Detection ofClostridium tyrobutyricumspores using polyclonal antibodies and flow cytometry

Abstract

Aims: The present work investigates the feasibility of using flow cytometry (FCM) combined with fluorescent‐labelled specific polyclonal antibodies for the detection and presumptive identification of Clostridium tyrobutyricum spores in bovine milk. Methods and Results: Two fluorescent molecules (fluorescein isothiocyanate and Alexa Fluor 488) were conjugated to antispores polyclonal antibodies. Side scatter and forward scatter profiles of the Cl. tyrobutyricum spores marked with fluorescent antibodies permitted the detection of spores and differentiated them from other related microbial species. The detection limit of this method was 103 spores per 100 ml of milk, and results could be achieved in 2 h. Conclusions: FCM combined with fluorochrome‐conjugated antibodies, especially Alexa Fluor, could be an efficacious means to detect and provide presumptive identification of Cl. tyrobutyricum spores, as well as differentiation from other Clostridium species that can also cause late blowing in cheese. Significance and Impact of the Study: This study describes the basis for the development of a method suitable for analysis of milk destined for cheese manufacture that would permit the detection of Cl. tyrobutyricum spores in a short period. This would enable the industry to use contaminated milk for dairy products other than cheese where Cl. tyrobutyricum does not cause a problem.

Clostridium Tyrobutyricum Protect Intestinal Barrier Function from LPS-Induced Apoptosis via P38/JNK Signaling Pathway in IPEC-J2 Cells

“…C.t , a Gram-positive and strictly anaerobic bacterium, has received much attention from the dairy and chemical synthesis industries because of its efficiency in butyric acid production [19, 20]. However, the role of C.t in animal models or mammalian cells has not yet been reported.…”

Genomic Sequence and Characterization of the Virulent Bacteriophage φCTP1 from Clostridium tyrobutyricum and Heterologous Expression of Its Endolysin

“…Early detection in either feeds or milk could form part of an effective control strategy before cheese manufacture, and research has shown that endolysins can form the basis of highly efficient and sensitive detection systems, using either the cell wall binding domains or synthetic peptides (16,33). Recent publications have demonstrated some innovative methods for C. tyrobutyricum spore detection (18,19). The endolysin may not detect spores, due to differences in cell wall structures, but should at least identify areas where C. tyrobutyricum levels, and thus potentially spore concentrations, are high.…”

Specific peptides as alternative to antibody ligands for biomagnetic separation of Clostridium tyrobutyricum spores

“…However, in our case, obtaining functionalized paramagnetic beads had to be implemented from preparation of appropriate ligands. Previously obtained antibodies and peptides with affinity to C. tyrobutyricum spores [9,29] have been used. Their application together with magnetic beads and biomagnetic separation of this microorganism was studied in the present work, since promising results using this technique had been observed in separation and detection of microorganisms in milk [35,36] and even in the detection of other substances, for example, antibiotics [37].…”

“…Specific antibodies were isolated from serum by immunoaffinity as previously described [9]-5 mL of antisera was incubated with 10 mL of an immunosorbent, prepared by insolubilization of C. tyrobutyricum spores with glutaraldehyde, with continuous stirring for 2 h at room temperature. Afterward, the gel was placed into a column and washed with 0.05 M NaH 2 PO 4 /Na 2 HPO 4 , 0.5 M NaCl, pH 7.4, until the absorbance of the eluate at 280 nm was below 0.03.…”

“…Thus, the existence of a fast (<24 h), specific, and extremely sensitive method to detect this bacterium before milk processing would be advantageous, specially to screen milk that is going to be used in the production of mature cheese, thus avoiding future problems. Some methods for detecting spores of C. tyrobutyricum, such as immunodetection assay after membrane filtration of milk [6], methods using specific DNA probes [7,8], or flow cytometry [9] have been developed. However, the reference method still used at the moment is the most-probablenumber (MPN), a semiquantitative method, which takes up to 7 days to yield results and whose specificity is often uncertain [10].…”