Hygiene Aspects of Premature Nutrition

Abstract

The very low birth weight and the not fully developed immune system make preterm infants especially susceptible to infections. Therefore microbiological food safety of preterm nutrition is a particular challenge. This is also due to the fact that breastfeeding is often not possible in these infants. There are several obstacles to breastfeeding, such as intensive care conditions and individual nutritional requirements of the newborn. The chapter covers the microbiological aspects of preterm nutrition, including quality requirements for commercial infant formulas, breastmilk fortifiers and extracted breast milk. The main pathogens of concern (e.g. Cronobacter spp. Salmonella spp. and Clostridium botulinum) are discussed in detail, including related food safety indicators. An important part of the chapter is devoted to the hygienic aspects of preterm formula preparation techniques, storage conditions and microbiological risks linked to certain feeding techniques (e.g. tube feeding). The risks associated with microorganisms found in commercial infant formula and in the prepared environment, as well as the risk of biofilm formation, are described. Options and requirements for risk mitigation are discussed in detail.

1. Introduction

The very low birth weight and the not fully developed immune system make preterm infants especially susceptible to infections. The development stage of the organ systems depends stringently on the gestation age of the newborn. This concerns also the intestinal tract and the immune system. Both are especially important for the resilience of infants to gastrointestinal infections. Regarding the intestinal tract, different aspects have a crucial influence on the immune defence. One critical aspect is that in mammals antibodies cannot pass the placenta. The newborn receives the first antibodies with the first milk of the mother, which is especially immunoglobulin enriched. Under circumstances of a mature digestion, proteins of the size of an immunoglobulin (150 to 1 Mio kDa) cannot pass the intestinal wall to enter the bloodstream. Therefore, the gut of mammals shows a different anatomy during their first days of life than the gut of adult individuals. The tight junctions seal usually the gaps between the enterocytes. These seals are quite open for the first three to four days of life to enable the antibodies of the colostrum to pass into the bloodstream. Although this is not necessary for human neonates, the mechanism is still observed phylogenetically and is even more pronounced in preterm infants. These open tight junctions provide an option for microorganisms and toxins like endotoxins to enter the bloodstream unhindered [1, 2].

Unlike the offspring of other mammals, immune globulins of primate species can pass the placenta and are present in the bloodstream of infants immediately after birth. This has the advantage that the colostrum in humans is not an absolute requirement for the survival of the newborn. However, the antibody-enriched colostrum also provides human infants with humoral protection against intestinal infections during the first days of life. In premature births, the breastmilk is often not available or does not meet the nutritional requirements of the preterm infant and definitely does not provide the quality of a fully developed colostrum [1, 2].

The virulence of microorganisms depends on the ability to overcome the non-specific and specific immune barriers of the host organisms, followed by the effect of specific pathogen factors, like toxins, on the target organ system or tissue. As explained before the specific and non-specific immune defences in very young infants are underdeveloped and weak. The lack of humoral immune protection is compounded by the underdeveloped acid barrier in the stomach of young children. This means pathogenic microorganisms can reach the small intestine, from where they can enter the blood system without passing the usual barriers. Thus, even bacteria with low virulence can cause significant harm to these premature organisms [1, 3].

The intestinal flora is competitive against pathogens that have reached the intestine, which provides an additional protection against gastrointestinal infections. Young children still need to develop their body flora including the gut flora. Their gut flora is not stable in the perinatal period [1]. Until now, it was assumed that the body of the newborn is sterile and that only during birth the first bacteria will colonise the skin and the enteral tract. For some years now, it has been discussed whether the fetus already comes into contact with microorganisms intrauterine [1, 4]. In any case, birth itself plays a decisive role in the colonisation of the body of the infant, as bacteria from the mother are transmitted intensively to the baby during the birth procedure [1]. Preterm infants are often not born in the natural way but by caesarean section. This way of birth already shows a retarded and different type of intestinal colonisation in term infants and is even more critical in preterm infants with regard to the formation of the intestinal microbiome.

The low birth weight and the awkward ratio of body surface to body weight make neonates more susceptible to toxin effects and low infection doses of microorganisms [5]. Moreover, the metabolic functionality in preterm infants is often not fully developed, which influences the neutralisation, turn over and excretion of bacterial toxins [6].

These special conditions meet the specific nutritional requirements of the preterm infants, often requiring individual formulas. In many cases, breastfeeding is not possible or has to be provided with extracted milk fortified with certain ingredients, and even the application of feeding tubes is also a regularly applied praxis. This requires specific hygiene precautions in the preparation, storage and application of food for preterm babies.

2. Types of preterm feedings

There are different types of formula designed for preterm infants. However, the most preferable option is the feeding of breastmilk. Due to the shortened gestation period, the breastmilk of the mothers alone is often not sufficient for the nutritional needs of the preterm child and certain supplements (breast milk fortifiers, thickeners) have to be added [1]. The addition of powdered breastmilk fortifiers to the liquid milk bears significant hygiene risks. The breastmilk fortifiers are not sterile but have similar microbiological properties as powdered formula. The feeding practices of breastmilk to preterm infants is another hygiene issue. Direct breastfeeding is usually not possible for these patients, so milk is applied by tube feeding, syringes or baby bottles. The extraction and storage of the breastmilk pose several hygiene risks, with the addition of fortifier being only one of many other contamination sources.

