positive result in the latex agglutination test (LAT) for C difficile toxin A (D-1). Similar concentrations of latex agglutinating antigen were produced by these strains in vivo. Positive reactions were also given by C sporogenes, proteolytic C botulinum Types A, B, and A/F, and Bacteroides assaccharolyticus. The latex agglutinating antigen was denatured by boiling for 10 minutes, but not by heating at 56 degrees C for 30 minutes. The reaction was abolished by incubation of test material with crude C difficile antitoxin but not with other clostridial antitoxins or specific antitoxin to C difficile toxin A. The latex agglutinating antigen present in C difficile eluted between 0.39% and 0.47% M sodium chloride, and that produced by the other clostridia, between 0.35% and 0.43% M sodium chloride by fast protein liquid chromatography. The latex agglutinating antigen of C difficile was neither cytotoxic nor mouse lethal and was distinct from toxin A and toxin B. In the analysis of faecal specimens from patients with diarrhoea the latex agglutination test correlated better with the presence of C difficile than with toxin B and detected both toxigenic and non-toxigenic strains. The latex agglutination test should only be used in the laboratory as an alternative to culture for C difficile and not as a method for the detection of C difficile toxins.     

Sporogenesis and toxin A production by Clostridium difficile

The kinetics of spore production by Clostridium difficile were not paralleled by release of C. difficile toxin A in vitro. Toxin A was not found to be associated with either purified whole spores or spore coats. Residual traces of toxin A detected in spore contents were almost certainly derived from contaminating vegetative cell debris. Thus, toxin A is unlikely to be a spore constituent or associated with sporogenesis.     

Rapid differentiation of Streptomyces from Nocardia by liquid chromatography

A liquid chromatographic method is described for analysis of aerobic actinomycetes for isomers of diaminopimelic acid. One or two colonies of organism were hydrolyzed with 6.0 mol of HCl per liter at 121 degrees C for 15 min. The hydrolysate was neutralized and buffered with an NaOH solution (3 mol/liter) containing 0.15 mol of sodium borate per liter. Precolumn derivatization with dansyl chloride was used to form a fluorescent product for detection. Analysis was performed by reversed-phase, ion-pair chromatography. The L-diaminopimelic acid isomer was detected in all 10 strains of Streptomyces tested, and the meso-diaminopimelic acid isomer was detected in all 10 strains of Nocardia tested. Liquid chromatography was compared simultaneously with thin-layer chromatography in the analysis of three strains of aerobic actinomycetes. Liquid chromatography required less growth of the organisms, and analysis was completed within 1 h, compared with the 3 to 5 days required by thin-layer chromatography.     

Inactivation of rabies virus in reagents used for the fluorescent rabies antibody test

Procedures for inactivating rabies virus in reagents used for the fluorescent rabies antibody test are described. Mouse brain adsorbing suspensions containing greater than or equal to 10(9) 50% lethal doses of virus per ml were rendered noninfectious by treatment with 0.1% beta-propiolactone or by heating at 56 degrees for greater than or equal to 30 min. Viable virus in tissue impression smears was inactivated by acetone fixation at 50 degrees C for greater than or equal to 30 min or by immersion in 0.1% beta-propiolactone at 37 degrees C for 2 h. Inactivated reagents gave specific and sensitive reactions in the fluorescent rabies antibody test.     

Rapid isolation of Yersinia spp. from feces.

