Effect of Clindamycin on Cytotoxin Production by Clostridium difficile

A total of 80 strains of Clostridium difficile, 33 toxigenic and 11 nontoxigenic clindamycin (CLDM)-sensitive (MIC less than 12.5 μg/ml), and 23 toxigenic and 13 nontoxigenic CLDM-resistant (MIC 200 to 6,400 μg/ml) were tested for cytotoxin production in the presence of CLDM. None of the 24 nontoxigenic strains produced cytotoxin regardless of the presence of CLDM and only six out of the 56 toxigenic strains showed 16- to 64-fold higher levels of cytotoxic activity in the presence of CLDM at the concentrations of 1/2 to 1/32 of the MIC than in the absence of CLDM; all of the six strains were CLDM sensitive. Further studies revealed that addition of CLDM to the culture caused enhanced cytotoxin synthesis, and that the maximum production of cytotoxin was obtained when CLDM was added to the medium at the time of inoculation or of the ensuing early logarithmic phase. Also, the influence of other antibiotics on the effect of CLDM was examined. Addition of metronidazole, vancomycin, chloramphenicol, cephaloridine, or penicillin, which induced cytotoxin to medium containing CLDM did not increase the effect of CLDM any further. Addition of CLDM to medium containing tetracycline, which inhibited cytotoxin production, induced cytotoxin production but not fully.     

Differentiation Between Clostridium sordellii and Clostridium bifermentans by Gas Chromatography

Clostridium bifermentans can be differentiated from Clostridium sordellii by gas chromatography on the basis of amines detected after growth for 6 hr in cookedmeat medium. Products detected after exposure of resting cells to amino acids gave evidence for the probable origin of the amines.     

Clostridium sordellii bacteriophage active on Clostridium difficile

Among five strains of Clostridium difficile and 39 strains of Cl. sordellii tested, one Cl. difficile phage and four Cl. sordellii phages were found to be lytic for Cl. difficille strain 2. The five phages were similar in morphology, showing a polyhedral head of 60 nm in diameter, a tail of 105–120 nm, a contractile tail sheath and a base plate. They were sensitive to heat (60°C/10 min) and stable at 4°C for at least 6 months. As the phage donor strains and the indicator strain were not cytotoxigenic, no phage-infected culture of Cl. difficile 2 was able to produce cytotoxin.     

Urease-negative strains of Clostridium sordellii.

Twenty-seven of 37 non-toxigenic, urease-negative strains originally identified as Clostridium bifermentans that were isolated in the Antarctic are reidentified as C. sordellii by the tests for DNA-DNA homology, by the absence of mannose in the cell wall, and by growth inhibition of mannose. The test for cell wall sugar components of urease-negative and -positive strains of C. sordellii revealed that glucose, mannose, and rhamnose could not be detected in any of eight urease-negative strains used by galactose was detectable in seven of the eight strains and that glucose or galactose or both of the two sugars were present in the urease-positive strains tested.     

Heat resistance of Clostridium sordellii spores

The thermal destruction kinetics of Clostridium sordellii spores was studied in this research. Decimal reduction times (D values) for C. sordellii ATCC 9714 spores ranged between 175.60 min for D₈₀ (the D value for spore suspensions treated at 80 °C) and 11.22 min for D₉₅. The thermal resistance (Z) and temperature coefficient (Q₁₀) values of spores were calculated to be as high as 12.59 °C and 6.23, respectively. At 95 °C, the relative thermal death rate and relative thermal death time of C. sordellii ATCC 9714 spores were found to be 0.0085/min and 118 min, respectively, indicating that the death rate of spores was 118 times lower at 95 °C than at 121.1 °C. Heat treatments at up to 85 °C for 120 min failed to cause a 100-fold destruction in spore populations of C. sordellii ATCC 9714. By contrast, spore counts were reduced by 2log₁₀ cycles within 73 min and 23 min at 90 °C and 95 °C, respectively. This is the first published report of thermal inactivation of C. sordellii spores; however, further studies are needed to confirm these results in real food samples.     

Spore Appendages and Taxonomy of Clostridium sordellii

Twenty-five strains of Clostridium sordellii were divided into two groups on the basis of spore fine structure. Sixteen strains formed spores with smooth tubular appendages, and nine strains formed spores which lacked appendages. The other properties of the 25 strains were relatively constant. Since the minor strain variability which was encountered did not correlate with spore appendage status, fragmentation of this species on the basis of spore appendage status is not advocated.     

Biohydrogenation of linoleic acid by Clostridium sporogenes, Clostridium bifermentans, Clostridium sordellii and Bacteroides sp.

Abstract Several strains of Clostridium bifermentans, Clostridium sporogenes and Clostridium sordellit and one strain of Bacteroides sp. hydrogenate linoleic acid into transvaccenic acid in vitro following the same pathway. Linoleic acid (18:2; 9- cis , 12- cis ) was first isomerised into 9- cis , 11- trans -octadecadienoic acid, after which the 9- cis double bond was reduced. These species also hydrogenated linoleic acid into an octadecenoic acid in vivo when mono-associated with gnotobiotic rats. Several other species of Clostridium and Bacteroides did not hydrogenate linoleic acid.     

Structural consequences of mono-glucosylation of Ha-Ras by Clostridium sordellii lethal toxin

Mono-glucosylation of Ha-Ras by Clostridium sordellii lethal toxin at effector region threonine 35 has diverse effects on the Ras GTPase cycle, the dominant one of which is the inhibition of Ras-Raf coupling, leading to complete blockade of Ras downstream signaling. To understand the structural basis of the functional consequences of glucosylation, the X-ray crystal structure of glucosylated Ras-GDP was compared with that of non-modified Ras. Glucosylated Ras exhibits a different crystal packing but the overall three-dimensional structure is not altered. The glucose group does not affect the conformation of the effector loop. Due to steric constraints, the glucose moiety prevents the formation of the GTP conformation of the effector loop, which is a prerequisite for binding to the Raf-kinase. The X-ray crystal data also revealed the alpha-anomeric configuration of the bound glucose, indicating that the glucose transfer proceeds under retention of the C-1 configuration of the d-alpha-glucose. Therefore, glucosylation preserves the inactive conformation of the effector loop independently of the nucleotide occupancy, leading to a complete inhibition of downstream signaling of Ras.     

Taxonomic position of lecithinase-negative strains of Clostridium sordellii

Eleven out of 43 strains of Clostridium sordellii from clinical sources did not produce lecithinase activity and were not toxic to mice. However, these strains did belong to the C. sordellii group and could readily be differentiated from C. bifermentans and C. difficile on the basis of DNA-DNA homologies, carbohydrate fermentation patterns, enzyme activities, GLC analysis of fatty acid fermentation products and the electrophoretic analysis of whole cell protein extracts.     

Inositol hexakisphosphate-dependent processing of Clostridium sordellii lethal toxin and Clostridium novyi alpha-toxin

Clostridium sordellii lethal toxin and Clostridium novyi α-toxin, which are virulence factors involved in the toxic shock and gas gangrene syndromes, are members of the family of clostridial glucosylating toxins. The toxins inactivate Rho/Ras proteins by glucosylation or attachment of GlcNAc (α-toxin). Here, we studied the activation of the autoproteolytic processing of the toxins by inositol hexakisphosphate (InsP(6)) and compared it with the processing of Clostridium difficile toxin B. In the presence of low concentrations of InsP(6) (<1 μM), toxin fragments consisting of the N-terminal glucosyltransferase (or GlcNAc-transferase) domains and the cysteine protease domains (CPDs) of C. sordellii lethal toxin, C. novyi α-toxin, and C. difficile toxin B were autocatalytically processed. The cleavage sites of lethal toxin (Leu-543) and α-toxin (Leu-548) and the catalytic cysteine residues (Cys-698 of lethal toxin and Cys-707 of α-toxin) were identified. Affinity of the CPDs for binding InsP(6) was determined by isothermal titration calorimetry. In contrast to full-length toxin B and α-toxin, autocatalytic cleavage and InsP(6) binding of full-length lethal toxin depended on low pH (pH 5) conditions. The data indicate that C. sordellii lethal toxin and C. novyi α-toxin are InsP(6)-dependently processed. However, full-length lethal toxin, but not its short toxin fragments consisting of the glucosyltransferase domain and the CPD, requires a pH-sensitive conformational change to allow binding of InsP(6) and subsequent processing of the toxin.     

