Prevalence of Cronobacter spp. and Salmonella in Milk Powder Manufacturing Facilities in the United States.

ABSTRACT: The U.S. Food and Drug Administration (FDA) conducted a sampling assignment in 2014 to ascertain the prevalence of Cronobacter spp. and Salmonella in the processing environment of facilities manufacturing milk powder. Cronobacter was detected in the environment of 38 (69%) of 55 facilities. The average prevalence of Cronobacter in 5,671 subsamples (i.e., swabs and sponges from different facility locations) was 4.4%. In the 38 facilities where Cronobacter was detected, the average prevalence of positive environmental subsamples was 6.25%. In 20 facilities where zone information of the sampling location was complete, Cronobacter was most frequently detected in zone 4, followed by zone 3, then zone 2, with zone 1 yielding the lowest percentage of positive samples. The prevalence of Cronobacter across the zones was statistically different (P < 0.05). There was no significant association between product type (i.e., lactose, whey products, buttermilk powder, and nonfat dried milk) and prevalence of Cronobacter in the facility. Salmonella was detected in the environment of three (5.5%) of the 55 facilities; all three facilities produced dried whey product. The overall prevalence of Salmonella in 5,714 subsamples was 0.16%. In facilities in which Salmonella was detected, the average prevalence was 2.5%. Salmonella was most frequently detected in zone 4, followed by zone 3. Salmonella was not detected in zone 1 or zone 2. The disparity between Salmonella and Cronobacter prevalence indicates that additional measures may be required to reduce or eliminate Cronobacter from the processing environment.

A top-down proteomics approach for differentiating thermal resistant strains of Enterobacter sakazakii.

Thermal tolerance has been identified as an important factor relevant to the pathogenicity of Enterobacter sakazakii in human neonates. To identify a biomarker specific for this phenotypic trait, intact protein expression profiles of 12 strains of E. sakazakii were obtained using liquid chromatography mass spectrometry. Proteins were extracted from the bacterial cells, separated by reversed-phase liquid chromatography and mass analyzed. At the end of the chromatography run, the uncharged masses of the multiply charged proteins were determined via automated software routines. The resulting data provided an accurate mass expression profile of the proteins found in the individual strains. From the individual expression profiles, it was possible to identify unique proteins corresponding to strains with thermal resistance. One protein found only in the thermal tolerant strains was sequenced and identified as homologous to a hypothetical protein found in the thermal tolerant bacteria, Methylobacillus flagellatus KT. The protein sequence of this protein was then used to reverse-engineer PCR primers for the gene sequence associated with the protein. In all cases, only thermal tolerant strains of E. sakazakii produced amplified PCR products, demonstrating the specificity of this biomarker.

Survival of Enterobacter sakazakii in a dehydrated powdered infant formula.

A quantity of dehydrated powdered infant formula was prepared to contain Enterobacter sakazakii strain 607 at approximately 106 CFU/ml when rehydrated according to the manufacturer's instructions. The survival of the microorganism in the dry formula was followed for 2 years, during which samples periodically were rehydrated and analyzed for viable E. sakazakii. During the initial 5 months of storage at room temperature, viable counts declined approximately 2.4 log cycles. During the subsequent 19 months, the concentration of viable E. sakazakii declined an additional 1.0 log cycle. These results indicate that a small percentage of E. sakazakii cells can survive for extended periods in dehydrated powdered infant formula.

Effect of prior growth conditions on the thermal inactivation of 13 strains of Listeria monocytogenes in two heating menstrua.

The thermal tolerance of 13 Listeria monocytogenes strains was tested using a submerged heating coil apparatus. The strains were grown individually for 18 h at 37 degrees C in acidogenic tryptic soy broth (without dextrose) supplemented with 1% glucose and 1% glutamine (TSB+G) or nonacidogenic tryptic soy broth supplemented with 1% glutamine but containing no glucose (dextrose) (TSB-G). The former medium results in cells induced for pH-dependent, stationary-phase acid resistance, whereas the latter medium allows L. monocytogenes to grow to high numbers in the absence of glucose, yielding cells that are not induced for pH-dependent, stationary-phase acid resistance. The average final pH values of the 18-h TSB+G and the TSB-G cultures were 4.7 and 6.7, respectively. The cells grown in the acid resistance-inducing and non-acid resistance-inducing media were then tested in two heating menstrua that consisted of brain heart infusion broth adjusted to pH 3.0 and water activity (a(w)) of 0.987 or pH 7.0 and a(w) 0.970. In 14 of the 26 menstruum-strain combinations tested, the acid resistance-induced strains were more heat resistant then the equivalent noninduced cultures. No difference in the pattern of thermal resistance in response to induction of acid resistance was apparent among the different serovars tested. The results suggest that the ability of prior induction of acid resistanceto enhance thermal resistance can vary substantially among L. monocytogenes strains.

Potential for internalization, growth, and survival of Salmonella and Escherichia coli O157:H7 in oranges.

Internalization potential, survival, and growth of human pathogens within oranges were investigated in a series of laboratory experiments. Submerging oranges into dye solutions at various temperature differentials was used to assess internalization potential. Conditions in which dye internalization was observed were further studied by applying Escherichia coli O157:H7 or Salmonella onto the stem scar, subjecting the oranges to a temperature differential, juicing, and measuring numbers of pathogens in the resulting juice. Pathogens for growth and survival studies were applied to or injected into simulated peel punctures. Oranges with small peel holes of selected sizes were also placed into solutions containing these pathogens. Bacterial survival was also evaluated in orange juice at 4 and 24 degrees C. Oranges internalized pathogens at a frequency of 2.5 to 3.0%, which mirrored dye internalization frequency (3.3%). Pathogens were internalized at an uptake level of 0.1 to 0.01% of the challenge applied. Bacteria grew within oranges at 24 degrees C, but not at 4 degrees C. Thirty-one percent of oranges with 0.91-mm surface holes showed pathogen uptake, whereas 2% of oranges with 0.68-mm holes showed pathogen uptake. Pathogens added to fresh orange juice and incubated at 24 degrees C declined 1 log CFU/ml within 3 days. These results suggest that internalization, survival, and growth of human bacterial pathogens can occur within oranges intended for producing unpasteurized juice.

Thermal inactivation of Enterobacter sakazakii in rehydrated infant formula.

The presence of low levels of Enterobacter sakazakii in dried infant formula have been linked to outbreaks of meningitis, septicemia, and necrotizing enterocolitis in neonates, particularly those who are premature or immunocompromised. In the current study, the ability of 12 strains of E. sakazakii to survive heating in rehydrated infant formula was determined at 58 degrees C with a submerged coil apparatus. The observed D58-values ranged from 30.5 to 591.9 s, with the strains appearing to fall into two distinct heat resistance phenotypes. The z-value of the most heat-resistant strain was 5.6 degrees C. When dried infant formula containing this strain was rehydrated with water preequilibrated to various temperatures, a more than 4-log reduction in E. sakazakii levels was achieved by preparing the formula with water at 70 degrees C or greater.