Identification and Typing of Clostridia in Raw Milk in Egypt

S ummary : Three spp. of clostridia were isolated from samples of raw cow and buffalo milk obtained near Cairo. Of 150 isolates of anaerobes, 108 were Clostridium perfringens , 30 were Cl. butyricum , and 12 were Cl. sporogenes. The Cl. perfringens isolates comprised 100 nonhaemolytic and 8 pathogenic haemolytic strains. The latter strains typed by neutralization tests, were of type A. Fifteen of the nonhaemolytic strains were also of type A; of these, 6 strains produced heat resistant spores and 9 strains produced heat susceptible spores. Feeding mice with these 15 nonhaemolytic strains caused marked reduction in intestinal passage time.     

The Influence of High Concentrations of Carbon Dioxide on the Germination of Bacterial Spores

The influence of carbon dioxide at 1–55 atm on the germination of Clostridium sporogenes, Clostridium perfringens and Bacillus cereus spores in a complex medium was studied. The germination studies at atmospheric pressure were done in the pH range 5.2–6.7. Controls at the same pH were done in 100% nitrogen. Carbon dioxide at atmospheric pressure (1 atm) inhibited the spore germination of B. cereus spores but strongly enhanced the germination rate of those of the clostridia. Spore germination of Cl. sporogenes and Cl. perfringens was inhibited completely at 10 atm and at 25 atm, respectively. The germination rate in carbon dioxide or nitrogen was generally higher at pH 6.7 than at 5.2–6.0.     

The effect of carbohydrates on the sporogenesis of Clostridium perfringens and Bacillus anthracis

The authors studied the effect of a number of carbohydrates on the sporogenesis of Clostridium perfringens and Bacillus anthracis (vaccine strain STI) as probable soil factors capable of influencing the duration of survival of these causative agents in the external environment. Differences in the effect of the same sugars on the formation of spores by these microorganisms and clearly expressed sporogenesis-inhibiting effect of glucose (and also of lactose in clostridia) have been demonstrated. The analysis of the peculiarities of sporogenesis under unadjusted and stabilized pH values provides a basis for regarding the "glucose effect" as repression of sporogenesis in the given causative agent, but not as inhibition resulting from considerable acidification of the culture medium. This is essential for the soil conditions characterized by high buffer capacity. The ecological value of substances of carbohydrate nature consists in their important role in the energetics and trophicity of microbial coenoses of the soil which cannot fail reflecting on the fate of pathogenic microorganisms in the soil.     

The abattoir source of culturable psychrophilic Clostridium spp. causing 'blown pack' spoilage of vacuum-packed chilled venison

AIMS: To identify the abattoir source(s) of culturable psychrophilic clostridia causing 'blown pack' spoilage of vacuum-packed chilled meats. METHODS AND RESULTS: Psychrophilic and psychrotolerant clostridia were isolated from hides, faeces and tonsils of deer slaughter stock, and from a meat plant environment. The isolates were differentiated using restriction fragment length polymorphism analysis of the 16S rDNA gene (PCR-RFLP) and 16S-23S rDNA internal transcribed spacer (ITS) analysis. PCR-RFLP group I clostridia were found to have restriction patterns indistinguishable from the patterns of 'blown pack'-causing Clostridium gasigenes DB1A(T) and R26. Gas production in packs inoculated with vegetative cells of PCR-RFLP group I clostridia was first evident after 14 days at 2 degrees C. The prevalence of these clostridia was similar in hide and faecal samples from slaughter animals, but these micro-organisms were absent from tonsils and the meat plant environment. Banding patterns of PCR-RFLP group II clostridia showed some cross-similarity with patterns of the 'blown pack'-causing micro-organism Cl. estertheticum DSM 8809(T) and Cl. estertheticum-like meat strains. The majority of clostridia in PCR-RFLP group II were found in the faeces of slaughter animals. Isolates representing PCR-RFLP group II did not, however, produce gas in vacuum packs stored at 2 degrees C for 84 days. CONCLUSIONS: The data suggest that soil particles attached to hide or present in faeces are the most probable primary reservoir from which 'blown pack' clostridia are introduced onto carcasses. Therefore, dressing procedure hygiene remains paramount in order to control the spread of psychrophilic Clostridium spp. in a meat plant. SIGNIFICANCE AND IMPACT OF THE STUDY: The paper provides information critical for controlling 'blown pack' spoilage in meat processing plants. It reports on the use of molecular techniques for determination of abattoir sources of 'blown pack'-causing clostridia.     

