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275Ann Ist super sAnIt 2008 | Vol. 44, no. 3: 275-280

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Summary. The Enterobacter sakazakii is considered an emerging pathogen and has been recentlyconnected to neonatal cases o necrotizing enterocolitis and meningitis due to use o contaminatedpowdered inant ormula. However its presence is not limited to powdered inant ormula; it canalso be ound in a broad range o oods and in water, in a variety o areas, including hospitals andhouses. Due to the gravity o the inections attributed to E. sakazakii, it is necessary to introducerigorous control measures to reduce the risks o contamination at various levels: industrial, to pre-vent rom production to marketing the contamination o products; at a domestic level by reducingthe risk o contamination, during preparation, handling, and storage, o reconstituted products;

and legislative by establishing guidelines and recommendations issued by competent authorities, toguarantee the saety o inant ood.

Key words:E. sakazakii, powdered inant ormula, inections, inants, control measures.

Riassunto(Enterobacter sakazakii: epidemiologia, maniestazioni cliniche e misure di controllo).Recentemente, lEnterobacter sakazakii, considerato un patogeno emergente, stato legato a casi dimeningite ed enterocoliti necrotizzanti neonatali dovuti al consumo di latte in polvere per linanziacontaminato. La sua presenza non comunque limitata al latte in polvere; esso pu, inatti, ritrovarsiin unampia variet di alimenti, acqua e ambienti inclusi ospedali e abitazioni. A causa della severitdelle inezioni sostenute da Enterobacter sakazakii necessaria una riduzione del rischio da esso rappre-sentato, intraprendendo una serie di rigorose misure di controllo, sia a livello industriale, per prevenirela contaminazione del prodotto lungo la liera produttiva no al consumatore, sia a livello domesticoper ridurre la contaminazione del prodotto ricostituito durante la preparazione, manipolazione e con-

servazione; nonch a livello legislativo per stabilire linee guida e raccomandazioni, emanati da organicompetenti, per garantire la sicurezza degli alimenti destinati allinanzia.

Parole chiave:E. sakazakii, latte in polvere per linanzia, inezioni, neonati, misure di controllo.

Enterobacter sakazakii: epidemiology,clinical presentation, prevention and control

Alfonsina Fiore, Maria Casale and Paolo AureliDipartimento di Sanit Pubblica Veterinaria e Sicurezza Alimentare,

Istituto Superiore di Sanit, Rome, Italy

INTRODUCTION

Enterobacter sakazakii (E. sakazakii) is agram-negative rod belonging to the amily oEnterobacteriaceae (Table 1).

Until 1980 it has been considered a yellowish vari-ant oEnterobacter cloacae, due to its peculiar char-

acteristic o orming colonies ranging in colour rombright yellow to pale yellow. In recent years, it has beenidentied as a distinct species and researchers namedit in honour o the Japanese bacteriologist RiichiSakazaki who greatly contributed in understandingEnterobacteriaceae [1]. In a recent study a reclassica-tion o this microorganism has been proposed, on thebasis o the creation o the genus Cronobacter. Thisgenus includes our species, two subspecies and onegenomospecies. Cronobacter sakazakiiis the type spe-cies o this proposed genus [2].

E. sakazakii is considered an ubiquitous microor-ganism: it has been in act isolated rom a great vari-

ety o sites [3]: oods, water and several areas, includ-ing houses and hospitals. A recent article reportedthe presence oE. sakazakiiin a lot o maternal milkstored in a milk bank [4].

It is also considered an emerging opportunisticpathogen, responsible o cases o neonatal inec-

tions (sepsis, meningitis, necrotizing enterocolitis)related to use o contaminated inant milk ormula,which can represent both the vehicle and the sourceo contamination. Inant milk ormulas are prepa-rations in which cows milk or soy milk is used tosubstitute, modiy and/or strengthen/ortiy humanmilk. The essential requirements o inant ormu-lae, in Europe, are reported in the Inant FormulaeDirective [5]. Powered inant ormula is basically anon-sterile product which can be, once rehydrated,a good medium or microorganisms. Its productionprocess [6], in act, unlike the one or liquid ormu-las, may be subject to bacterial contamination, lack-

Address or correspondence: Paolo Aureli, Dipartimento di Sanit Pubblica Veterinaria e Sicurezza Alimentare, IstitutoSuperiore di Sanit, Viale Regina Elena 299, 00161 Rome, Italy. E-mail: p.aureli@iss.it.

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276 Alonsina Fiore, Maria Casale and Paolo Aureli

ing in the required conditions guaranteeing a sterileend product.