In many cases, breastmilk is not available or specific nutritional needs require the use of infant formula products. From a hygiene point of view, the use of sterilised ready-to-feed formula is the most recommendable option. If used as a sole source of nutrition, the contamination risks during handling and feeding are minimised. The sterilisation process guarantees the inactivation of all infectious microorganisms including spore-forming bacteria and viruses. However, the bacterial debris of these inactivated microorganisms is still in the formula but the amount is very low due to the advanced hygiene standard during the manufacturing process. A quality parameter for this kind of formula is the level of endotoxin. Endotoxins are lipopolysaccharides, which can be found as part of the cell wall of gram-negative bacteria. The group of gram-negatives includes Enterobacteriaceae, but also the non-fermenters, which are often detected as water bacteria in process water. Endotoxins in food are usually not an issue, as the transfer from the gut into the bloodstream is very limited. The tight junctions between the enterocytes seal the enteron very effectively, and the small amount of endotoxins, which still pass the intestinal wall, are efficiently neutralised in the liver. In young infants and especially in preterm infants, the situation is different. On one hand, the tight junctions are still open as explained earlier and on the other hand, the detoxification ability of the liver is not fully developed. If the level of endotoxins in the formula is too high, these lipopolysaccharides can enter the bloodstream. Monocytes would recognise them and start a cytokine-mediated immune response. These endotoxin levels can cause symptoms from mild sub-clinical disorders to febrile temperature. As the health status of preterm infants is quite fragile in many cases, the endotoxin-related burden is an additional factor that could influence the development of the newborn.

Powdered formula for preterm infants is available in a range of formats to meet the broad variety of the nutritional needs of prematurely born babies. Basically, the conventional starter formulas are also applicable for preterm infants. However, based on their gestational development stage, the responsible medical staff has to decide about the requirement of individually tailored diets. A variety of vitamin, mineral protein and calorie enriched formulas and fortifiers are available on the market to meet the individual nutritional needs of preterm infants. In some cases, the underdeveloped gastrointestinal tract of the baby is not able to break down proteins and carbohydrates, so the infant has to be supplied with free amino acids. In many cases, reflux might be a problem, which requires the addition of thickeners to the diet. These products are often combined or added to breastmilk or ready-to-feed formula.

Powdered infant formula is manufactured under the highest hygiene standards but is not sterile. The microbiological burden of the powder is usually low, but the reconstituted formula is an optimal growth medium where bacteria can multiply rapidly. The powdered formula differs from conventional milk powder in its composition. From a microbiological point of view, the elevated fat content is most important, because it protects bacteria in the dry environment and promotes the biofilm formation on contact surfaces. The powdered formula is produced in several steps based on milk ingredients like skim milk powder and whey protein concentrate. These ingredients have been spray dried and are usually delivered as bulk products to the baby food manufacturer. The spray drying process is conducted in a counter flow of falling droplets against a hot air stream of more than 70°C. However, the bacteria in the droplets are protected against high temperatures due to the evaporative cooling. Therefore, spray drying is not a sufficient heat treatment to kill off microorganisms. These bulk ingredients are mixed with a range of specific nutrients like minerals, vitamins and lipids. The dosing of the especially important micro-ingredients is often done manually and bears a certain risk of contamination from the operating staff. Fat blends are often stored at higher temperatures, which make the survival of vegetative bacteria impossible, but the introduction of bacteria spores can be linked to this type of ingredient. The blending and packing operation of the baby powder is usually a fully automated step, nevertheless, contamination risks are not fully excluded. There are a number of bacteria species, which have adapted to the dry environment and are found as process contaminants in many powder factories.

The intestinal flora is an important part of the defence against gastrointestinal infections and plays an irreplaceable role in digestion and metabolism. The gut flora is labile in all infants during the first 12 months of life, but in preterm infants, this is an issue of special concern. The underdeveloped intestinal flora is seen as a factor that increases the risk for necrotising enterocolitis (NEC) and late-onset sepsis (LOS) [7]. The addition of probiotic strains to the diet of preterm infants is widely discussed. In a number of randomised clinical trials, the prophylactic effect against NEC has been shown and no adverse effects of the probiotics have been reported [8]. It is not clear whether the risk for NEC is only significantly reduced in preterm infants who receive breastmilk supplemented with probiotics. An advantage is not observed in infants fed with probiotic-enriched formula. It seems that there is a shortage of some bioactive ingredients in the formula, which are present in breastmilk [7]. However, the risk of probiotic sepsis remains one of the concerns linked to this kind of supplementation, especially in preterm infants [9, 10, 11, 12]. Invasive diseases linked to probiotics are reported rarely and have never been seen in a randomised clinical trial [8]. However, 49 case reports on invasive diseases in children caused by probiotics have been published in the scientific literature between 1995 and 2021 according to D’Agostin et al. [9]. About 55% of the cases occurred in preterm infants and the majority developed septicaemia. All kinds of probiotics were involved in cases of invasive disease. In most cases, the outcome was favourable but in three cases there was a fatal outcome caused by Limosilactobacillus reuteri (formerly Lactobacillus reuteri), Saccharomyces boulardii and Bacillus clausii [9].

Therefore, the use of probiotics in preterm infants requires a careful assessment of benefits and risks for the individual case. Important risk factors for probiotic bacteremia are e.g. intestinal comorbidity and intravenous catheters [9].

Full-term infants who are exclusively breastfed usually have no need for additional liquid supply [13]. The fluid and electrolyte management of preterm infants is much more complex and in most cases, glucose solutions and water are supplied. In Europe, however, it is common practice to feed newborn infants herbal teas to supply the baby with fluid and relieve intestinal colic, although herbal teas are not recommended for young infants because they impair iron absorption due to the polyphenols they contain [13].

Furthermore, herbal teas often contain high levels of different bacteria species that are not eliminated during preparation and teas serve as an excellent growth medium for microorganisms.

3. Microbiological contaminants in preterm feed

Microorganisms get into the preterm feed from different sources. Some originate from dairy ingredients and survived the different processing steps. These bacteria are, in most cases, spore formers and can survive high temperatures, but Enterococci may also survive the processing steps along the entire line. Another important contamination route are blending operations in powdered infant formula manufacture. A specific flora of microorganisms has become adapted to the dry environment in powder production facilities. These bacteria can survive in the powdered formula for a long time. A high risk of contamination is posed by bacteria that get into the formula during preparation. The germs have the opportunity to multiply rapidly in the dissolved formula. These microorganisms may come from the hands of the person preparing the formula, from insufficiently cleaned feeding equipment or from the environment.

