Draft Genome Sequence of Clostridium cochlearium Strain AGROS13, Isolated from a Sheep Dairy Farm in New Zealand

ABSTRACT We report the draft genome sequence of a new Clostridium cochlearium strain, AGROS13, which was isolated from a sheep dairy farm environment in New Zealand. The genome is 2.7 Mbp, with a GC content of 28.2%. The genome sequence was found to be closely related to that of Clostridium cochlearium ATCC 17787. The new strain harbors a biosynthetic gene cluster coding for an unknown sactipeptide.

Intrinsic Class D β-Lactamases of Clostridium difficile

ABSTRACT Clostridium difficile is the causative agent of the deadly C. difficile infection. Resistance of the pathogen to β-lactam antibiotics plays a major role in the development of the disease, but the mechanism of resistance is currently unknown. We discovered that C. difficile encodes class D β-lactamases, i.e., CDDs, which are intrinsic to this species. We studied two CDD enzymes, CDD-1 and CDD-2, and showed that they display broad-spectrum, high catalytic efficiency against various β-lactam antibiotics, including penicillins and expanded-spectrum cephalosporins. We demonstrated that the cdd genes are poorly expressed under the control of their own promoters and contribute only partially to the observed resistance to β-lactams. However, when the cdd1 gene was expressed under the control of efficient promoters in the antibiotic-sensitive Clostridium cochlearium strain, it produced high-level resistance to β-lactams. Taken together, the results determined in this work demonstrate the existence in C. difficile of intrinsic class D β-lactamases which constitute a reservoir of highly potent enzymes capable of conferring broad-spectrum, clinically relevant levels of resistance to β-lactam antibiotics. This discovery is a significant contribution to elucidation of the mechanism(s) of resistance of the clinically important pathogen C. difficile to β-lactam antibiotics. IMPORTANCE C. difficile is a spore-forming anaerobic bacterium which causes infection of the large intestine with high mortality rates. The C. difficile infection is difficult to prevent and treat, as the pathogen is resistant to many antimicrobial agents. Prolonged use of β-lactam antibiotics for treatment of various infectious diseases triggers the infection, as these drugs suppress the abundance of protective gut bacteria, allowing the resistant C. difficile bacteria to multiply. While resistance of C. difficile to β-lactam antibiotics plays the major role in the development of the disease, the mechanism of resistance is unknown. The significance of our research is in the discovery in C. difficile of β-lactamases, enzymes that destroy β-lactam antibiotics. These findings ultimately can help to combat deadly C. difficile infections.

Influence of Long-Chain Polyphosphate and Heat Treatment on Clostridium cochlearium and Clostridium sporogenes Isolated from Processed Cheese Spread

The outgrowth of Clostridium spp. spores causes spoilage in processed cheese products due to gas and off-odor formation. The present study focuses on the response of spores of Clostridium sporogenes and Clostridium cochlearium at 25°Cto polyphosphate, both alone and in combination with heat treatment. The two strains used were isolated from spoiled cheese spread. The addition of 1.5% polyphosphate but not 0.75% polyphosphate totally inhibited the growth of C. sporogenes SIK4.3; in contrast, 0.75% polyphosphate was sufficient to totally inhibit C. cochlearium CCUG 45978. The highest polyphosphate concentration tested (1.5%) was sporicidal for C. sporogenes SIK4.3 but not for C. cochlearium CCUG 45978. When 0.75% polyphosphate Bekaplus FS was combined with a holding time of 5 min at 98°C, no survival or growth of C. sporogenes SIK4.3 was detected; however, the same effect was not achieved through heating alone or through application of polyphosphate alone. C. cochlearium CCUG 45978 was more heat tolerant, as shown by higher D-values. In conclusion, the results strongly suggest that polyphosphate Bekaplus FS has the potential to restrict the growth of C. sporogenes and C. cochlearium in cheese spread stored at ambient storage temperature. Experiments with cheese are needed in order to verify this effect.

Native Corrinoids from Clostridium cochlearium Are Adeninylcobamides: Spectroscopic Analysis and Identification of Pseudovitamin B12 and Factor A

ABSTRACT The corrinoids from the obligate anaerobe Clostridium cochlearium were extracted as a mixture of Coβ-cyano derivatives. From 50 g of frozen cells, approximately 2 mg (1.5 μmol) of B12 derivatives was obtained as a crystalline sample. Analysis of the corrinoid sample ofC. cochlearium by a combination of high-pressure liquid chromatography and UV-Vis absorbance spectroscopy revealed the presence of three cyano corrinoids in a ratio of about 3:1:1. The spectroscopic data acquired for the sample indicated the main components to be pseudovitamin B12(Coβ-cyano-7"-adeninylcobamide) (60%) and factor A (Coβ-cyano-7"-[2-methyl]adeninylcobamide) (20%). Authentic pseudovitamin B12 was prepared by guided biosynthesis from cobinamide and adenine. Both pseudovitamin B12 and its homologue, factor A, were subjected to complete spectroscopic analysis by UV-Vis, circular dichroism, mass spectrometry, and by one- and two-dimensional 1H,13C-, and 15N nuclear magnetic resonance (NMR) spectroscopy. The third component was indicated by the mass spectra to be an isomer of factor A and is likely (according to NMR) to be 7"-[N 6-methyl]-adeninylcobamide, a previously unknown corrinoid. C. cochlearium thus biosynthesizes as its native “complete” B12 cofactors the 7"-adeninylcobamides and two homologous corrinoids, in which the nucleotide base is a methylated adenine.