Keeping baby bug free

Powdered milk formula is not sterile and microbial contamination can occur – here we learn how to detect potentially hazardous bacteria in baby’s’ favorite treat

THE bacteria Salmonella and Enterobacter sakazakii have been associated with illness attributed to contaminated powdered infant formula, and are cited by the European Food Safety Authority’s (EFSA) Scientific Panel on Biological Hazards as the pathogens of greatest concern to newborn babies.

Although infections with these bacteria from formula milk are relatively rare, powdered infant formula is not sterile and microbial contamination can cause serious illness. In December 2006, the Department of Health (DH) and the Food Standards Agency (FSA) issued revised guidance for health professionals on the safe preparation, storage and handling of powdered infant formula milk to reduce these risks.

Good hygiene practices are essential in preparing and storing feeds made from powdered formula. EFSA, DH and the FSA recommend allowing boiled water to cool to no less than 70°C, which in practice means using water that has been covered for less than 30 minutes after boiling. After reconstitution, the powdered infant formula should be cooled to an appropriate feeding temperature. It is recommended that feeds are used within two hours of preparation, as the length of time for which reconstituted formula is stored increases the risk of bacterial growth and therefore infection. In order to further refine advice regarding the microbiological safety of powdered infant formula, the FSA is supporting studies examining optimal storage times and conditions in further detail.

Dr Steven Forsythe’s research group at Nottingham Trent University is investigating how quickly Enterobacteriaceae such as Salmonella, Enterobacter sakazakii, Escherichia coli, Klebsiella pneumonia and Citrobacter freundii can grow in reconstituted powdered infant formula. In order to study the growth rates of these organisms over a range of temperatures, they are using the Rapid Automated Bacterial lmpedance Technique (RABIT) from Don Whitley Scientific.
The RABIT is used to measure changes in the culture medium’s electrical conductance that occur when charged metabolites are produced by the bacteria of interest. Dr Forsythe’s group is using six separate modules at different temperatures to measure samples at set time intervals over a range of dilutions. A calibration curve is produced for each organism, where the time to detection is directly proportional to the growth rate and indicative of real-time microbial activity.

Allowing data export to all standard spreadsheet and database programs, the automated system’s intuitive software simplifies sample entry and results analysis. Quality-assured results are available more quickly than with traditional microbiological methods, improving both sample throughput and laboratory efficiency.

This approach is used in Dr Forsythe’s laboratory instead of inoculating large numbers of culture plates with different incubation times and temperatures, and is much less labour intensive and less time consuming. Using the manual plate method, samples at each time point would need to be collected, diluted, plated, incubated overnight and counted the following day.
Dr Forsythe’s research is due to be completed in September 2008 and the final report will be available via the Agency’s website (http://www.food.gov.uk).

Salmonella
• Salmonella is a bacterium which is one of the commonest causes of food poisoning worldwide. There are over 2,000 different types of salmonella, but with the exception of the few which cause typhoid or paratyphoid fever, the illness they cause is similar. Salmonella bacteria cause food poisoning, typhoid fever and paratyphoid fever. More than 2500 different strains of salmonella have been identified.
• Anyone can get salmonella, but young children, the elderly and people whose immune systems are not working properly have a greater risk of becoming severely ill.
• Salmonella is a genus of rod-shaped Gram-negative enterobacteria. It was named after Daniel Elmer Salmon, an American veterinary pathologist, although it was his partner Theobald Smith (better known for his work on anaphylaxis) who first discovered the bacterium in 1885 from pigs.
• Disease-causing Salmonella species have recently been re-classified into a single species, Salmonella enterica. Salmonella Typhi causes typhoid fever. Other salmonellae are frequent causes of foodborne illness, especially from poultry and raw eggs and more generally from food that has been cooked or frozen and not eaten straight away.

Enterobacter sakazakii
• Enterobacter sakazakii is a Gram-negative rod-shaped pathogenic bacterium of the genus Enterobacter. It is a rare cause of invasive infection with historically high case fatality rates (40–80%) in infants.It is an opportunistic pathogen widely found in the environment.
• During many years the only published data on the isolation of E. sakazakii were cases of neonatal meningitis or necrotising enterocolitis related to powdered infant formulae. This led to the establishment of a causal link. In some later cases the organism was isolated on utensils such as mixers used in bottle kitchens. Even though in some cases E. sakazakii could not be isolated from the infant formulae, a causal relationship was assumed.
• It is, however, only very recently that information on the widespread occurrence of E. sakazakii has become available. Recent publications have demonstrated that this microorganism can be found in a wide variety of foods, water and environments including homes and hospitals.
• It can cause bacteraemia, meningitis and necrotising enterocolitis. E. sakazakii infection has been associated with the use of infant formula.
• E. sakazakii was defined as a new species in 1980 by Farmer et al. 1980. DNA-DNA hybridization showed that E. sakazakii was 53–54% related to species in two different genera, Enterobacter and Citrobacter. However diverse biogroups were described and Farmer et al. suggested that these may represent different species.

Escherichia coli
• E. coli is Gram-negative, facultative anaerobic and non-sporulating. It can live on a wide variety of substrates. E. coli uses mixed-acid fermentation in anaerobic conditions, producing lactate, succinate, ethanol, acetate and carbon dioxide.
• Most E. coli strains are harmless, but some, such as serotype O157:H7, can cause serious food poisoning in humans, and are occasionally responsible for costly product recalls.
• The harmless strains are part of the normal flora of the gut, and can benefit their hosts by producing vitamin K2, or by preventing the establishment of pathogenic bacteria within the intestine.
• E. coli was discovered by German pediatrician and bacteriologist Theodor Escherich in 1885, and is now classified as part of the Enterobacteriaceae family of gamma-proteobacteria.
• Virulent strains of E. coli can cause gastroenteritis, urinary tract infections, and neonatal meningitis. In rarer cases, virulent strains are also responsible for peritonitis, mastitis, septicemia and Gram-negative pneumonia. Recently it is thought that E. coli and certain other foodborne illnesses can sometimes trigger serious health problems months or years after patients survived that initial bout

By Sam Hinchcliffe.Sam is a product specialist at Don Whitley Scientific, with particular responsibility for RABIT. She has an honours degree in Biomedical Sciences from Manchester Metropolitan University and proven technical ability in a number of molecular and microbiological techniques.