**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 [26–29]. 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 [32–34].

Between 20 and 80% of the infants do not survive a *Cronobacter* infection [32–34]. 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.

#### *Hygiene Aspects of Premature Nutrition DOI: http://dx.doi.org/10.5772/intechopen.107861*

*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-liketoxin 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

#### *Maternal and Child Health*

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

*Hygiene Aspects of Premature Nutrition DOI: http://dx.doi.org/10.5772/intechopen.107861*

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:


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 microorganisms 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

#### *Hygiene Aspects of Premature Nutrition DOI: http://dx.doi.org/10.5772/intechopen.107861*

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 temperaturecontrolled (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]:


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]:
