The objective of the present study was to create a collection of Lux-tagged Cronobacter strains to determine whether bioluminescence could be used to monitor growth of this pathogen in infant milk formula (IMF). Nine Cronobacter strains (seven C. sakazakii , one C. malonaticus , and one C. muytjensii ) were transformed with plasmid p16S lux , and integration of the plasmid at the desired site on the chromosome was confirmed by PCR. The integrated plasmid was stable in the absence of antibiotic selection, and growth of the Lux-tagged strains was similar to that of their nontagged counterparts. Growth of Lux-tagged strains was monitored in real time in 10 commercial brands of IMF by measuring light emission with a luminometer. Although all of the IMF samples tested were able to support the growth of the Cronobacter strains, differences were observed among IMF brands. Variations in the amount of light emitted by individual Cronobacter strains were also noted. Monitoring light emission with a combination of two strains that produced higher and lower than average relative light readings was a good surrogate for evaluating the entire collection of Lux-tagged strains.

Thirty-three antimicrobial agents, including antimicrobial peptides (nisin, lacticin 3147, isracidin), organic acids, emulsifiers (organic acid esters), glycine, lysozyme, tocopherol, EDTA, milk fat globule membrane, and the lactoperoxidase system (LPOS) were screened for anti– Cronobacter sakazakii activity. The compounds were initially screened individually in parallel in synthetic media. Those showing antimicrobial activity were then tested in reconstituted whole milk and finally in reconstituted powdered infant formula (PIF), using mild temperatures of reconstitution and prolonged storage at room temperature. Propionic acid and monocaprylin (as POEM M-100) in combination showed inhibitory activity at sufficiently low concentrations (0.1 to 0.2%) in milk to be considered as potential antimicrobial additives for the inhibition of C. sakazakii in reconstituted PIF. More interestingly, LPOS, when combined with the broad-spectrum bacteriocins nisin or lacticin 3147, inhibited outgrowth of C. sakazakii at 37°C for 8 h. The combined effects of POEM M-100 and either acetate or propionate and LPOS with lacticin 3147 or nisin were evaluated under the Food and Agriculture Organization of the United Nations–World Health Organization high-risk scenario for PIF, i.e., low temperature of reconstitution and long storage or feeding times at ambient temperature. In the presence of LPOS and lacticin 3147, growth of Cronobacter spp. was inhibited for up to 12 h when the PIF was rehydrated at 40 or 50°C. These results highlight the potential of combinatory approaches to improving the safety of infant milk formula.

Cronobacter species are opportunistic pathogens, and a mortality rate of 40 to 80% is associated with infections. This pathogen can cause a range of serious diseases such as meningitis, septicemia, necrotizing enterocolitis, and brain abscesses and has been responsible for a variety of sequelae such as quadriplegia. Although Cronobacter can cause disease in both adults and infants, infant infections associated with powdered formula are the focus of this review. Since the first reported Cronobacter infection outbreak in 1958, powdered infant formula has been identified as a major source of these outbreaks, resulting in many recalls of powdered infant formula worldwide. This contamination has created an immense problem for the powdered infant formula industry. In this review, we discuss the taxonomy of Cronobacter species, the natural habitat of Cronobacter and its presence in foods, the physiology, pathogenicity, and virulence of Cronobacter species, and available detection methods. We also discuss reported cases of Cronobacter infection linked to powdered infant formula consumption and then focus specifically on the official World Health Organization guidelines for preparation of powdered infant formula.

The aim of this study was to examine the potential of using a lux -tagged Cronobacter sakazakii strain to monitor growth of the bacterium in various liquids. C. sakazakii was transformed with plasmid p16S lux, and integration of the plasmid at the desired site on the chromosome was confirmed by PCR. The growth of the lux -tagged strain was similar to that of the non– lux -tagged strain, and the integrated plasmid was stable when cells were cultured in the absence of antibiotic. Growth of the lux -tagged strain was monitored in real time in Luria-Bertani broth, skim milk, and infant milk formula by using both the Luminoskan luminometer and the Xenogen IVIS imager. Bioluminescence could be detected when the lux -tagged strain was cocultured with other bacteria. The effect of monocaprylin and nisin on the growth of C. sakazakii in milk was monitored by measuring bioluminescence. In conclusion, growth of a lux -tagged C. sakazakii can be monitored in real time in both clear and opaque liquids by measuring bioluminescence. lux -tagged C. sakazakii strains could be potentially used in high-throughput assays to monitor the effects of various infant milk formula compositions on growth of the bacterium.

