scholarly journals Development and Application of a Novel Peptide Nucleic Acid Probe for the Specific Detection of Cronobacter Genomospecies (Enterobacter sakazakii) in Powdered Infant Formula

2009 ◽  
Vol 75 (9) ◽  
pp. 2925-2930 ◽  
Author(s):  
C. Almeida ◽  
N. F. Azevedo ◽  
C. Iversen ◽  
S. Fanning ◽  
C. W. Keevil ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Infant Formula ◽  
Peptide Nucleic Acid ◽  
Powdered Infant Formula ◽  
Enterobacter Sakazakii ◽  
Specific Detection ◽  
Peptide Nucleic Acid Probe ◽  
Bacterial Contaminants ◽  
Specificity And Sensitivity

ABSTRACT Here, we report a fluorescence in situ hybridization (FISH) method for rapid detection of Cronobacter strains in powdered infant formula (PIF) using a novel peptide nucleic acid (PNA) probe. Laboratory tests with several Enterobacteriaceae species showed that the specificity and sensitivity of the method were 100%. FISH using PNA could detect as few as 1 CFU per 10 g of Cronobacter in PIF after an 8-h enrichment step, even in a mixed population containing bacterial contaminants.

scholarly journals Fluorescence In Situ Hybridization Method Using a Peptide Nucleic Acid Probe for Identification of Salmonella spp. in a Broad Spectrum of Samples

2010 ◽  
Vol 76 (13) ◽  
pp. 4476-4485 ◽  
Author(s):  
C. Almeida ◽  
N. F. Azevedo ◽  
R. M. Fernandes ◽  
C. W. Keevil ◽  
M. J. Vieira
Keyword(s):  
Nucleic Acid ◽  
Broad Spectrum ◽  
Peptide Nucleic Acid ◽  
Bacterial Species ◽  
Powdered Infant Formula ◽  
Fish Method ◽  
Specificity And Sensitivity ◽  
Pna Fish ◽  
Enrichment Step

ABSTRACT A fluorescence in situ hybridization (FISH) method for the rapid detection of Salmonella spp. using a novel peptide nucleic acid (PNA) probe was developed. The probe theoretical specificity and sensitivity were both 100%. The PNA-FISH method was optimized, and laboratory testing on representative strains from the Salmonella genus subspecies and several related bacterial species confirmed the predicted theoretical values of specificity and sensitivity. The PNA-FISH method has been successfully adapted to detect cells in suspension and is hence able to be employed for the detection of this bacterium in blood, feces, water, and powdered infant formula (PIF). The blood and PIF samples were artificially contaminated with decreasing pathogen concentrations. After the use of an enrichment step, the PNA-FISH method was able to detect 1 CFU per 10 ml of blood (5 × 109 ± 5 × 108 CFU/ml after an overnight enrichment step) and also 1 CFU per 10 g of PIF (2 × 107 ± 5 × 106 CFU/ml after an 8-h enrichment step). The feces and water samples were also enriched according to the corresponding International Organization for Standardization methods, and results showed that the PNA-FISH method was able to detect Salmonella immediately after the first enrichment step was conducted. Moreover, the probe was able to discriminate the bacterium in a mixed microbial population in feces and water by counter-staining with 4′,6-diamidino-2-phenylindole (DAPI). This new method is applicable to a broad spectrum of samples and takes less than 20 h to obtain a diagnosis, except for PIF samples, where the analysis takes less than 12 h. This procedure may be used for food processing and municipal water control and also in clinical settings, representing an improved alternative to culture-based techniques and to the existing Salmonella PNA probe, Sal23S10, which presents a lower specificity.

scholarly journals Visualization of the Mycelia of Wood-Rotting Fungi by Fluorescence in Situ Hybridization Using a Peptide Nucleic Acid Probe

2013 ◽  
Vol 77 (2) ◽  
pp. 405-408 ◽  
Author(s):  
Yuji NAKADA ◽  
Satoshi NAKABA ◽  
Hiroshi MATSUNAGA ◽  
Ryo FUNADA ◽  
Makoto YOSHIDA
Keyword(s):  
Nucleic Acid ◽  
Peptide Nucleic Acid ◽  
Peptide Nucleic Acid Probe

Enterobacter sakazakii in Dried Infant Formulas and Milk Kitchens of Maternity Wards in São Paulo, Brazil

2009 ◽  
Vol 72 (1) ◽  
pp. 37-42 ◽  
Author(s):  
GABRIELA PALCICH ◽  
CINTIA de MORAES GILLIO ◽  
LINA CASALE ARAGON-ALEGRO ◽  
FRANCO J. PAGOTTO ◽  
JEFFREY M. FARBER ◽  
...  
Keyword(s):  
Intensive Care Unit ◽  
Intensive Care ◽  
Neonatal Intensive Care Unit ◽  
Infant Formula ◽  
Neonatal Intensive Care ◽  
Room Temperature ◽  
Sao Paulo ◽  
Powdered Infant Formula ◽  
São Paulo ◽  
Enterobacter Sakazakii

This study was the first conducted in Brazil to evaluate the presence of Enterobacter sakazakii in milk-based powdered infant formula manufactured for infants 0 to 6 months of age and to examine the conditions of formula preparation and service in three hospitals in São Paulo State, Brazil. Samples of dried and rehydrated infant formula, environments of milk kitchens, water, bottles and nipples, utensils, and hands of personnel were analyzed, and E. sakazakii and Enterobacteriaceae populations were determined. All samples of powdered infant formula purchased at retail contained E. sakazakii at <0.03 most probable number (MPN)/100 g. In hospital samples, E. sakazakii was found in one unopened formula can (0.3 MPN/100 g) and in the residue from one nursing bottle from hospital A. All other cans of formula from the same lot bought at a retail store contained E. sakazakii at <0.03 MPN/100 g. The pathogen also was found in one cleaning sponge from hospital B. Enterobacteriaceae populations ranged from 101 to 105 CFU/g in cleaning aids and <5 CFU/g in all formula types (dry or rehydrated), except for the sample that contained E. sakazakii, which also was contaminated with Enterobacteriaceae at 5 CFU/g. E. sakazakii isolates were not genetically related. In an experiment in which rehydrated formula was used as the growth medium, the temperature was that of the neonatal intensive care unit (25°C), and the incubation time was the average time that formula is left at room temperature while feeding the babies (up to 4 h), a 2-log increase in levels of E. sakazakii was found in the formula. Visual inspection of the facilities revealed that the hygienic conditions in the milk kitchens needed improvement. The length of time that formula is left at room temperature in the different hospitals while the babies in the neonatal intensive care unit are being fed (up to 4 h) may allow for the multiplication of E. sakazakii and thus may lead to an increased health risk for infants.

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