Requirements for Germination of Clostridium sordellii Spores In Vitro

Clostridium sordellii is a spore-forming, obligately anaerobic, Gram-positive bacterium that can cause toxic shock syndrome after gynecological procedures. Although the incidence of C. sordellii infection is low, it is fatal in most cases. Since spore germination is believed to be the first step in the establishment of Bacilli and Clostridia infections, we analyzed the requirements for C. sordellii spore germination in vitro. Our data showed that C. sordellii spores require three structurally different amino acids and bicarbonate for maximum germination. Unlike the case for Bacilli species, d-alanine had no effect on C. sordellii spore germination. C. sordellii spores germinated only in a narrow pH range between 5.7 and 6.5. In contrast, C. sordellii spore germination was significantly less sensitive to temperature changes than that of the Bacilli. The analysis of the kinetics of C. sordellii spore germination showed strong allosteric behavior in the binding of l-phenylalanine and l-alanine but not in that of bicarbonate or l-arginine. By comparing germinant apparent binding affinities to their known in vivo concentrations, we postulated a mechanism for differential C. sordellii spore activation in the female reproductive tract.Clostridium sordellii is an anaerobic, Gram-positive, spore-forming bacterium that is commonly found in soil and in the intestines of animals (4). Many C. sordellii strains are nonpathogenic; however, virulent strains cause lethal infections in several animal species, such as hemorrhagic enteritis in foals, sheep, and cattle (5, 10, 16, 28), omphalitis in foals (43), and wound infection in humans (4, 35).C. sordellii also can cause life-threatening necrotizing infections after gynecological procedures (4). In addition, fatal cases of C. sordellii endometritis following medical abortion with a mifepristone-misoprostol combination have been reported recently (13, 19, 56). The increased use of mifepristone-misoprostol for medical abortion may result in larger numbers of C. sordellii infections (38, 40).Although C. sordellii rarely has been identified in the genital tract, a correlation between gynecological procedures and C. sordellii-mediated toxic shock syndrome is apparent (19). Pregnancy, childbirth, or abortion may predispose some women to acquire C. sordellii in the vaginal tract (19). Under these conditions, C. sordellii infections result in an almost 100% mortality rate.Since there is no national system for tracking and reporting complications associated with gynecological procedures, the identification of the true rates of reproductive tract infections in women is not readily available (8). Therefore, the number of known C. sordellii-associated infections, although low, may be underreported (19, 29). Furthermore, unsafe abortion practices in developing countries cause large mortality rates due to complicating infections (24, 34). In many cases, however, the causative agent of the abortion-associated sepsis have not been characterized (24). Thus, the worldwide morbidity and mortality associated with C. sordellii infections is not currently known.C. sordellii produces several virulence factors. The two major toxins are the lethal toxin (TcsL) and the hemorrhagic toxin (37, 46). The lethal toxin produced by C. sordellii is causally involved in enteritis of domestic animals and in systemic toxicity following infections of humans (46). Furthermore, TcsL is associated with rapid mortality in C. sordellii endometritis rodent models (26). Interestingly, TcsL cytopathic effects are increased at low pH, a characteristic found in the vaginal tract (48). The hemorrhagic toxin is not well characterized, but it has been reported to cause dermal and intestinal necrosis in guinea pigs (6, 52).C. sordellii, like other Bacilli and Clostridia species, has the ability to form metabolically dormant spores that are extremely resistant to environmental stresses, such as heat, radiation, and toxic chemicals (42, 55). Upon encountering a suitable environment, spores germinate into vegetative cells, the form that is responsible for toxin production and disease onset (39, 54).In most cases, the germination process initially is triggered by the detection of low-molecular-weight