A combination of direct viable count and fluorescence in situ hybridization for specific enumeration of viable Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus

Abstract

Aims: We have developed a direct viable count (DVC)‐FISH procedure for quickly and easily discriminating between viable and nonviable cells of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains, the traditional yogurt bacteria. Methods and Results: direct viable count method has been modified and adapted for Lact. delbrueckii subsp. bulgaricus and Strep. thermophilus analysis by testing different times of incubation and concentrations of DNA‐gyrase inhibitors. DVC procedure has been combined with fluorescent in situ hybridization (FISH) for the specific detection of viable cells of both bacteria with specific rRNA oligonucleotide probes (DVC‐FISH). Of the four antibiotics tested (novobiocin, nalidixic acid, pipemidic acid and ciprofloxacin), novobiocin was the most effective for DVC method and the optimum incubation time was 7 h for both bacteria. The number of viable cells was obtained by the enumeration of specific hybridized cells that were elongated at least twice their original length for Lactobacillus and twice their original size for Streptococcus. Conclusions: This technique was successfully applied to detect viable cells in inoculated faeces. Significance and Impact of the Study: Results showed that this DVC‐FISH procedure is a quick and culture‐independent useful method to specifically detect viable Lact. delbrueckii subsp. bulgaricus and Strep. thermophilus in different samples, being applied for the first time to lactic acid bacteria.

Streptococcus thermophilus NCIMB 41856 ameliorates signs of colitis in an animal model of inflammatory bowel disease

“…Fluorescence in situ hybridisation (FISH) to S. thermophilus was carried out as previously described (Garcia-Hernandez et al, 2012) on sections of formalin-fixed colon using a Cy3-conjugated probe (5'-CATGCCTTCGCTTACGCT-3') specific to 23S rRNA. Total levels of bacteria were quantified using a combination of three FITC-conjugated eubacteria probes to 16S rRNA (EUB338 5'GCT GCC TCC CGT AGG AGT-3', EUB 338 II 5'GCA GCC ACC CGT AGG TGT-3, and EUB338 III 5'GCT GCC ACC CGT AGG TGT-3') as previously described (Daims et al, 1999).…”

Evolution of microbiological analytical methods for dairy industry needs

“…Propionibacteria were detected and enumerated in cheese with good correlations with bacterial counts (Babot et al, 2011). García-Hernández et al (2012) have combined a direct viable count method with FISH to enumerate viable yogurt strains inoculated in feces. FISH allows highly specific bacterial detection and, unlike other molecular techniques, provides information about spacial distribution of bacteria in their environment due to the use of imaging.…”

In Vivo Study of the Survival of Lactobacillus delbruecki subsp. bulgaricus CECT 4005T and Streptococcus thermophilus CECT 801 by DVC‐FISH after Consumption of Fermented Milk

“…LAB viability has been classically determined by plate counting, although it shows some disadvantages. Thus, it is time‐consuming and the number of viable bacteria can be underestimated due to the irregular distribution of microorganisms in the sample and the loss of culturability under stress conditions (Garcia‐Hernandez and others 2012). There is a clear need for culture‐independent methods to quantify viable LAB in microbial communities like fecal samples (Del Campo and others 2005).…”

“…Viable bacteria incubated in presence of nutrients and a gyrase inhibitor (antibiotic) are able to elongate and increase their cellular metabolic activity being their division inhibited by the presence of antibiotic. Viable cells can then be easily discriminated from nonviable cells, due to differences in their respective sizes (Garcia‐Hernandez and others 2012). The combination of DVC incubation which increases intracellular rRNA levels, with FISH performed on rRNA‐targeted sequences could prove useful in detecting and identifying viable cells in mixed microbial communities.…”

“…This technique has been recently developed for L. delbruecki subsp. bulgaricus CECT 4005T and S. thermophilus CECT 801 (Garcia‐Hernandez and others 2012) for their detection in both inoculated broths and inoculated fecal samples.…”