A large part oE. sakazakiiinections occur in hos-pitals, in the neonatal intensive care units. Keepingreconstituted ormula at room temperature or longperiods o time, in act, increases the risk o inection.That is why improving training and hygiene in hospi-tals is dramatically important in avoiding E. sakazakiioutbreaks.

The International Commission or MicrobiologicalSpecication or Foods [7], due to the seriousness o

pathologies associated with E. sakazakii, dened it aserious danger or a segment o the population, withchronic eects which may be long-lasting, capable oconstituting a threat or lie. As a consequence, E.sakazakii has been included among the most com-mon pathogens such as Listeria monocytogenes, TypeAClostridium botulinum andCryptosporidium parvum[3]. Inants are the type o population most seriouslyaected, particularly those hospitalized in neonatalintensive care units and ed with powdered ormu-las. For this reason, the Codex Committee on FoodHygiene o FAO/WHO, is considering a revision othe Recommended International Code o Hygienic

Practices or Foods or Inants and Children, and

the creation o a risk prole or E.sakazakiiin inantmilk ormulas.

E. sakazakiiis generally detected in oods, environ-ment (i.e., dust) and in clinical samples by using tra-ditional and molecular biology methods (Table 2).

EPIDEMIOLOGY

E. sakazakiimay cause inections in all age groupsand particularly in subjects rom 0 to 12 months.Among this last population, premature born ater

less than 36 weeks, underweight inants, inantswith immunodeciency, inants whose mothers areHIV-positive and inants hospitalised in intensivecare units are more at risk o inection. The reasonis that they are usually ed with ormulae, which arethe most common vehicle o transmission o the mi-croorganism.

Only 50% o inected adults suer a severe courseo illness (even i so ar no cases o meningitis havebeen reported); in such cases the vehicle o trans-mission and the inectious dose are unknown.

The number o reported cases o inection is verylow, but nevertheless it recently slightly increased.

Nowadays the reported cases worldwide are less

Table 2 | Methods or enumeration, detection and typing o E. sakazakii in ood samples*

Current methodsor

DNA-based methodsor

Enumeration Ri. Detection Ri. Detection Ri. Typing Ri.

MPN [8]

[9]

Detection [11] BAX-PCR [12] Pulsed-feld gel

electrophoresis (PFGE)

[14]

[15]

Colony - count [10] Real-time PCR [13] Random amplifcation o

polymorphic DNA (RAPD-PCR)

[16]

Ribotyping [17]

*detection oE. sakazakii in environmental samples [18]; detection oE. sakazakii in biological samples [19]

Table 1 | Characteristic eatures o E. sakazakii

General eatures Gram negative rods, acultative anaerobic, no spore orming, mesophilic, yellow pigmented, osmotholerant,generally motile, peritrichous, provided o capsule

Maximum temperature o growth: 41-45 C

Minimum temperature o growth: 5.5-8.0 C

No growth at 4 C

Distinguished into 16 bio groups

Generation times 40 min at 23 C

4.18-5.52 h at 10 C75 min a 25 C in reconstituted inant milk powder

Signifcant biochemical eatures Catalase positive, oxidase negative, ala-glucosidase positive, phosphoamidase negative

Habitat Foods, environment, animals, biological liquid

Virulence actory Enterotoxin

Antimicrobial susceptibility Tetracycline, aminoglycosides, beta-lactam antibiotics, chinolone, antiolates, chloramphenicol, nitrourantoine

Chemical compounds susceptibility Chitosans, oligomers o chitosans, monocaprylin (caprylic acid ester)

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277EntErobactErsakazakii

than 60 [20]; anyway, it needs to be noted that thisnumber may be underestimated since not all clini-cal analysis laboratories carry out research on thepathogen and not all countries have a system or re-porting diseases.

In the past years, mortality due to inections rom E.sakazakiiwas higher than 50%; in recent years it hasdecreased, even though it is still quite high 20% [21].

By and large, cases so ar reported can be dividedinto two groups: sporadic and epidemic [22].

The rst two cases o meningitis related to E. saka-zakii, where reported in 1961, when the microorgan-ism was still called E. cloacae [23]. Since then, caseso inections related to E. sakazakiihave been largelyreported worldwide [24] with an increase o reportedcases in recent years. This increase is probably due toa higher number o subjects at risk and to improveddiagnostic ability [22].