germinants by a sensitive biosensor (39, 54). This sensor consists of a proteinaceous germination (Ger) receptor encoded, in general, by a tricistronic operon. Spore germination requirements have been studied most extensively for Bacilli and can be initiated by a variety of factors, including amino acids, sugars, and nucleosides (20, 30).Spore germination in the Clostridia generally requires combinations of multiple germinants. The germination of spores of proteolytic Clostridium botulinum types A and B was triggered by a defined three-component mixture comprised of l-alanine (or l-cysteine), l-lactate (or sodium thioglycolate), and sodium bicarbonate (3). In contrast, the optimum germination of spores of nonproteolytic C. botulinum types B, E, and F required binary combinations of l-alanine-l-lactate, l-cysteine-l-lactate, and l-serine-l-lactate (45).Clostridium difficile is a human pathogen that can cause fulminant colitis (11). Interestingly, C. difficile does not encode any known Ger receptors (53). However, it is likely that germination receptors exist, because C. difficile spores must germinate in order to complete their life cycle. While C. difficile germination receptors remain elusive, the spores of C. difficile germinate in rich medium supplemented with bile salts (62). More recently, taurocholate (a bile salt) and glycine (an amino acid) were shown to act as cogerminants for C. difficile spore germination (57, 61).Clostridium bifermentans is a close relative of C. sordellii (14). The minimum requirement for C. bifermentans spore germination was the presence of l-alanine, l-phenylalanine, and l-lactate (59). In addition, an unknown factor present in yeast extract was suggested to enhance germination (59). However, the Ger receptors involved in C. bifermentans spore germination are not known.Even though many Bacilli and Clostridia species use similar metabolites as germinants, the mechanisms of germinant recognition remain to be elucidated. Unfortunately, the multimeric interactions of Ger receptor complexes and the hydrophobic nature of the Ger receptor subunits have hindered our understanding of the mechanism of germinant recognition.To understand the molecular determinants of germinant recognition, we recently applied kinetic methods to study bacterial spore germination (1, 2, 18). Spore germination can be analyzed quantitatively by fitting optical density (OD) decreases to the Michaelis-Menten equation (2). The kinetic parameters obtained allow the determination of the apparent binding affinity (K_m) of spores for the different cogerminants and the maximum rate of spore germination (V_max). In these instances, K_m refers to the concentration of substrate required to reach half of the maximal germination rate. These parameters can, in turn, be used to determine the mechanism of germination and potential interactions between germination receptors. Furthermore, by comparing apparent K_m values to germinant concentrations in vivo, models for spore-germinant complex distribution can be proposed, and rate-limiting steps for the germination process can be derived. Thus, kinetic analysis can yield information on spore activation even if the identities of the germination receptors are not known.Using this procedure, we were able to determine the mechanism for Bacillus anthracis germination with inosine and l-alanine. In turn, this information was used to design nucleoside analogs that inhibit B. anthracis spore germination in vitro and protect macrophages from anthrax cytotoxicity (2).Since C. sordellii germination receptors have not been identified, we used chemical probes and kinetic methods to investigate the conditions necessary for spore germination. We found that C. sordellii spores germinate better at slightly acidic pH. Furthermore, germination rates varied slightly from 25 to 40°C. We also found that C. sordellii spores have an absolute requirement for a small amino acid, a basic amino acid, an aromatic amino acid, and bicarbonate (NaHCO₃) for efficient germination. Kinetic analysis showed allosteric interaction for the putative l-phenylalanine and l-alanine germination receptors. In contrast, l-arginine or bicarbonate recognition followed typical Michaelis-Menten kinetics. The implication of germinant recognition and host environment is discussed.