In 2004, microbiology experts categorised potential pathogens, based on the available evidence of a clear link between infant illness and the presence of certain pathogens or bacterial toxins in powdered infant formula. Three categories have been defined, from category A (clear evidence of causality) over category B (causality plausible, but not yet demonstrated) and C (causality less plausible, or not yet demonstrated). Only Salmonella spp. and Cronobacter spp. were identified as bacteria with a clearly proven link between illness and formula contamination [14].

Salmonella spp. are gram-negative rods. Their natural reservoir is the intestinal tract of animals. Salmonella shows a temperature range for growth from 5–45°C with an optimum temperature between 35° and 42°C [15, 16]. A pH below 4.0 reduces the number of viable Salmonella cells. Therefore, the acid barrier of the stomach forms an effective line of defence against Salmonella infections. Infants have a higher pH in the stomach than adults and the milk-based diet further protects against Salmonella during the gastric passage. This makes infants and premature babies especially vulnerable to Salmonella infections [17, 18]. A heat treatment of 2 minutes at 70°C sufficiently kills Salmonella. They are not able to form spores [19].

Salmonella causes severe gastrointestinal infections with diarrhoea, abdominal pains, chills, fever, vomiting and dehydration. The onset of symptoms is usually within 72 h. The severity of the disease depends, among other things, on the virulence of the Salmonella strain and the amount ingested, but all serotypes are potentially pathogenic to humans. Infants and especially preterm infants are among the most vulnerable individuals, where even small infectious doses of less than 100 cfu can lead to potentially fatal infections [16, 19, 20].

Cronobacter spp. formerly known as Enterobacter sakazakii belongs to the genus Enterobacteriaceae [21]. The growth optimum of the microorganism is between 25° and 45°C. There is no multiplication below 5° C and over 45° C [22]. Under optimal growth conditions, the generation time is about 20 minutes and drops to 2 h at room temperature [23].

In dry environments, Cronobacter spp. can survive up to 2 years [24, 25]. The reservoir of Cronobacter spp. includes a wide variety of environmental and food sources [26, 27, 28, 29]. Biofilm formation was observed on different surfaces, which is particularly important for the hygiene management in the care environment of preterm infants [23, 30]. Cronobacter spp. infections are rare and occur especially in neonates and very young children. Since 1958, reports on about 180 infections have been published [31]. In 95% of all Cronobacter cases, infants are affected during their first 2 months of life and the risk for infections is particularly high for preterm infants [31]. Cronobacter spp. cause meningitis, septicaemia and NEC [32, 33, 34].

Between 20 and 80% of the infants do not survive a Cronobacter infection [32, 33, 34]. Among those who recover from the disease, many suffer from lifelong sequelae [31]. The infection dose, for Cronobacter infections, is estimated to be between 100 and 10,000 bacterial cells per meal [35]. This makes bacterial growth in the prepared food a necessary prerequisite for an infection, because these high numbers of Cronobacter cells are not reached in any kind of formula or extracted breastmilk. Although powdered infant formula is in most cases the source of the infection, it has to be regarded that the bacterium can also derive from utensils for formula preparation like bottles or teats, water and handling failures [31]. Biofilm formation is a risk factor for Cronobacter infections if feeding is done via stomach tubes for medical purposes [30, 31, 36]. Stojanovic et al. (2011) analysed 150 herbal teas and found 32% of the teas Cronobacter-positive [37]. Teas are part of the usual diet even of very young infants as early as the first week of life [38]. The teas are often kept over hours at the bedside of babies or patients and are used for oral hygiene or perfusion of stomach tubes [39, 40]. This makes teas a potential Cronobacter-infection risk.

Microorganisms for which World Health Organisation (WHO) experts see a causality between powdered infant formula and infections, but which has not yet been proven, are summarised in category B [14]. These are:

Pantoea agglomerans, Escherichia vulneris, Hafnia alvei, Klebsiella pneumoniae, Citrobacter koseri, Citrobacter freundii, Klebsiella oxytoca, Enterobacter cloacae, Escherichia coli, Serratia spp., and Acinetobacter spp.

Besides these species, other members of the Enterobacteriaceae have been reported to cause gastrointestinal infections such as Edwardsiella trada, Proteus mirabilis, Providencia alcalifaciens, Morganella morganii, Moellerella wisconsensis [16].

The group comprises Enterobacteriaceae with a known potential for opportunistic infections, which makes them particularly important for vulnerable patients like young children and preterm infants. The category has been extended to Acinetobacter spp., which does not belong to the genus Enterobacteriaceae but is regarded as a similar hazard for nosocomial infections. Category B has similarity to the so-called bile tolerant gram-negative bacteria (BTGNB), a method- and risk-based classification that is used by the US Pharmacopoeia. The BTGNB group includes all gram-negative bacteria that grow in the presence of bile salts and can utilise glucose [41]. The most important species in this group is Escherichia (E.) coli, which includes a number of enteropathogenic variants.

The other listed species include more particular pathotypes [36].

All Enterobacteriaceae are thermolabile and a rather mild heat treatment at 70°C over 2 min will eliminate them if the initial levels do not exceed an amount, which is usually assured by general hygiene measures. However low levels of Enterobacteriaceae can be expected in powdered infant formula even when these products have been manufactured according to all hygiene requirements. These nosocomial pathogens require fairly high infection doses (e.g. 108 and 1010 cfu per g for enterotoxic E. coli in adults) to cause illness [42]. In many cases, the infections are linked to tube feeding or catheter treatment. In most cases, multiplication of these bacteria in the feed is necessary to reach the necessary dose for oral infections. This group of bacteria is often involved in the formation of biofilms [43, 44] which are a particular risk in constant feeding via tubes where giving sets can harbour biofilms. A feed contaminated with high numbers of bacteria poses a significant risk for handling contamination of intravenous catheters, which might cause a septicemic infection.