Maximum shields: the assembly and function of the bacterial spore coat

Spores produced by bacilli and clostridia are surrounded by a multilayered protein shell called the coat. As the armor-like appearance of the coat suggests, this structure, along with others within the spore, confers the remarkable resistance properties that make Bacillus anthracis spores such potent biological weapons. Here, I review recent studies of coat assembly in the model organism Bacillus subtilis, and explore the implications of these findings for coat assembly in B. anthracis and for defense against biological weapons.     

Diversity of mesophilic clostridia in Costa Rican soils

Costa Rica is located in the Tropic, one of the most biologically diverse regions of the world; its soil is an important epicenter of biodiversity and Clostridium spp. are among the most frequent bacteria. The diversity of clostridia in Costa Rican soils and its possible association with geographic zone, pH or type of soil was studied in 117 soil samples: 18 from the Atlantic Zone, 30 from the Central Plateau, 30 from the Dry Pacific, 13 from the North Zone, and 26 from the South Pacific. The pH and the mesophilic clostridia species were determined for each sample. For bacterial isolation, a selective methodology for spores and pre-reduced anaerobically sterilized media were used. A total of 1945 strains of clostridia were isolated, 98% were identified and corresponded to 54 species; the most frequent species were C. subterminale (56%), C. oceanicum (51%), C. bifermentans and C. glycolicum (50%, each), C. sporogenes (49%), and C. sordellii (42%). An average of 7.1 species per sample was obtained; the Atlantic Zone showed the greatest diversity: 8.6 species per sample and a total of 45 species. Except for C. chauvoei, all described toxigenic clostridia species were isolated; C. sordellii (42%) and C. perfringens (38%) were the most frequent. No statistical relation could be established between geographic zone or type of soil and any species, showing that clostridia had a high adaptation capability to grow in different soil conditions; only some clostridia were isolated from very acidic samples while others from soils with a wide range of pH. In general, a uniform distribution of most species and a high variety of clostridia in Costa Rican soils were observed, in agreement with the high biodiversity described for other living beings in this country.     

Arbuscular mycorrhizal fungal propagules in a salt marsh

The tolerance of indigenous arbuscular mycorrhizal fungi (AMF) to stressful soil conditions and the relative contribution of spores of these fungi to plant colonization were examined in a Portuguese salt marsh. Glomus geosporum is dominant in this salt marsh. Using tetrazolium as a vital stain, a high proportion of field-collected spores were found to be metabolically active at all sampling dates. Spore germination tests showed that salt marsh spores were not affected by increasing levels of salinity, in contrast to two non-marsh spore isolates, and had a significantly higher ability to germinate under increased levels of salinity (20) than in the absence of or at low salinity (10). Germination of salt marsh spores was not affected by soil water levels above field capacity, in contrast to one of the two non-marsh spore isolates. For the evaluation of infectivity, a bioassay was established with undisturbed soil cores (containing all types of AM fungal propagules) and soil cores containing only spores as AM fungal propagules. Different types of propagules were able to initiate and to expand the root colonization of a native plant species, but spores were slower than mycelium and/or root fragments in colonizing host roots. The AM fungal adaptation shown by this study may explain the maintenance of AMF in salt marshes.     

Iron reduction and gley formation by nitrogen-fixing clostridia

Summary Studies on iron reduction and the mechanism of gley formation by nitrogen-fixing clostridia are reported. Up to 10⁶ cells/g soil of anaerobic, nitrogen-fixing clostridia, capable of reducing iron (III)-oxide, were counted in samples taken from various top soils. In a gleyed subsoil as many as 10⁵ bacteria per g soil, capable of reducing and fixing nitrogen, were enumerated using the most probable number technique. In general, the ratio of the auxotrophic iron reducing clostridia (glucose+yeast extract fermenters) to the prototrophic iron reducing flora (glucose fermenters) was found much larger in the top soil samples than in those derived from various gleyed subsoils.An enrichment method for the isolation of nitrogen-fixing, iron reducing clostridia of the butyric-butyl type is described. The iron reducing capacity of this type of clostridia as well as of Clostridium pasteurianum was determined quantitatively. Generally, the presence of soil or soil extract enhanced the amount of dissolved ferrous iron, both with butyric acid fermenters and with Cl. pasteurianum.When enriched iron reducing clostridia were incubated anaerobically under N₂-atmosphere in a sterile, red-colored, lateritic type of soil with glucose, intense gleying occurred within a few days. Microscopic observations indicated the presence of sporeforming bacteria of the Clostridium butyricum type or related species.The biological and chemical mechanism of gley formation is discussed.This research was started at the Institut für Landwirtschaftliche Mikrobiologie, Justus Liebig-Universität, Giessen, Germany.     