Direct plating or cold enrichment or both have been used to isolate Yersinia spp. from feces. Freeze-shock double enrichment and KOH treatment have been recommended for recovery of Yersinia enterocolitica from surface waters and food, respectively. These techniques were evaluated as alternatives for rapid recovery of Yersinia spp. from feces. Stool samples were homogenized in buffered saline and autoclaved. Escherichia coli. Klebsiella pneumoniae, and Pseudomonas aeruginosa were each added to the suspension at a final concentration of 1.5 x 10(6) colony-forming units per ml. Yersinia cells were then added to a final concentration of 1.5 x 10(3), 1.5 x 10(4), 1.5 x 10(5), or 1.5 x 10(6) colony-forming units per ml. A total of 21 strains of Y. enterocolitica, 2 of Yersinia kristensenii, and 1 each of Yersinia intermedia and Yersinia fredriksenii were tested. For freeze-shock double enrichment, seeded stool samples were frozen overnight (-70 degrees C), transferred successively to m-tetrathionate broth (6 h. 37 degrees C) and selenite broth (2 h 37 degrees C), and plated on MacConkey, salmonella-shigella, and cellobiose-arginine-lysine agars for quantitation. For KOH treatment, seeded stool samples were mixed with 0.5% KOH at a ratio of 1:2 for 2 min and plated as described above. E. coli, K. pneumoniae, and P. aeruginosa were virtually eliminated after either method was used. All Yersinia strains were recovered after KOH treatment even at the lowest initial concentration (1.5 x 10(3) colony-forming units per ml). However, after freeze-shock double enrichment, not all strains were retrievable, and those isolates which were recovered were grown only from samples containing the highest number of Yersinia strains (1.5 x 10(6) colony-forming units per ml). KOH treatment of stool samples seems to be a viable substitute for more protracted methods of recovering Yersinia spp.     

Use of a selective enrichment broth to recover Clostridium difficile from stool swabs stored under different conditions

The recovery of Clostridium difficile from the stools of patients with C. difficile-associated diarrhea was evaluated by use of an enrichment broth (cycloserine-cefoxitin fructose broth supplemented with 0.1% sodium taurocholate [TCCFB]) and was compared to that from selective agar (cycloserine-cefoxitin fructose agar [CCFA]) and alcohol shock followed by inoculation onto blood agar (AS-BA). TCCFB was superior to CCFA and AS-BA, and neither the storage time nor the storage temperature affected the recovery rate.     

Detection of a vascular permeability factor in the extracellular products of Renibacterium salmoninarum.

The presence of vascular permeability factors in the extracellular products (ECP) of 10 strains of Renibacterium salmoninarum with different geographical origin and serological characteristics are reported. All the ECP produced haemorrhagic and/or oedematous zones at the injection site with a diameter ranging from 10-30 mm. However, the ECP samples did not display toxic effect in fish at the same dose as inoculated in rabbit (180-400 micrograms protein/0.1 ml). No differences were observed in the production of this dermatotoxic factor between the two antigenic groups found in this microorganism. Whereas heating (80 and 100 degrees C/15 min) the ECP samples resulted in a complete loss of their proteolytic activity, only a decrease (but not total inactivation) of the dermatotoxic effects was detected. Therefore, although proteases could be implicated in the permeability factor, they are not totally responsible for this activity.     

Sterilization of bacterial spores by using supercritical carbon dioxide and hydrogen peroxide

It was hypothesized that supercritical carbon dioxide (SC-CO(2)) treatment could serve as an alternative sterilization method at various temperatures (40-105 degrees C), CO(2) pressures (200-680 atm), and treatment times (25 min to 6 h), and with or without the use of a passive additive (distilled water, dH(2)O) or an active additive (hydrogen peroxide, H(2)O(2)). While previous researchers have shown that SC-CO(2) possesses antimicrobial properties, sterilization effectiveness has not been shown at sufficiently low treatment temperatures and cycle times, using resistant bacterial spores. Experiments were conducted using Geobacillus stearothermophilus and Bacillus atrophaeus spores. Spore strips were exposed to SC-CO(2) in commercially available supercritical fluid extraction and reaction systems, at varying temperatures, pressures, treatment times, and with or without the use of a passive additive, such as dH(2)O, or an active additive, such as H(2)O(2). Treatment parameters were varied from 40 to 105 degrees C, 200-680 atm, and from 25 min to 6 h. At 105 degrees C without H(2)O(2), both spore types were completely deactivated at 300 atm in 25 min, a shorter treatment cycle than is obtained with methods in use today. On the other hand, with added H(2)O(2) (<100 ppm), 6 log populations of both spore types were completely deactivated using SC-CO(2) in 1 h at 40 degrees C. It was concluded from the data that large populations of resistant bacterial spores can be deactivated with SC-CO(2) with added H(2)O(2)at lower temperatures and potentially shorter treatment cycles than in most sterilization methods in use today.     