E. coli is a member of the family of Enterobacteriaceae. The term coliforms, which is often found in this context, comprises a larger group of Enterobacteriaceae that have the ability to form acid from lactose and glucose. The group of coliforms includes E. coli, Citrobacter spp., Klebsiella spp., Enterobacter spp. and others [19, 45].

E. coli is part of the intestinal flora of humans and animals. E. coli indicates, therefore, faecal contaminations [45]. Usually, E. coli strains found in the human bowel are harmless commensals but an increasing number of sero- and pathotypes have been identified to be linked to diarrhea and, in some cases, severe complications [45]. A broad range of symptoms has been described, next to diarrhoea, fever, headache, abdominal spasm and nausea. Several types of pathogenic E. coli exist with different pathomechanisms based on adhesins, toxins, invasion proteins and defence mechanisms against host immunity. The main E. coli pathogen groups are:

Enteropathogenic E. coli (EPEC), which are an important causative agent of diarrhoea in infants and young children under 1 year of age with fever, vomiting or abdominal pain. Incubation times of 17 to 72 hours have been reported. The illness lasts between 6 hours and 72 hours. The adhesions structures bundle forming pili (Bfp) and the intimate attachment (EaeA-gene coded) are the underlying patho-mechanisms. EPEC generally do not produce any enterotoxins. A destruction of the intestine brush border microvilli (attachment and effacing lesions) is the consequence of the infection.

The Enteroaggregative E. coli (EAEC) carry a plasmid that enables them to produce fimbriae, short pilus-like structures that promote specific aggregation and adherence of the bacteria to the gut cells.. The EAEC are able to produce a heat-stable enterotoxin. These features result in a prolonged diarrhoea of more than 14 days, especially in children [19].

Enteroinvasive E. coli (EIEC): The illness caused by this group develops within 2–48 hours. The EIEC strains invade the cells of the large intestine. Typical symptoms are bloody and non-bloody diarrhoea or dysentery with fever, headache, muscular pain and abdominal spasm [19].

Enterotoxic E. coli (ETEC): ETEC colonises the small intestine but do not enter the host cells. They produce toxins of a heat-labile and a heat stable type. The strains cause diarrhoea in infants but can also be pathogenic for adults, presenting as travellers diarrhoea. The infection dose in adults is about 108 to 1010 cells and for infants some log units lower [19, 42].

The Enterohemorrhagic E. coli (EHEC) are also known as Verotoxin or Shiga-like-toxin producing E. coli (VTEC or STEC, respectively). The strains attach to the cells of the large intestine and cause lesions and bloody hemorrhagic colitis, the produced toxins (verotoxin, enterohemolysin) enter the bloodstream to cause kidney failure (hemolytic uremic syndrome - HUS) and can damage the blood cells (thrombotic thrombocytopaenic purpura - TTP). The detection of the Shiga toxin gene indicates always the potential of an EHEC infection including HUS and TTP. These diseases are very severe and particularly in infants, the risk of a fatal outcome or permanent kidney failure is high. The infection doses have been reported as low as 10 cells. The incubation period may range from 3 to 9 days and duration of the illness from 2 to 9 days. EHEC are able to survive pH 2, which enables them to pass the gastric acid barrier [16, 19, 45].

A number of other pathogenic E. coli cause diseases of the urogenital system, meningitis and sepsis. These strains are usually not spread through the consumption of food [16, 46].

In category C, WHO experts included microorganisms for which they considered the causal relationship to be less plausible or for which it has yet not been possible to demonstrate that there is a stringent link between infant infections and powdered formula. In some cases, infections in infants were reported but the concerned microorganism has not been isolated from infant formula, in other cases, the bacterium has been found in infant formula but was not linked to illness in infants. However, these organisms are well-known food pathogens and there is no reason that infants would not share the risk of other vulnerable groups [14]. The category C organisms are:

Bacillus cereus, Clostridium perfringens, Clostridium difficile, Clostridium botulinum, Listeria monocytogenes, Staphylococcus aureus, and coagulase-negative Staphylococci.

Bacillus (B.) cereus is a spore-forming bacterium and a well-known enteropathogen. It appears as gram-positive rod and is able to grow aerobically and anaerobically [47]. Some strains produce different toxins: a heat-sensitive enterotoxin, which causes diarrhea and is formed in the small intestine, although preformation in food is also possible. The extremely heat stable emetic toxin (cereulide) is performed in the affected food and causes an intoxication with symptoms occurring within 1 to 5 hours. The symptoms last for up to 24 h with nausea, vomiting and occasional diarrhoea. The numbers of bacteria cells found in food associated with B. cereus poisoning have usually been as high as 106 g⁻¹. However, occasionally numbers as low as 103 g⁻¹ were observed and should be regarded as a potential risk for infants especially. In most cases of B. cereus intoxication, a storage of the food at elevated temperatures made an excessive growth of the bacterium in the food possible and resulted in a preformation of toxins. The heat stability of the emetic toxin bears the risk that during bacterial growth emetic toxin is formed and subsequent process steps reduce the B. cereus counts, but the toxin concentration remains at high levels. A classical microbiological analysis would not reveal the problem. The toxins are detectable only with more elaborated methods [45]. B. cereus is found in a wide range of environmental and food sources like soil, dust, surface water, cereals, milk and dairy products. B. cereus has been found in 9–12% of fresh milk and in 35–87% of pasteurised milk samples. In powdered milk, B. cereus is a common contaminant and 50% of the powdered infant formula was found to be positive for B. cereus on low levels (10–100 g⁻¹). While this does not pose an acute risk, problems can occur if there is temperature abuse or prolonged storage time after reconstitution of the formula, which allows bacteria to grow to higher numbers [16, 48]. Bacteria of the B. cereus group have a growth range between 10 and 50°C, with an optimum temperature of 28–35°C. Some psychotropic strains are capable to grow at 4°C. The minimum pH of growth is 5.5, below 4.5 vegetative cells start to die off [16]. B. cereus spores are heat resistant and can, therefore, be expected after spray-drying of milk-based infant formula.

