Specific detection of Pseudomonas spp. in milk by fluorescence in situ hybridization using ribosomal RNA directed probes

Gunasekera, Thusitha S.; Dorsch, M.; Slade, Martin B.; Veal, Duncan

doi:10.1046/j.1365-2672.2003.01930.x

Cited by 76 publications

(52 citation statements)

References 21 publications

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“…A level in the range 10 5 -10 6 cfu/g is common for this kind of cheeses at around 2-3 months of ripening [36,40,45], but the concentration of Enterbacteriaceae greatly decreases after that period [11,29,38], since their growth is affected by the stressing cheese conditions. Even though pseudomonads, the most frequently psychrotrophic bacteria of raw milk [28], due to their intense proteolytic and lypolytic activities [10] are implicated with spoilage of milk and milk-derivates [44], they are not generally searched in ripened cheeses. In case of milk, the spoilage occurs when their concentrations are around 10 7 cfu/mL and, for this reason, Pseudomonas spp.…”

Section: Discussionmentioning

confidence: 99%

Effect of different salting technologies on the chemical and microbiological characteristics of PDO Pecorino Siciliano cheese

Todaro

Francesca

Reale

et al. 2011

Eur Food Res Technol

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The present work was carried out to evaluate the effect of two salting technologies [dry salting (DS) and the combined dry-brine salting (DBS)] on the chemicophysical and microbiological characteristics of PDO Pecorino Siciliano cheeses of different final weight (6 and 12 kg). Dry matter was significantly influenced by both salting process and final size. Twelve kilogram cheeses treated by DBS showed higher protein content with higher soluble nitrogen per cent than 6 kg cheeses. Salt content was in the range 3.1-4.0% on dry matter. The colour did not show significant differences for any of the factors, but 12 kg cheeses subjected to DS showed higher yellow index than the other cheeses. The resistance at 30% of strain was influenced by cheese size, with 6 kg cheeses showing higher resistances than 12 kg cheeses. All cheeses were dominated by coccus LAB, but pseudomonads and Enterobacteriaceae showed comparable levels of about 10 5 cfu/g. Significant microbiological differences were evidenced only for enterococci and yeasts concerning the final cheese size. Thirteen species of LAB, belonging to five genera (Enterococcus, Lactobacillus, Lactococcus, Pediococcus and Streptococcus), were identified, but several spoilage/ pathogenic species were also identified, especially Pseudomonas putida, Citrobacter freundii and Stenotrophomonas maltophilia. LAB isolates were preliminary evaluated for their physiological characteristics in view of developing autochthonous starters to improve the microbiological quality of PDO Pecorino Siciliano cheese.

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Section: Discussionmentioning

confidence: 99%

Effect of different salting technologies on the chemical and microbiological characteristics of PDO Pecorino Siciliano cheese

Todaro

Francesca

Reale

et al. 2011

Eur Food Res Technol

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“…An approach is the fluorescence in situ hybridization technique, where a Pseudomonas spp. rRNA-specific probe has been designed and demonstrated for UHT-milk inoculated with bacteria (Gunasekera et al 2003). Another approach is the flow cytometric Gram staining technique for milk-associated bacteria in milk (Holm and Jespersen 2003).…”

Section: Discussionmentioning

confidence: 99%

A flow cytometric technique for quantification and differentiation of bacteria in bulk tank milk

Holm

Mathiasen²,

Jespersen³

2004

J Appl Microbiol

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Aims: The present study describes a flow cytometric technique for quantification and differentiation of bacteria in bulk tank milk according to the main cause of elevated counts. Methods and Results: A total of 75 Danish bulk tank milk samples exceeding the grading level of 3AE0 · 10 4 CFU ml )1 were examined by both flow cytometry and traditional microbiological analyses. The correlation coefficient (r) between the two methods was 0AE71. For the differential analyses of the dominant bacterial populations four different parameters were used to give a species-characteristic pattern. The four parameters were as follows: staining with Oregon Green Ò conjugated wheat germ agglutinin that binds to the cell wall of bacteria, staining with hexidium iodide that binds to all bacterial DNA, the flow cytometric forward scatter and the flow cytometric side scatter. Three regions in the flow cytometric plot were defined: region 1 includes bacteria mainly associated with poor hygiene, region 2 includes psychrotrophic hygiene bacteria and region 3 includes bacteria mainly related to mastitis. The ability of the flow cytometric technique to predict the main cause of elevated bacterial counts on routine samples was examined. Comparing these results with results obtained by traditional microbiological analyses for identification showed that for 81% of the samples the two techniques agreed on the main cause of an elevated bacterial count. Conclusions:The ability of the presented flow cytometric technique to enumerate and differentiate bacteria in bulk tank milk according to the main cause of elevated counts was demonstrated. Significance and Impact of the Study: This study described the first step in development of a technique suitable for routine analyses of bulk tank milk samples. A technique indicating the main cause of an elevated count will enable the farmer to eliminate the contamination source within a short time limit.

