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Evaluation of the Native Microbiota and Comparative Analysis of a Known Cronobacter Sakazakii and a Newly Isolated Bacillus Cereus Strain in Powdered Infant Formula
Title
Evaluation of the Native Microbiota and Comparative Analysis of a Known Cronobacter Sakazakii and a Newly Isolated Bacillus Cereus Strain in Powdered Infant Formula
Creator
Patil, Sonali Prashant
Date
2024
Description
There have been numerous reports of Powdered Infant Formula (PIF) recalls and outbreaks due to the absence of a kill step in the post...
Show moreThere have been numerous reports of Powdered Infant Formula (PIF) recalls and outbreaks due to the absence of a kill step in the post-pasteurization processing, improper handling pre and post processing and/or reconstitution, and lack of effective sanitization and cleaning of the food contact surfaces in the manufacturing facilities. The objectives of this present study were to 1) survey and identify background microflora in commercial PIF products through microbiological analysis, 16S rRNA, and whole genome sequencing (WGS); 2) compare the survival rate of a known Cronobacter sakazakii and a newly isolated Bacillus cereus DFPST-SP1 in PIF under a humidity level of 33 ± 3% over a period of 28 d; 3) examine the relative resistance of these two strains to thermal treatments at temperatures 40, 70, and 100 °C followed by storage at room temperature (RT) for 30 min; and 4) evaluate the bactericidal effect of 70% ethanol on the two artificially deposited bacterial strains on stainless steel (SS) and plastic coupons. Three biological trials were conducted for each study. To determine whether the increase, decrease, or difference in the bacterial populations and other parameters like water activity (aw) was statistically significant, a T-test was performed (p ≤ 0.05 was considered significant). Results of 16S rRNA sequencing revealed the presence of certain bacterial species in PIF, such as Lactococcus lactis, B. cereus, Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica, etc. distributed across a relative abundance of >25%, <25%, and ≤3%. After the enrichment and isolation as per Bacteriological Analytical Manual (BAM), C. sakazakii or S. enterica were not detected, while colonies exhibiting a blue-green appearance resembling Listeria spp. and certain Bacillus spp. were subjected to WGS for species-level identification. The assembly_1 from formulation 1 was confirmed as B. cereus sequence type 2255 and was renamed as B. cereus DFPST-SP1 in the contribution of this thesis work. The storage study conducted on PIF inoculated with C. sakazakii and B. cereus DFPST-SP1 at 33% RH showed that there was 0.25-0.27 log CFU/g reduction towards the end of 28 d, but no significant difference was observed between the two strains. The thermal challenge study revealed that the newly isolated B. cereus strain and C. sakazakii used in this study were highly thermotolerant. Based on the sanitizer challenge study, 70% ethanol was significantly more effective in reducing populations of C. sakazakii as compared to B. cereus. Moreover, higher log reductions of C. sakazakii 587 populations on stainless steel coupons compared to plastic coupons were observed, indicating that bacteria adhere more tightly to plastic surfaces than stainless steel (SS). Overall, the findings of this study shed new light on bolstering the safety standards of PIF and highlight the need for improved cleaning and sanitization procedures within manufacturing facilities in order to ensure the safety of reconstituted PIF, thereby enhancing public health, particularly infants and neonates.
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Evaluation of the Native Microbiota and Comparative Analysis of a Known Cronobacter Sakazakii and a Newly Isolated Bacillus Cereus Strain in Powdered Infant Formula
Title
Evaluation of the Native Microbiota and Comparative Analysis of a Known Cronobacter Sakazakii and a Newly Isolated Bacillus Cereus Strain in Powdered Infant Formula
Creator
Patil, Sonali Prashant
Date
2024
Description
There have been numerous reports of Powdered Infant Formula (PIF) recalls and outbreaks due to the absence of a kill step in the post...