In newborn and preterm infants, clinical manifestations such as septicemia, respiratory tract infection, enterocolitis, hepatitis, endocarditis, endophthalmitis and encephalitis with cerebral abscess have been reported in infections with B. cereus. The severity of these infections in neonates ranges from symptomless gut colonisation to fatal outcomes in 40% of the cases. The role of bacterial exotoxins in these diseases is not clear [49]. Infection with B. cereus in infants is rare but severe. Between 1977 and 2018 only 50 cases have been reported in the scientific literature. In most cases, the source of infection has not been proven and there have been suspicions about respiratory support equipment, umbilical catheterization, gastric feeding tubes, dried formulas, extracted human breastmilk, linens, heating, ventilation and air conditioning systems [50]. Although the link between infection and extracted human breastmilk has never been proven, B. cereus remains a major concern in the hygiene management of both extracted breast milk and infant formula. About 10% of the breastmilk bank amount is discarded in 9 of 10 cases due to contamination with B. cereus. Standard procedures for extracted human breastmilk handling include a pasteurisation step at 62.5°C for 30 min but B. cereus in spore form can survive this treatment. Additionally, a post-pasteurisation contamination is possible as B. cereus is often found in the hospital environment [49].

Clostridia are gram-positive rods that grow only under strictly anaerobic conditions [19]. They are found ubiquitously in air, soil, water, faeces, milk and other foods [16, 45]. Foodborne illnesses can be caused by several species, including Clostridium (Cl.) perfringens, Cl. botulinum Cl. butyricum, Cl. sphenoides, Cl. sordelli, Cl. spiroforme, Cl. difficile and Cl. baratii [45].

Cl. perfringens has been found in low numbers in many types of processed food and is part of the physiological gut flora in low numbers. Cl. perfringens is able to grow at unusually high temperatures. The optimal growth temperature is 43–45°C where the generation time is rather short (7 minutes). The growth temperature range reaches from 15 to 50°C. The pH optimum for growth is quite narrow and stretches from pH 6 to 7; very little growth occurs below pH 5 or above pH 8.3 and in the presence of oxygen [16]. Cl. perfringens enterotoxins can cause food poisoning. Viable vegetative cells in large numbers (>105 g⁻¹) in foods are necessary for foods to cause food poisoning [16]. The toxin is normally formed in the human intestine. Onset of symptoms is 8–24 hours after ingestion of the contaminated food. The illness causes diarrhoea and severe abdominal pain. A full recovery is normally seen within 24 h. Complications and death from Cl. perfringens have been reported among vulnerable consumers. In preterm infants, the bacterium has been linked to NEC. About one-third of the preterm neonates have been seen colonised 3 weeks after birth. A colonisation was unlikelier with prolonged breastfeed, antibiotic treatments and oxygen support. The positive effect of breastmilk has been seen only for breast-fed infants and infants fed with extracted milk from their own mother. In pasteurised donor milk, the effects have not been observed [16, 51]. An antibiotic treatment can also cause imbalances of the gut flora causing severe health problems caused by Cl. perfringens or Cl. difficile. Cl. difficile belong to the normal gut flora but may cause also severe symptoms in neonates like diarrhoea with dehydration and electrolyte imbalance, abdominal pain and distension and poor weight gain sometimes with fatal outcome.

Cl. botulinum is much less often found then Cl. perfringens. Cl. botulinum is able to form seven different toxins. Responsible for most human botulism cases are the toxins A, B, E and F to lesser extent. There is a mesophilic group that is proteolytic and able to produce heat-resistant spores and a psychotropic group, which is often non-proteolytic and whose spores have only a low heat resistance [16]. Growth is seen from 20 to 40°C under strictly anaerobic conditions. The toxins of Cl. botulinum, which are pre-formed in food, initially cause vomiting and diarrhoea after ingestion and end with double vision, difficulty in breathing and paralysis. The toxins work by interference with nerve stimuli. The Cl. botulinum toxin is the most potential natural poison. Its lethal dose varies between 0.005 and 0.5 μg. 0.1 g of food in which Cl. botulinum has grown is sufficient to cause botulism [16, 45, 52].

A special form of botulism is infant botulism which may be caused by the ingestion of only a few spores. These multiply in the gut of the infant and produce toxins. The disease causes progressive paralysis that starts with constipation and develops into respiratory paralysis and death if untreated. The illness affects only infants below 1 year of age. Adults are protected by their normal, inhibitory gut flora. Infant botulism has been caused by honey in which 80 spores g⁻¹ were present [53].

In 2001, the case of a 5-month-old infant with infant botulism with a possible link to powdered infant formula has been reported. Cl. botulinum type A was found in faeces. In a powdered infant formula package fed to the baby and an unopened package of the same batch Cl. botulinum type B has been found. Therefore, no stringent link could be made between the formula and the illness but the case shows that infant feed is a potential source for infant botulism [54, 55].