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“…In this study, two kinds of Cy5-labeled oligonucleotide probes based on 16S rRNA sequences were used; one was the probe for the detection of P. putida, specifically designed by DuTeau et al (5Ј-to-3Ј sequence, TTG CCA GTT TTG GAT GCA GT) (7), and the other was the probe for the detection of Pseudomonas spp., specifically designed by Gunasekera et al (5Ј-to-3Ј sequence, GAT CCG GAC TAC GAT CGG TTT) (8). These hybridization and washing conditions were modified for the detection of CTC-stained cells by CTC-FISH.…”

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confidence: 99%

Enumeration of Respiring Pseudomonas spp. in Milk within 6 Hours by Fluorescence In Situ Hybridization following Formazan Reduction

Kitaguchi

Yamaguchi

Nasu

2005

Appl Environ Microbiol

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Respiring Pseudomonas spp. in milk were quantified within 6 h by fluorescence in situ hybridization (FISH) with vital staining. FISH with an oligonucleotide probe based on 16S rRNA sequences was used for the specific detection of Pseudomonas spp. at the single cell level. 5-Cyano-2,3-ditolyl tetrazolium chloride (CTC) was used to estimate bacterial respiratory activity. The numbers of respiring Pseudomonas cells as determined by FISH with CTC staining (CTC-FISH) were almost the same or higher than the numbers of CFU as determined by the conventional culture method.Conventional culture methods are commonly used for the microbiological quality assurance of food and drink. These methods are simple but generally require more than 24 h to yield reliable results. Underestimation of bacterial numbers sometimes occurs, because the cells which are viable but no longer culturable by culture methods are difficult to detect. Therefore, rapid and simple culture-independent methods are required.Several culture-independent methods are used for the detection of bacteria in food. PCR is one of the most useful techniques because of its high sensitivity. Reverse transcription-PCR, in particular, is a potentially valuable technique for the detection of viable bacteria (13, 27); however, these techniques require the extraction of nucleic acid from samples, and PCRs are sometimes inhibited by components of food, such as lipids, proteins, and salts (30). Enzyme-linked immunosorbent assay (ELISA) is also used frequently for the detection of bacteria in food (28); however, it is often used in conjunction with culture methods and incurs the limitations of culture techniques in enumerating bacterial cells. Fluorescence in situ hybridization (FISH) is widely used and is also suitable for the specific detection of targeted bacteria phylogenetically at species, genus, and family levels (2, 4), because the databases of rRNA sequences are publicly available.We attempted to enumerate Pseudomonas spp. with physiological activity in milk by combining FISH with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) staining (CTC-FISH). Pseudomonas spp. are among the most important spoilage bacteria in milk (5, 6). They grow actively during refrigerated storage and produce enzymes, such as protease and lipase, which cause the degradation of milk compounds and reduce its shelf life. CTC is a redox dye widely used for the evaluation of bacterial respiratory activity (23,24). CTC staining can be applied to anaerobic as well as aerobic bacteria (3). CTC is reduced to red fluorescent CTC-formazan crystals in actively respiring cells. However, the crystals are easily dissolved in organic solvents used in the main processes of FISH, and CTC-stained cells are no longer visible with the conventional FISH procedure.We optimized the CTC concentration and the procedure for FISH in order to retain CTC-formazan crystals inside cells and detect bacteria with a specific rRNA sequence simultaneously. In addition, the numbers of viable Pseudomonas cells determined by CTC-FI...

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Specific detection of Pseudomonas spp. in milk by fluorescence in situ hybridization using ribosomal RNA directed probes

Cited by 76 publications

References 21 publications

Effect of different salting technologies on the chemical and microbiological characteristics of PDO Pecorino Siciliano cheese

Effect of different salting technologies on the chemical and microbiological characteristics of PDO Pecorino Siciliano cheese

A flow cytometric technique for quantification and differentiation of bacteria in bulk tank milk

Enumeration of Respiring Pseudomonas spp. in Milk within 6 Hours by Fluorescence In Situ Hybridization following Formazan Reduction

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