In situ detection of antibiotic-resistance elements in single Bacillus cereus spores

Rapid detection of Bacillus spores is a challenging task in food and defense industries. In situ labeling of spores would be advantageous for detection by automated systems based on single-cell analysis. Determination of antibiotic-resistance genes in bacterial spores using in situ labeling has never been developed. Most of the in situ detection schemes employ techniques such as fluorescence in situ hybridization (FISH) that target the naturally amplified ribosomal RNA (rRNA). However, the majority of antibiotic-resistance genes has a plasmidic or chromosomal origin and is present in low copy numbers in the cell. The main challenge in the development of low-target in situ detection in spores is the permeabilization procedure and the signal amplification required for detection. This study presents permeabilization and in situ signal amplification protocols, using Bacillus cereus spores as a model, in order to detect antibiotic-resistance genes. The permeabilization protocol was designed based on the different layers of the Bacillus spore. Catalyzed reporter deposition (CARD)-FISH and in situ polymerase chain reaction (PCR) were used as signal amplification techniques. B. cereus was transformed with the high copy number pC194 and low copy number pMTL500Eres plasmids in order to induce resistance to chloramphenicol and erythromycin, respectively. In addition, a rifampicin-resistant B. cereus strain, conferred by a single-nucleotide polymorphism (SNP) in the chromosome, was used. Using CARD-FISH, only the high copy number plasmid pC194 was detected. On the other hand, in situ PCR gave positive results in all tested instances. This study demonstrated that it was feasible to detect antibiotic-resistance genes in Bacillus spores using in situ techniques. In addition, in situ PCR has been shown to be more sensitive and more applicable than CARD-FISH in detecting low copy targets.