Low Persistence of Bacillus thuringiensis Serovar israelensis Spores in Four Mosquito Biotopes of a Salt Marsh in Southern France

We studied the persistence of Bacillus thuringiensis serovar israelensis (Bti) in a typical breeding site of the mosquito Ochlerotatus caspius in a particularly sensitive salt marsh ecosystem following two Bti-based larvicidal applications (Vectobac 12AS, 1.95 L/ha). The treated area was composed of four larval biotopes that differed in terms of the most representative plant species (Sarcocornia fruticosa, Bolboschoenus maritimus, Phragmites australis, and Juncus maritimus) and the physical and chemical characteristics of the soil. We sampled water, soil, and plants at various times before and after the applications (from spring to autumn, 2001) and quantified the spores of B. thuringiensis (Bt) and Bacillus species. The B. cereus group accounted for between 0% and 20% of all Bacillus spp. before application depending on the larval biotope. No Bti were found before application. The variation in the quantity of bacilli during the mosquito breeding season depended more on the larval biotope than on the season or the larvicidal application. More bacilli were found in soil (10(4)-10(6) spores/g) than on plant samples (10(2)-10(4) spores/g). The abundance in water (10(5) to 10(7) spores/L) appeared to be correlated to the water level of the breeding site. The number of Bti spores increased just after application, after declining; no spores were detected in soil or water 3 months after application. However, low numbers of Bti spores were present on foliage from three of the four studied plant strata. In conclusion, the larvicidal application has very little impact on Bacillus spp. flora after one breeding season (two applications).     

Clostridium perfringens type A: certain characters of epidemiologic significance.

Survival of spores of Cl. perfringens type A was significantly greater than that of vegetative cells in acid pH (pH 1.2). Survival of spores in soil varied from strain to strain. Time required for 5 million spores to be reduced to 500 per gram of soil varied from 2 to 8 months with an average of 4.5+/-2.3 months. Quantitative and qualitative heat resistance studies revealed that a majority of the Indian and all the American strains tested were heat-sensitive. These characters of Cl. perfringens have an important bearing on the epidemiology of food poisoning due to this agent.     

A medium for the isolation, enumeration and rapid presumptive identification of injured Clostridium perfringens and Bacillus cereus

A blood-free egg yolk medium (BCP) containing pyruvate, inositol, mannitol and a bromocresol purple indicator in a nutrient agar base has been developed to initiate the growth of Clostridium perfringens. It is comparable to blood agar for the growth of normal, chilled stored vegetative cells and heat-injured spores of Cl. perfringens and Bacillus cereus. It has the advantage over blood agar in exhibiting presumptive evidence of Cl. perfringens (production of lecithinase and inositol fermentation) after an overnight incubation at 43 degrees - 45 degrees C. Pyruvate, catalase and other hydrogen peroxide degraders were found to remove toxins rapidly formed in media exposed to air and light. Free radical scavengers of superoxide, hydroxyl ions and singlet oxygen were ineffective. Without scavengers the formation of 10-20 micrograms/ml hydrogen peroxide in the exposed medium was indicated and found lethal to injured Cl. perfringens. The BCP medium has been used successfully for the rapid identification and enumeration of Cl. perfringens in foods and faeces from food poisoning outbreaks and cases of suspected infectious diarrhoea. Greater recovery of severely injured vegetative Cl. perfrigens could be obtained by pre-incubation at 37 degrees C of inoculated media for 2-4 h followed by overnight incubation at 43 degrees - 45 degrees C. Tryptose-sulphite-cycloserine and Shahidi-Ferguson-perfringens agar base were found to inhibit the growth of several strains of injured vegetative Cl. perfringens. This was not completely overcome by the addition of pyruvate. The inclusion of mannitol also allows the medium to be used for the presumptive identification of B. cereus. Growth and lecithinase activity are profuse on BCP. Heat-injured spores are recovered equally well on BCP and blood agar. A scheme for the identification of some other clostridia on BCP is presented.     

Quantitative aspects of exchangeable calcium in spores of Bacillus megaterium

Rode, L. J. (The University of Texas, Austin), and J. W. Foster. Quantitative aspects of exchangeable calcium in spores of Bacillus megaterium. J. Bacteriol. 91:1589-1593. 1966.-More than 90% of the calcium in Ca(45)-labeled native spores was released from the cells during germination. Some 95% of the spore calcium was not exchangeable when ungerminated native spores were titrated to pH 4 with HNO(3). Ca, Mg, Na, Si, and Fe were displaced from the spores by H(+). The adsorption of Ca(45) by H-spores and its subsequent release were studied under a variety of conditions. The isolated "coat fraction" of spores adsorbed substantial amounts of Ca(45). Release of the adsorbed Ca was achieved with various reagents.     