Carnitine has beneficial vitamin-like qualities and functions as a compatible solute. The presence of L-carnitine in infant formula significantly promoted the growth of Listeria monocytogenes at 7°C, whereas disruption of the genes for carnitine and glycine betaine uptake compromised growth of the L. monocytogenes mutant in formula containing carnitine that was stored at 7°C. Thus, addition of carnitine to baby formula may pose a potential food safety risk.

Salmonella carriage in pigs represents a serious health problem that undoubtedly contributes to the spread of human disease. Thus, the efficient and reliable testing of farm animals for bacteria such as Salmonella is an important aspect of any efficient control strategy. Serological analysis of 15 meat juice samples detected antibodies against Salmonella in some, but not all, of the animals identified bacteriologically as harboring the pathogen, indicating a lack of correlation between the bacteriological and serological methods used for Salmonella detection. The results suggest that testing by enzyme-linked immunosorbent assay is appropriate at the herd level, with culture methods preferable for individual animal analysis. A novel culture protocol detected Salmonella in the cecal contents of 15 pigs, whereas a method based on the European Standard identified only 9 pigs as being Salmonella -positive. During the study, an unusual finding was the relatively high incidence of Salmonella London carriage in the pigs tested.

The glutamate decarboxylase (GAD) acid resistance system of the foodborne pathogen Listeria monocytogenes plays a major role in its survival at low pH. It was found that survival of the wild-type strain, LO28, in acidified reconstituted skim milk, diluted to reduce free glutamate levels, improves in response to supplementation with monosodium glutamate. A mutant, in which the two listerial GAD homologs have been deleted (and in which there is no discernible GAD activity), did not respond to glutamate supplementation and displayed greatly enhanced sensitivity in a number of low pH foods, even when levels of free glutamate were as low as 0.22 mM. We thus show that the GAD system plays a major role in the survival of L. monocytogenes in acidic foods even when levels of free glutamate are low.

Lacticin 3147 is a novel heat-stable bacteriocin, produced by Lactococcus lactis DPC 3147, that exhibits a broad-range inhibition spectrum similar to nisin. In this study, the effect of lacticin 3147 and nisin on the shelf life of fresh pork sausage and their ability to control pathogens ( Clostridium perfringens DSM 756, Salmonella Kentucky AT1) and nonpathogenic Listeria innocua DPC 1770 was investigated. The following preservative regimens were evaluated, both in broth and sausage systems: (i) 450 ppm of sodium metabisulphite; (ii) 500 IU g −1 or ml −1 of nisin, (iii) 2500 arbitary units (AU) g −1 or ml −1 of lacticin 3147; (iv) 2% sodium lactate and 500 IU of nisin; (v) 2% sodium citrate and 500 IU g −1 or ml −1 of nisin; (vi) 2% sodium lactate and 2500 AU g −1 or ml −1 of lacticin 3147, (vii) 2% sodium citrate and 2500 AU g −1 or ml −1 of lacticin 3147, (viii) 2% sodium lactate, and (ix) 2% sodium citrate. There was no significant difference in the activity of nisin and lacticin 3147 against any of the target strains used, both bacteriocins performing significantly better than sodium metabisulfite against gram-positive strains in broth systems. Trends indicate that the combination of organic acids with either bacteriocin enhanced its activity against Salmonella Kentucky and L. innocua and was particularly effective in the inhibition of C. perfringens in fresh pork sausage. In addition, lacticin 3147 combined with either sodium citrate or sodium lactate maintained significantly lower ( P < 0.05) total aerobic plate counts for the duration of the trials and may function as an alternative to sodium metabisulfite in the preservation of fresh pork sausage.