Clostridium difficile in gnotobiotic mice.

Germfree mice associated with Clostridium difficile developed intestinal disease characterized by polymorphonuclear cell infiltration of the lamina propria, diarrhea, and cecal cytotoxin concentrations positive at a 10(-6) dilution. The numbers of viable bacteria never exceeded 10(10) colony-forming units per g (dry weight). Despite the high toxin levels and chronic inflammation over a 30-day period, the mortality rate was low (less than 2%). Daily treatment of these animals with two oral doses of 2 mg of vancomycin resulted in stool levels of greater than 200 micrograms/ml, well in excess of the minimum inhibitory concentration for C. difficile. This therapy decreased viable cell density by 2 to 3 logs and increased the spore counts from 10(5.8) to 10(7.8) colony-forming units per g (dry weight) by day 7, and animals were free of detectable toxin. However, once therapy was stopped, viable bacteria and spore counts and cytotoxin concentrations returned to previous levels. Treatment of mice with concentrations of clindamycin shown to be inhibitory in vitro had no effect on C. difficile toxin titers or bacterial counts, although the appearance of a clindamycin-resistant population was noted. These data indicate that vancomycin, given orally, decreases the concentration of toxin, but C. difficile survive as spores. By contrast, large populations of vegetative cells and high cytotoxin levels persist when clindamycin is used, even at an inhibitory concentration.     

Molecular and microbiological characterization of Clostridium difficile isolates from single, relapse, and reinfection cases

In this study, we investigated the correlation between the microbiological characteristics of Clostridium difficile clinical isolates and the recurrence of C. difficile-associated disease (CDAD). Twenty C. difficile isolates recovered from 20 single infection cases and 53 isolates from 20 recurrent cases were analyzed by pulsed-field gel electrophoresis (PFGE) and PCR ribotyping, and the cytotoxicity, antimicrobial susceptibility, and sporulation/germination rates of the isolates were examined. Recurrent cases were divided into relapse or reinfection cases by the results of C. difficile DNA typing. Among the 20 recurrent cases, 16 cases (80%) were identified to be relapse cases caused by the initial strain and the remaining 4 cases (20%) were identified to be reinfection cases caused by different strains. All 73 isolates were susceptible to both vancomycin and metronidazole, but resistance against clindamycin, ceftriaxone, erythromycin, and ciprofloxacin was found in 87.7%, 93.2%, 87.7%, and 100% of the isolates, respectively. No correlations between DNA typing group, cytotoxicity, and sporulation rate of isolates and infection status, i.e., single, relapse, or reinfection, were observed. However, the isolates recovered from relapse cases showed a significantly higher germination rate when incubated in medium lacking the germination stimulant sodium taurocholate. These results indicate that the germination ability of C. difficile may be a potential risk factor for the recurrence of CDAD.     

Comparison of the effects of acid and base hydrolyses on hydroxy and cyclopropane fatty acids in bacteria

The cellular fatty acid compositions of Legionella oakridgensis, Brucella suis, Pseudomonas aeruginosa, and Francisella tularensis were compared after base hydrolysis (saponification), acid hydrolysis, and acid methanolysis procedures were used to release the fatty acids. The branched-chain, unsaturated, saturated, and ester-linked hydroxy acids were released as effectively with saponification at 100 degrees C for 30 min as with acid hydrolysis or acid methanolysis at 85 degrees C for 16 h. Although the amide-linked hydroxy acids were released more effectively by acid hydrolysis or acid methanolysis, these methods degraded the cyclopropane fatty acids, producing a number of new peaks or artifacts in the chromatograms. Cyclopropane fatty acids were not degraded by saponification, and at least 50% of the hydroxy acids were released when the cells were saponified with 15% NaOH in 50% aqueous methanol. Thus, the results show that saponification for 30 min at 100 degrees C with 15% NaOH, followed by methylation is an excellent method for routine fatty acid analysis of bacteria and for screening cultures whose identity and fatty acid composition are unknown.     