L. monocytogenes is a non-sporing, gram-positive rod. The bacterium is facultatively anaerobic with growth temperatures from 1 to 45°C and a growth pH between 4.6 and 9.2. The range for optimal growth is 30 to 37°C and a pH of 7. It can cause listeriosis with symptoms such as fever, headache, gastrointestinal problems and vomiting, which can develop into meningitis or septicaemia, especially in vulnerable groups like neonates. Pregnant women may develop a flu-like disease, which could cause miscarriage or diaplacentar can harm the foetus. The infection can reach mortality rates of 30 to 40% in vulnerable groups. An infection dose of 106–109 cells is necessary to cause infections in immunocompromised persons. However, in most cases, the human infections with L. monocytogenes remain clinically inapparent. Foods involved in the foodborne infections are raw milk products, raw vegetables and meat products. Pasteurisation temperatures can kill Listeria reliably, therefore a contamination of powdered infant formula from raw materials is unlikely but a recontamination from the environment remains possible [45]. In neonates, a listeriosis is seen in two forms. The granulomatosis infantiseptica listeriosis is an intra-uterine acquired infection with early onset (< 7 days of life). The incubation time for a listeriosis infection ranges between 3 and 70 days [45]. The onset of symptoms (fever, respiratory, circulatory and liver distress) of the early onset infection is usually seen within 2 days after birth. A second form is the late onset listeriosis with symptoms like hyperexcitability, vomiting, cramps and pneumonia. This infection is acquired during birth from the mother or after birth from the environment. Listeria infections can be transmitted directly in person-to-person contact or via food or other vehicles. Nevertheless, to our knowledge, a transmission with infant feed has not been reported so far [56, 57].

Staphylococcus (S.) aureus is a gram-positive coccus, which can grow aerobically or anaerobically. S. aureus grows between 7 and 48°C, with an optimum of 35–37°C and a pH range from 4.0 to 9.8 (optimum 6.0–7.5). S. aureus is inactivated by pasteurisation [45]. The bacterium can cause food intoxication when a heat stable enterotoxin is produced. The toxin is performed in the food when the bacteria have the chance to grow to high numbers. Staphylococci are poor competitors and do not grow well in the presence of other microorganisms. In food with considerable numbers of competitive flora, the presence of S. aureus may be unproblematic [16]. However, infant feed is a food with a very low bacterial flora, which gives contaminants, such as Staphylococci, enough room to multiply. There are eight different enterotoxins types, which can be produced by S. aureus. Most common in food intoxication is enterotoxin A. 0.1 to 1 μg toxin in food can cause food poisoning [16, 19, 45]. The enterotoxin production is linked to the bacterial growth and the amount of toxin produced depends on the strain and growth conditions (pH, temperature, water activity). Levels of 105–106 cells per g of food have to be reached for relevant toxin concentrations. The symptoms of Staphylococcus-intoxication include nausea, vomiting, abdominal spasm and diarrhoea and headache and muscle spasm. The symptoms start abruptly within 2–8 hours and recovery is normally seen within a few hours [16, 45].

In food microbiology, the focus is on coagulase positive Staphylococci which also include S. intermedius and S. hyicus. These two species can produce the enterotoxins also. The methicillin-resistant Staphylococci (MRSA) are a topic of concern in the health care environment. MRSA have the same potential to produce enterotoxins as any methicillin-sensitive strain. In 2002, an outbreak of gastrointestinal illness has been linked to MRAS for the first time [58].

In very low-birthweight neonates infections with coagulase negative Staphylococci are a matter of concern. These Staphylococci are responsible for more neonatal infections than S. aureus. S. epidermidis is the predominant species in these infections but there are also reports of neonatal infections with S. haemolyticus, S. hominis, S. warneri, S. saprophyticus, S. cohnii and S. capitis [14, 59, 60].

The reservoir of S. aureus and other Staphylococci is the human mucosa in the nose and throat. S. aureus can be found on the nasopharyngeal mucosa of 20–40% of the healthy population in Germany and the Netherlands. The bacteria are often transferred by handling food from the skin of the operators [18, 45].

4. Microbiological quality parameters of infant food

Powdered infant formula is manufactured under high-standard hygiene conditions. Therefore, the products have an extremely low bioburden but are not sterile. To control the microbiological quality of the powders, quality parameters have been laid down in different legislations and recommendations. The European legal standards consist of three types of parameters. These are, on the one hand, direct tests for pathogens or safety criteria (Cronobacter, Salmonella, Listeria, S. aureus, B. cereus, E. coli and C. perfringens) and, on the other hand, the following two process hygiene criteria:

1. the index microorganisms that point to the possible occurrence of pathogenic organisms in foods (Enterobacteriaceae, sulphite reducing Clostridia).

2. indicator organisms that are used for the validation and control of the process integrity and hygiene (total viable count (TVC), yeast and moulds and Enterococci).

The pathogens have been discussed in detail above. Quality control tests for Salmonella and Cronobacter are required in most legislations. Commission Regulation (EC) No 2073/2005 [46] requires to prove the absence of Salmonella in 30 times 25 g and Cronobacter in 10 times 30 g for dried infant formula and products for special medical purposes for infants under 6 months of age. Additionally, ready-to-eat foods for infants are required to be negative for L. monocytogenes in 10 times 25 g.

Similar requirements are found in a number of national and international standards. For Cronobacter and Salmonella criteria identical to the Commission Regulation (EC) No 2073/2005 [46] are found in the Codex Alimentarius documents [61] and the US-FDA requirements [62].

The process hygiene criteria are used to indicate the possible presence of an underlying contamination with severe pathogens. The Commission Regulation (EC) No 2073/2005 [46] has defined process hygiene criteria. Presumptive B. cereus is an indicator for toxigenic B. cereus, which has to be tested 5 times in 1 g (counts exceeding 500 cfu/g are not acceptable but one sample with counts between 50 and 500 cfu/g is accepted). Enterobacteriaceae have to be absent 10 times in 10 g. The presence of Enterobacteriaceae indicates an elevated risk for the presence of nosocomial pathogens of the BTGNB-group including Salmonella and Cronobacter. The Commission Regulation (EC) No 2073/2005 [46] lays it in the hands of the infant formula manufacturer to prove a stringent link between Enterobacteriaceae and Cronobacter. If this is possible, parallel tests for both species are not required as long as tests for Enterobacteriaceae are negative.

Another group of bacteria with index function are sulphite reducing Clostridia (SRC). The bacteria are gram-positive anaerobic rods and can reduce sulphite to H2S, which is of interest as an analytical characteristic for differentiating Clostridia from competing flora. The SRC are used as indicators for pathogenic Clostridia. These microorganisms are spore formers and can survive heat treatments such as pasteurisation steps. Therefore, the SRC also serve as good indicators of the microbiological quality of the processed raw materials [63]. The International Commission on Microbiological Specifications for Foods (ICMSF) regards SRC as a valuable parameter to indicate pathogenic Clostridia. A proposed limit of 100 cfu/g could show that the established hygiene control measures are sufficient to keep the risk for Cl. botulinum negligible [64].

Enterococci are used as an indicator bacterium for faecal contamination, often together with E. coli or coliforms. In powdered infant formula, Enterococci can be a useful indicator for severe hygiene failures as they have a higher heat resistance than gram-negative non-spore-forming rods and may reflect better the hygiene history of the production and raw material quality. Moreover, Enterococci are opportunistic pathogens and might pose a direct health hazard to vulnerable consumer groups like preterm infants [65]. Enterococci are usual contaminants in powdered infant formula and do not pose a direct health hazard in low numbers [66]. Elevated levels of Enterococci might indicate shortages in the production or handling hygiene of infant feed.

An obvious link between process and handling hygiene provides the total aerobic plate count. If elevated total aerobic plate counts are found this may be due to the poor quality of raw materials, inadequate cleaning of processes, the growth of micro-organisms during manufacturing or recontamination after heat treatment. The Codex Alimentarius committee recommends a microbiological limit for mesophilic plate count of 5000 cfu/g with five samples to be tested of which two are allowed to range between 500 cfu/g and 5000 cfu/g [61].

The count of yeast and mould can support the assessment of the process quality. These microorganisms are found in powdered formula in small numbers, and numbers exceeding 100 cfu/g might indicate a hygiene problem [67]. A number of moulds are able to produce toxins, which could be e.g. carcinogenic. Therefore, an elevated level of moulds in the powdered formula is always a matter of concern.

5. Hygiene aspects in handling infant food

Certain microorganisms can survive the manufacturing process of powdered infant formula and may be present in low concentrations. Although the bacteria are not able to proliferate in the formula, some of them may remain viable for a long time. In addition, pathogens can also get into the formula afterwards, e.g. through contaminated preparation utensils or through inadequate hygiene. In turn, bacteria can multiply quickly in the ready-prepared formula if it is not properly cooled. Therefore, infant starter formula should be fed directly after preparation (i.e. within 2 hours) [68]. This recommendation applies in private households without exception.

Boiled drinking water has to be used for infant formula for children during their first months of life. Local sources of water contamination as well as long holding times of the water in pipes or the formation of biofilms on taps can result in higher levels of microbes and pathogens even in drinking water. Sterile filters are sometimes used as an alternative to the boiling of water. However, a recontamination may take place downstream of the filter. The process of boiling water serves to reduce and eliminate microbial risks but at the same time can lead to additional health risks to the infant, such as scalding or burn injuries. Feeding insufficiently cooled formula can cause scalding in the mouth of the infant. In order to avoid the latter, a few drops of the prepared formula from the baby bottle should be applied to the inside of the wrist to test the temperature. These drops of formula should not feel warm and certainly not hot. If the temperature control is done with thermometers, the contamination risk has to be regarded. Therefore, the use of a contactless infrared thermometer is recommended. The widespread practice of parents testing the temperature by drinking the infant’s formula by themselves has to be avoided since a transfer of microbes from the oral flora of the parents to the infant will occur and that could later cause problems such as dental caries. The general hygiene criteria for the preparation of powdered infant starter formula also apply to preterm infants who have reached the stage where they can be fed in the same way as full-term infants. The handling requirements must also be complied with after the discharge from the hospital in private households, childcare facilities and daycare centres.

In the professional care environment (e.g. in day nurseries or hospitals) some deviating practices have been established like the preparation of larger quantities of formula in advance if preparation just before consumption is not possible. However, this approach requires an effective hygiene regimen and precise temperature monitoring. Sterile (aseptic) conditions are required in the facility used for preparation, and both the storage and transportation of the prepared formula must be temperature-controlled (no more than 24 hours at below 5°C) [68]. For these reasons, the preparation of formula in advance should only be practised in a professional environment, and even here, the alternative option of using commercial sterile liquid formula should be considered, since the storage of prepared infant formula always involves a greater risk [68].

When mixing powdered infant formula with water, the temperatures specified by the manufacturer must be strictly observed. Water temperatures ranging from roughly 20–50°C are appropriate for preparation. The formula must have cooled down to drinking temperature before feeding. The following hygiene rules must be observed during preparation [69]:

• Washing the hands thoroughly with soap and hot water (under a running tap) before preparation

• Thoroughly cleaning of bottles, spoons and teats with detergent and hot water, and afterwards proper drying. Boiling these utensils or immersing them in boiling water for at least 2 minutes provides an extra level of safety. The use of a commercial steriliser for baby bottles is also an option. These kinds of heat deactivation techniques are especially recommended for the care of infants, which have been born prematurely and in professional childcare environments.

• Powdered infant formula must never be prepared at the same time or space with the preparation of other foods, especially raw foods. A separation from equipment cleaning activities has to be applied as well.

• Powdered milk formula has to be stored in a dry, cool place and sealed tightly. Additional information is provided by the manufacturer.

• The bottles should only be prepared shortly before feeding and cooled to drinking temperature as quickly as possible (max. 15 min) to avoid the proliferation of microbes.

• The feeding should be finished within 2 hours and any remains of prepared formula have to be disposed of immediately.

• If a formula is allowed to dry in the equipment, cleaning is much more difficult. Therefore, bottles and teats should be rinsed with drinking water directly after use.

• Measuring scoops should be stored separately from the powder in a closed container and touched only by the handle. If the scoop is provided in the powder, it should be removed from the package with a clean set of tweezers.