The broad-spectrum bacteriocin lacticin 3147, produced by Lactococcus lactis DPC3147, is inhibitory to a wide range of gram-positive food spoilage and pathogenic organisms. A 10% solution of demineralized whey powder was fermented with DPC3147 at a constant pH of 6.5. The fermentate was spray dried, and the resulting powder exhibited inhibitory activity. The ability of the lacticin 3147–enriched powder to inhibit Listeria monocytogenes Scott A and Staphylococcus aureus 10 was assessed in buffer at both acidic (pH 5) and neutral (pH 7) pH. In addition, the ability of the powder to inhibit L. monocytogenes Scott A in an infant milk formulation was assessed. Resuspension of approximately 10 8 midexponential phase L. monocytogenes Scott A cells in a 10% solution of the lacticin 3147–enriched powder resulted in a 1,000-fold reduction in viable cells at pH 5 and pH 7 after 3 h at 30°C. In the case of S. aureus 10, resuspension of 2.5 × 10 7 midexponential phase cells in a 15% solution of the lacticin 3147–enriched powder at pH 5 resulted in only a 10-fold reduction in viable cell counts, compared with a 1,000-fold reduction at pH 7, following incubation for 3 h at 30°C. The use of the lacticin 3147 powder in an infant milk formulation resulted in greater than a 99% kill of L. monocytogenes within 3 h at 30°C. These results suggest that this bioactive lacticin 3147 food ingredient may find applications in many different foods, including those with pH close to neutrality.

Log-phase Listeria monocytogenes cells become tolerant to a variety of environmental stresses following acid adaptation at pH 5.5. We demonstrated that adapted cells also exhibit increased tolerance to nisin and, to a lesser extent, lacticin 3147. At nisin concentrations of 100 and 200 IU/ml the survival of acid-adapted cells was approximately 10-fold greater than nonadapted cells. However, acid adaptation had only a moderate effect on the tolerance of L. monocytogenes to lacticin 3147, a phenomenon that possibly reflects the distinct mode of action of this bacteriocin. Analysis of the fatty acid composition of the bacterial membrane indicated that straight-chain fatty acids C14:0 and C16:0 were significantly increased in acid-adapted cells while levels of C18:0 decreased. The results indicate that stress mechanisms that are induced in mildly acidic conditions provide protection against the antimicrobial action of bacteriocins. This increased resistance of acid-adapted L. monocytogenes could cause increased survival of this pathogen in food products in which nisin or other bacteriocins are used as preservatives.

Enterocin 1146, a bacteriocin produced by Enterococcus faecium . DPC1146, has a rapid bactericidal effect on Listeria in buffer systems, broth, and milk. In trypticase soy broth, increasing the bacteriocin dose and/or decreasing the pH extended the lag phase of Listeria innocua . A logarithmic relationship was found between response (as proportion of survivors or growth compared to a control) and dose. Increasing the inoculum level of the indicator reduced the effectiveness of enterocin 1146. Log-phase cells of L. innocua were more resistant than stationary-phase cells in both broth and buffer systems. In milk treated with 250 arbitrary units of enterocin 1146/ml and inoculated with 10 3 or 10 5 CFU/ml of Listeria monocytogenes Scott A, populations reached only 5–14% of the control after 24 h at 30°C, with numbers exceeding 10 7 , while at 6°C a slow decrease in population was found.

Enterococcus faecium DPC 1146 produces a bacteriocin, enterocin 1146, which is inhibitory to Listeria monocytogenes . Enterocin 1146 was produced in GM17 and in milk. The bacteriocin was partially purified by ammonium sulfate precipitation. Its molecular weight, estimated by SDS-PAGE, was 3.0 kDa. It could be stored at −20°C without loss of activity, but pH had a marked effect on enterocin 1146, which was more stable at both high (up to 120°C) and low temperatures (4°C) at pH 5 than at pH 7 and 9. The sensitivity of 57 strains belonging to 35 different species was studied using a critical dilution assay. L. monocytogenes and L. innocua were most sensitive; enterocin 1146 had a bactericidal effect on Listeria . Starter and nonstarter lactic acid bacteria (except Lactobacillus sake )were insensitive or relatively resistant to the bacteriocin. Genetic determinants for bacteriocin production and immunity do not appear to be plasmid borne.