Isolation and sources of 'blown pack' spoilage clostridia in beef abattoirs

Aims:  (i) To evaluate methods for isolation and molecular detection of blown pack spoilage (BPS) clostridia and (ii) to survey beef abattoirs for sources and distributions of Clostridium estertheticum and Cl. gasigenes .
Methods and Results:  Molecular detection and conventional isolation methods were used to detect and recover BPS associated clostridia ( Cl. estertheticum and Cl. gasigenes ), from four commercial Irish beef abattoirs and their environments, during a one year study. DNA-based methods detected 218 Cl. estertheticum and 300 Cl. gasigenes , from 1680 samples, whereas culture-methods only yielded 17 Cl. estertheticum and 176 Cl. gasigenes isolates. BPS Clostridia were frequently detected in beef abattoirs and their environments, especially at areas prior to hide removal. The study noted a higher percentage of positive samples during the month of May (38·6%).
Conclusions:  (i) DNA-based techniques are the most reliable ways to determine the presence of these organisms in various samples and (ii) hides and faeces are the main reservoirs of BPS clostridia in the abattoirs.
Significance and Impact of the Study:  This paper provides useful information to detect BPS organisms, as well as to develop a science-based control strategy of the problem.     

Polymerase chain reaction for detection of Clostridium botulinum types A, B and E in food, soil and infant faeces

The application of the polymerase chain reaction (PCR) for detection of Clostridium botulinum types A, B and E in foods, environmental and clinical samples was evaluated and compared to the mouse bioassay. Samples inoculated with 10, 100 and 1000 spores of Cl. botulinum types A and B included pasteurized milk, UHT milk, infant formula, infant faeces, meat juice, canned tuna, mushrooms, blood sausage and soil. Clostridium botulinum type E spores were inoculated into fish eggs, canned tuna, picked herring, raw fish and soil at similar levels. Spores were added to 2.5 g of each sample with the exception of soil which was inoculated in 10 g samples. The presence of Cl. botulinum in sample enrichments was determined by both PCR and the bioassay. An overall correlation of 95.6% was observed between PCR results and the mouse bioassay. Of the total of 114 samples tested there was disparity between the mouse bioassay and the PCR in three samples of soil inoculated with 100 type A or E spores and 10 type B spores per 10 g, respectively, and two samples of infant faeces inoculated with 10 type A or B spores per 2.5 g. All of these samples gave negative animal results and positive PCR results.     

Development of a Selective Medium for the Isolation of Clostridium sporogenes and Related Organisms

An attempt was made to develop a selective isolation medium for Clostridium sporogenes and related organisms based on the ability of these organisms to obtain their energy for growth by means of coupled oxidation-reduction reactions between appropriate pairs of amino acids (Stickland reaction). Using a semi-defined basal medium containing various combinations of amino acids, it was found that Cl. sporogenes utilized a wider range of amino acid pairs than strains of five other species of clostridia known to carry out a Stickland-type fermentation.
With alanine and proline as the principal energy sources and the medium solidified with agar. it was shown that reference strains of Cl. sporogenes and proteolytic Cl. botulinum types A, B and F could be recovered almost quantitatively, with or without prior heating at 80 °C for 10 min. By contrast, growth of test strains of Streptococcus faecalis, Strep. faecium , 'saccharolytic' Cl. botulinum types B, C, D, E and F and 'proteolytic' strains of types C and D was suppressed on this medium, as were strains of 26 other species of clostridia.
Addition of 50 μg/ml of polymyxin to the agar medium had no detectable effect on the recovery of Cl. sporogenes or Cl. botulinum. When samples of soil and mud were plated on the antibiotic-containing medium, 63.1% of 225 isolates thus obtained were identified as Cl. sporogenes/botulinum.     

Spore germination of the psychrotolerant, red meat spoiler, Clostridium frigidicarnis

Aims: To determine germination triggers of Clostridium frigidicarnis, an important spoilage bacterium of chilled vacuum‐packed meat. Methods and Results: Germination of Cl. frigidicarnis spores in the presence of a range of potential nutrient and non‐nutrient germinants was tested by monitoring the fall in optical density and by phase‐contrast microscopy. The amino acid l ‐valine induced strong germination when paired with l ‐lactate in sodium phosphate under anaerobic conditions. Several other amino acids promoted germination when paired with l ‐lactate in sodium phosphate and the co‐germinants NaHCO₃ and l ‐cysteine. Heat activation, while not necessary for germination, increased the rate of germination. Spore germination was not observed when spores were incubated aerobically. Conclusions: Spores of psychrotolerant Cl. frigidicarnis germinated in the presence of l ‐valine in combination with l ‐lactate in sodium phosphate buffer under anaerobic conditions. Significance and Impact of the Study: Anaerobic conditions, l ‐valine and l ‐lactate, have been identified as triggering germination in Cl. frigidicarnis, and are all present in packs of fresh, vacuum‐packaged, red meat. This new information adds to what is known about red meat spoilage by cold tolerant clostridia and can be used to develop intervention strategies to prevent meat spoilage.     