• As an alternative to a formula preparation in advance for occasions like feeding infants at night, travelling with infants or preparing daily portions of formula for later feeding in childcare facilities, the best approach is to portion the powder into clean and dry bottles, store the boiled drinking water in a clean and sealed thermos flask and mix both only shortly before feeding. Professional childcare facilities like hospitals have particular strict conditions for the preparation of formula, they make a storage at below 5°C for a maximum of 24 h possible. The formula has to be portioned into separate bottles under the same strict hygiene condition immediately after preparation. Before storing in the refrigerator, the bottles have to be cooled down to room temperature as quickly as possible e.g. under running water. In such cases, the refrigerator temperature must be documented at regular intervals. At temperatures above 5°C, slow but constant bacterial growth is possible in the formula.

• Additives like fortifiers have to be added immediately before feeding if necessary.

• When brought to drinking temperature (max. 37°C) with a bottle warmer as fast as possible (max. 15 min) the formula can be fed to the infant within 2 hours. These temperatures offer ideal conditions for bacterial growth during prolonged feeding.

Preterm infants require special care and a high hygiene standard during their first days or weeks of life. The infants are usually nursed in hospitals during this critical period. In the hospital environment, special hygienic requirements have to be met. Therefore, it is recommended to establish a dedicated milk preparation room [70]. The hygiene requirements for the milk preparation room should be described in detail in a hygiene plan that covers the following aspects as a minimum [69]:

Structural requirements:

• The milk preparation room should be a defined environment with conditioned air (defined temperature and humidity). Only authorised staff should get access to the milk preparation room, so access has to be regulated. The staff has to be trained on hygiene rules on a regular basis. The traffic routes in the facility have to be defined to minimise the risk of cross-contamination. A one-directional flow from powdered formula to the prepared bottles or feeding containers is recommended and crossing traffic of waste, formula for disposal or equipment for cleaning with prepared formula and sterilised equipment has to be avoided. The handling of fresh formula should be organised in a “white” area which is separated from the grey area used for the handling of bottles and equipment for cleaning.

Equipment:

• The equipment for use has to be defined. For extremely critical techniques like tube feeding, one-way material should be used consequently. This kind of material is for single use only and must not be reused. This applies also to relatively short time frames. The materials have to be disposed of in a way that a mix-up with new unused material and unintended reuse can be excluded. Re-useable material has to be cleaned in a validated cleaning procedure.

Procedures:

• A risk assessment for the different types of feed should be done. There are types of feed with elevated hygiene risks like tube feeding and unfreezing and supplementing extracted human milk. The procedures for preparation must be specified in detail. A picture board is recommended to illustrate the different preparation procedures for the staff. The preparation of especially critical feeds can be done under a laminar airflow. Tools for risk management like a HACCP (Hazard Analysis Critical Control Point) concept should be applied and a detailed documentation of the preparation process must be established. This should include the key facts of the prepared formula such as batch number of formula, preparation staff, preparation time, preparation amount and storage temperatures. A retention sample of each prepared batch has to be kept for at least 5 days, preferably deep frozen.

Cleaning and disinfections:

• The cleaning process has to be validated for the different materials in use (glass, polycarbonate, etc.) for the temperature applied, detergents and disinfectants, the application times and the used disinfection equipment.

• Hygiene monitoring in the milk preparation room:

• The milk preparation room must be part of the general environmental monitoring of the hospital. Microbiological surface swabs have to be taken from critical spots. General hygiene parameters such as total plate count should be included as well as critical parameters such as Salmonella, Cronobacter and Enterobacteriaceae.

Requirements for personnel:

• The personal hygiene of the employed staff has to be defined according to hand washing and disinfection. The hand washing and disinfection should be monitored by microbiological hand swabs on regular basis. Jewellery, skin piercings artificial fingernails and nail polish are not accepted. Behaviour rules for the milk preparation room have to be defined e.g. the use of cell phones is not allowed and people with gastric or respiratory infections are not allowed in the room. The work clothing has to be worn exclusively in the milk preparation room. Hair and beards have to be covered and for critical operations, a mouth mask and possibly disposable gloves should be worn.

Quality controls (microbiological monitoring):

• The prepared formula, extracted human milk and the used materials like powders and equipment should be monitored on a regular basis. Details on the quality parameters of infant formula have been described before.

6. Conclusions

The personnel employed in the milk preparation room must have the appropriate qualifications to ensure compliance with these special hygienic requirements. The in-house monitoring systems and hygiene management measures have to be the subject of regular instructions and training. Personal hygiene, like regular, hygienic hand washing, and wearing of hygienic clothing, including a head covering and possibly disposable gloves, is the personal responsibility of each employee. The hygienic approach to infant formula preparation and compliance with all guidelines, together with the full documentation and reporting of deviations from these rules is a management task for the concerned section.

In the case of premature babies and immunocompromised infants, the assessment of the individual nutritional requirements is the responsibility of the medical practitioner. This includes also the risk assessment for lactogenic viruses. There are some viruses, which can be transferred from the lactating mother to the newborn with human milk. The risks of infection have to be assessed individually regarding the clinical situation of the mother and the newborn [71].

In cases where breastmilk is not available sterile ready-to-feed formula products are the best alternative from a hygienic point of view. However, often these formulae do not meet the individual nutritional needs of an especially susceptible group of infants to the fullest extent. Accordingly, these products or the breastmilk must often be enriched with food supplements like breastmilk fortifiers. Their addition poses a hygiene risk and has to be done as hygienically as possible immediately before use as described above.

Powdered infant formula provides some more flexibility to combine the feed according to the individual requirements of the infant. However, the powders are not sterile. The reconstitution of the formula with water at temperatures above 70°C has often been recommended to reduce the microbiological risk. But the procedure has been found to be very inefficient for this purpose. Therefore, a case-by-case decision should be made weighing the potentially damaging effects of heat on the individually formulated nutrient concentrate against the benefits of reducing microbiological risks.