Show moreThere have been numerous reports of Powdered Infant Formula (PIF) recalls and outbreaks due to the absence of a kill step in the post-pasteurization processing, improper handling pre and post processing and/or reconstitution, and lack of effective sanitization and cleaning of the food contact surfaces in the manufacturing facilities. The objectives of this present study were to 1) survey and identify background microflora in commercial PIF products through microbiological analysis, 16S rRNA, and whole genome sequencing (WGS); 2) compare the survival rate of a known Cronobacter sakazakii and a newly isolated Bacillus cereus DFPST-SP1 in PIF under a humidity level of 33 ± 3% over a period of 28 d; 3) examine the relative resistance of these two strains to thermal treatments at temperatures 40, 70, and 100 °C followed by storage at room temperature (RT) for 30 min; and 4) evaluate the bactericidal effect of 70% ethanol on the two artificially deposited bacterial strains on stainless steel (SS) and plastic coupons. Three biological trials were conducted for each study. To determine whether the increase, decrease, or difference in the bacterial populations and other parameters like water activity (aw) was statistically significant, a T-test was performed (p ≤ 0.05 was considered significant). Results of 16S rRNA sequencing revealed the presence of certain bacterial species in PIF, such as Lactococcus lactis, B. cereus, Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica, etc. distributed across a relative abundance of >25%, <25%, and ≤3%. After the enrichment and isolation as per Bacteriological Analytical Manual (BAM), C. sakazakii or S. enterica were not detected, while colonies exhibiting a blue-green appearance resembling Listeria spp. and certain Bacillus spp. were subjected to WGS for species-level identification. The assembly_1 from formulation 1 was confirmed as B. cereus sequence type 2255 and was renamed as B. cereus DFPST-SP1 in the contribution of this thesis work. The storage study conducted on PIF inoculated with C. sakazakii and B. cereus DFPST-SP1 at 33% RH showed that there was 0.25-0.27 log CFU/g reduction towards the end of 28 d, but no significant difference was observed between the two strains. The thermal challenge study revealed that the newly isolated B. cereus strain and C. sakazakii used in this study were highly thermotolerant. Based on the sanitizer challenge study, 70% ethanol was significantly more effective in reducing populations of C. sakazakii as compared to B. cereus. Moreover, higher log reductions of C. sakazakii 587 populations on stainless steel coupons compared to plastic coupons were observed, indicating that bacteria adhere more tightly to plastic surfaces than stainless steel (SS). Overall, the findings of this study shed new light on bolstering the safety standards of PIF and highlight the need for improved cleaning and sanitization procedures within manufacturing facilities in order to ensure the safety of reconstituted PIF, thereby enhancing public health, particularly infants and neonates.
Show less
Evaluation of the Native Microbiota and Comparative Analysis of a Known Cronobacter Sakazakii and a Newly Isolated Bacillus Cereus Strain in Powdered Infant Formula
Title
Evaluation of the Native Microbiota and Comparative Analysis of a Known Cronobacter Sakazakii and a Newly Isolated Bacillus Cereus Strain in Powdered Infant Formula
Creator
Patil, Sonali Prashant
Date
2024
Description
There have been numerous reports of Powdered Infant Formula (PIF) recalls and outbreaks due to the absence of a kill step in the post...
Show moreThere have been numerous reports of Powdered Infant Formula (PIF) recalls and outbreaks due to the absence of a kill step in the post-pasteurization processing, improper handling pre and post processing and/or reconstitution, and lack of effective sanitization and cleaning of the food contact surfaces in the manufacturing facilities. The objectives of this present study were to 1) survey and identify background microflora in commercial PIF products through microbiological analysis, 16S rRNA, and whole genome sequencing (WGS); 2) compare the survival rate of a known Cronobacter sakazakii and a newly isolated Bacillus cereus DFPST-SP1 in PIF under a humidity level of 33 ± 3% over a period of 28 d; 3) examine the relative resistance of these two strains to thermal treatments at temperatures 40, 70, and 100 °C followed by storage at room temperature (RT) for 30 min; and 4) evaluate the bactericidal effect of 70% ethanol on the two artificially deposited bacterial strains on stainless steel (SS) and plastic coupons. Three biological trials were conducted for each study. To determine whether the increase, decrease, or difference in the bacterial populations and other parameters like water activity (aw) was statistically significant, a T-test was performed (p ≤ 0.05 was considered significant). Results of 16S rRNA sequencing revealed the presence of certain bacterial species in PIF, such as Lactococcus lactis, B. cereus, Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica, etc. distributed across a relative abundance of >25%, <25%, and ≤3%. After the enrichment and isolation as per Bacteriological Analytical Manual (BAM), C. sakazakii or S. enterica were not detected, while colonies exhibiting a blue-green appearance resembling Listeria spp. and certain Bacillus spp. were subjected to WGS for species-level identification. The assembly_1 from formulation 1 was confirmed as B. cereus sequence type 2255 and was renamed as B. cereus DFPST-SP1 in the contribution of this thesis work. The storage study conducted on PIF inoculated with C. sakazakii and B. cereus DFPST-SP1 at 33% RH showed that there was 0.25-0.27 log CFU/g reduction towards the end of 28 d, but no significant difference was observed between the two strains. The thermal challenge study revealed that the newly isolated B. cereus strain and C. sakazakii used in this study were highly thermotolerant. Based on the sanitizer challenge study, 70% ethanol was significantly more effective in reducing populations of C. sakazakii as compared to B. cereus. Moreover, higher log reductions of C. sakazakii 587 populations on stainless steel coupons compared to plastic coupons were observed, indicating that bacteria adhere more tightly to plastic surfaces than stainless steel (SS). Overall, the findings of this study shed new light on bolstering the safety standards of PIF and highlight the need for improved cleaning and sanitization procedures within manufacturing facilities in order to ensure the safety of reconstituted PIF, thereby enhancing public health, particularly infants and neonates.
Show less