Isolation and Characterization of Uracil-Degrading Clostridia from Soil

Five strains of uracil-degrading clostridia were isolated from soil using an enrichment medium containing uracil as the principal energy source. All the isolates resembled Clostridium glycolicum , although they lacked the characteristic ability to ferment ethylene glycol.
Both representative isolates and a reference strain of Cl. glycolicum degraded uracil and produced β-alanine when grown in a semi-defined medium. Thus, the uracil-degrading activity was similar to that described previously for Cl. uracilicum.     

Influence of exchangeable ions on germinability of bacterial spores

Rode, L. J. (The University of Texas, Austin), and J. W. Foster. Influence of exchangeable ions on germinability of bacterial spores. J. Bacteriol. 91:1582-1588. 1966.-Native spores of Bacillus megaterium Texas, and H-spores produced by titration of native spores to pH 4 with mineral acid, did not germinate in a solution of alanine and inosine unless a strong electrolyte was present. Ca-spores prepared from either H-spores or native spores did germinate efficiently in the same solution without a strong electrolyte. Of several other bivalent cations tested, only strontium and barium could substitute for calcium in conditioning spores for subsequent germination in the absence of an electrolyte. Variable responses were obtained with different metal ion forms of 62 unidentified soil isolates and several stock species of Bacillus. Although the pattern of response was not uniform in all organisms, ions played a crucial role in the germinability of the great majority of strains tested.     

PCR detection of psychrophilic Clostridium spp. causing 'blown pack' spoilage of vacuum-packed chilled meats

AIMS: To develop a practical molecular procedure that directly, without isolation, and specifically detects the presence of clostridia which cause 'blown pack' spoilage of vacuum-packed meat. METHODS AND RESULTS: Primer sets and PCR amplification procedures were developed that detect the presence of 16S rDNA gene and/or 16S-23S rDNA internal transcribed spacer fragments of 'blown pack' causing clostridia in meat. The specificity of the developed procedures was evaluated with DNA obtained from close phylogenetic neighbours of 'blown pack' causing clostridia, food clostridia and common meat spoilage microorganisms. The sensitivity of detection was assessed in non-enriched and low-temperature-enriched beef mince inoculated with serially diluted pure cultures of Clostridium estertheticum DSMZ 8809T and Cl. gasigenes DB1AT. The efficacy of detection procedures was evaluated for naturally contaminated vacuum-packed meat samples. Three primer sets, 16SE, 16SDB and EISR, produced amplicons of the expected size with DNA templates from target clostridia, but failed to yield PCR products with DNAs from any other microorganisms tested. With 16SE and 16SDB primers, minimum levels of detection were 104 CFU g(-1) for non-enriched, and 102 CFU g(-1) for enriched meat samples. Based on the established specificity of these primers, as well as DNA sequencing of amplicons, Cl. gasigenes was confirmed as the causative agent of 'blown pack' spoilage in two packs, and Cl. estertheticum as the causative agent in the third. CONCLUSIONS: The developed method can be used for rapid detection of 'blown pack' causing clostridia in commercial blown packs, or following low temperature enrichment, for detection of these microorganisms in meat containing as few as 100 clostridial cells per gram. SIGNIFICANCE AND IMPACT OF THE STUDY: The paper reports practical procedures that can be used for rapid confirmation of the causative agents of clostridial 'blown pack' spoilage in commercial spoiled packs, or for detection of psychrophilic clostridia in epidemiological trace back of 'blown pack' spoilage incidents in meat processing plants.     

The Detection of Clostridium welchii in the Differential Reinforced Clostridial Medium Technique

S ummary . The enumeration of sparse populations of clostridia by a total clostridia count technique in a fluid medium (DRCM) gives small estimates of the numbers of Clostridium welchii. This organism may be readily detected in mixed 'presumptive total' clostridia cultures by streaking on lactose-egg yolk-milk agar before pasteurizing these cultures. The results of screening poultry carcases and processing plants with this technique are presented and the incidence of Cl. welchii on 3 sites of poultry carcases (breast, vent and neck skin) are compared for 3 processing plants.