Studies of the influence of nicotinamide on the development of heat resistance in Bacillus cereus T

Effects of nicotinamide (NA) on the development of heat resistance and sporulation in Bacillus cereus T were studied. It was observed that NA produces more than 95% heat labile spores (80°C for 30 min). The growth and the utilization of poly-β-hydroxybutyrate (PHB) is not affected. The heat labile spores were morphologically comparable to untreated spores but the levels of spore specific proteins and dipicolinic acid (DPA) were only 15–20% of the maximum. NA-treated spores germinate poorly ( 10%) and a storage of 2 months at 4°C causes complete loss of germination characteristics. However, the exogenous addition of DPA or l-kynurenine completely reverses all effects indicating a possible involvement of L-tryptophan metabolism in the development of heat resistance.     

Enhancement of recovery of Legionella pneumophila from contaminated respiratory tract specimens by heat.

Heating of Legionella pneumophila and other Legionella spp. was studied to determine whether this technique could be used as a selective technique with contaminated clinical specimens. Studies of 13 different strains of Legionella spp. showed heterogeneous heat survival; heating at 60 degrees C for 1 to 2 min did not affect the survival of the majority of strains. Heating of four Pseudomonas aeruginosa strains at 60 degrees C for 2 min reduced bacterial counts by 98% or greater. Enterococci were heat tolerant, with virtually no inhibition under the same conditions. No inoculum effect was noted for any of the organisms tested. Heating of eight contaminated clinical specimens before plating on buffered charcoal-yeast extract medium reduced the numbers of contaminants on most plates but increased by only one the number of specimens yielding L. pneumophila. Plating the same specimens on selective media with or without heat pretreatment yielded L. pneumophila in every case. Heating of clinical specimens at 60 degrees C for 1 to 2 min before plating may occasionally increase the recovery of L. pneumophila from contaminated specimens, but this technique should not be generally used.     

Differential fall in ATP accounts for effects of temperature on hypoxic damage in rat hippocampal slices

Intracellular recordings, ATP and cytosolic calcium measurements from CA1 pyramidal cells in rat hippocampal slices were used to examine the mechanisms by which temperature alters hypoxic damage. Hypothermia (34 degrees C) preserved ATP (1.7 vs. 0.8 nM/mg) and improved electrophysiologic recovery of the CA1 neurons after hypoxia; 58% of the neurons subjected to 10 min of hypoxia (34 degrees C) recovered their resting and action potentials, while none of the neurons at 37 degrees C recovered. Increasing the glucose concentration from 4 to 6 mM during normothermic hypoxia improved ATP (1.3 vs. 0.8 nM/mg) and mimicked the effects of hypothermia; 67% of the neurons recovered their resting and action potentials. Hypothermia attenuated the membrane potential changes and the increase in intracellular Ca(2+) (212 vs. 384 nM) induced by hypoxia. Changing the glucose concentration in the artificial cerebrospinal fluid primarily affects ATP levels during hypoxia. Decreasing the glucose concentration from 4 to 2 mM during hypothermic hypoxia worsened ATP, cytosolic Ca(2+), and electrophysiologic recovery. Ten percent of the neurons subjected to 4 min of hypoxia at 40 degrees C recovered their resting and action potentials; this compared with 60% of the neurons subjected to 4 min of normothermic hypoxia. None of the neurons subjected to 10 min of hypoxia at 40 degrees C recovered their resting and action potentials. Hyperthermia (40 degrees C) worsens the electrophysiologic changes and induced a greater increase in intracellular Ca(2+) (538 vs. 384 nM) during hypoxia. Increasing the glucose concentration from 4 to 8 mM during 10 min of hyperthermic hypoxia improved ATP (1.4 vs. 0.6 nM/mg), Ca(2+) (267 vs. 538 nM), and electrophysiologic recovery (90 vs. 0%). Our results indicate that the changes in electrophysiologic recovery with temperature are primarily due to changes in ATP and that the changes in depolarization and Ca(2+) are secondary to these ATP changes. Both primary and secondary changes are important for explaining the improved electrophysiologic recovery with hypothermia.