Between October and December 2004, ve caseso inections, two o which atal, and ve cases ointestinal colonization, were diagnosed in the paedi-atric units o ve French hospitals, and attributed toE. sakazakii. Nine o the inants aected had beened with the milk usually administered to prematureinants. Ater these events, Ireland decided to pro-hibit the use o inant ormula, although the lot hadbeen sold only in France and in Algeria [25]. Duringthe same year, in New Zealand, another case o E.sakazakiiinection had a atal outcome, the micro-organism aected and killed a hospitalized inanted with milk ormula [26].

In July 2006, in a medical care centre in India, atwo month inant developed nosocomial E. sakaza-kiibacteraemia. The vehicle o contamination wasunclear and transmission rom hospital environ-ment cannot be excluded [27].

The most recent case oE. sakazakiiinection hasbeen reported in 2007 by the paediatric unit o a hos-pital in Bilbao, Spain [28], where a premature andunderweight inant had contracted neonatal sepsis.The source o inection was unclear: the inant hadnot been ed by inant ormula but with breast milkand a liquid ormula or premature inants.

CLINICAL PRESENTATION

Enterobacteriaceae is the most important amily ogram-negative bacteria. Bacteria o this amily are re-sponsible o inections aecting bile ducts, prostate,the urinary tract, and o the most common noso-comial inections. The main source o diusion andtransmission to sensitive sites is the intestine [29].

E. sakazakii, one o the bacteria o the Enterobac-teriaceae amily, is considered an invasive opportun-istic pathogen responsible o sepsis, meningitis, andnecrotizing enterocolitis (NEC), especially amongnewborns (premature, underweight, aected by im-munodeciency) and o nosocomial inections, es-pecially in paediatric intensive care units where itcauses almost 50% o inections. The most severeinection caused by E. sakazakii is meningitis. The

pathway the pathogen ollows to reach the cerebralspinal liquid and cause meningitis is unknown. It iswidely assumed that the route o inection is the ca-rotid plexus; paracellular and transcellular mecha-nisms are involved. Bacterial glycopeptides, endo-

toxins, proteases, and elastases seem to induce per-meability o haematic and cerebral barriers openingthe way to the pathogen. The most common patho-logical symptoms in advanced stages o meningi-tis, are: ventriculitis, cerebral abscess, ormation ocysts, hydrocephalus, quadriplegia, retarded neuraldevelopment and inarction. Patients who recoverrom the disease suer rom physical and mentalretardation.

E. sakazakii, in the same way as Pneumococcus,Haemophilus and Meningococcus, which cause men-ingitis among children below 5, has a particular tro-pism or the central nervous system. On the basis

o its tendency to cause cerebral lesions, it has beencompared to Citrobacter diversus.

Neonatal necrotizing enterocolitis is characterizedby necrosis and intestinal pneumatosis. The diseaseaects 13% o underweight inants.

Essential prerequisites or the pathogenesis othis disease seem to be: neonatal intestinal ischae-mia, microbial colonization o the intestine and ex-cess o proteins in the intestinal lumen associatedwith administration o inant ormula. The pres-ence o necrotizing enterocolitis among inants edwith articial milk is ten times higher than amongbreasted inants; this is probably due to the pres-

ence o class A protective immunoglobulins in ma-ternal milk.Necrotizing enterocolitis as well as meningitis

cause high mortality. In particular, with respect tonecrotizing enterocolitis, the percentage varies be-tween 10% and 55%, while in respect to meningitis itvaries between 40% and 80%.

Neonatal inections due to E. sakazakiicould alsobe contracted directly rom the mother at birth, butno cases o intestinal and genital inections havebeen reported among mothers o inected newborns.Cases o neonatal inections ater a caesarean sec-tion have been reported [29]. Nevertheless, coloniza-

tion o inants, especially i premature, with humanand environmental bacteria is almost inevitable.The signs and symptoms o the initial phase oE.

sakazakii inection are: anorexia, irritability, jaun-dice, paleness, cyanosis, collapse, spasms and tem-perature instability [30].

PREVENTION AND CONTROL

In order to reduce the risk associated to consump-tion o contaminated ormulas, correct inormationand education regarding good practices requiredduring preparation and handling o this product arerequired.

It is unknown i specic techniques or prepara-tion and preservation o rehydrated milk in hospi-tals and day care centres do actually exist.

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278 Alonsina Fiore, Maria Casale and Paolo Aureli

USA competent authorities ensured that hospitalunits which prepare and handle powdered milk ol-low the guidelines issued by the American DieteticAssociation [31]. These recommendations includeusing ready-to-eed ormulas, complying with the

rules or aseptic preparation and rerigeration at 2-3C o reconstituted milk or a lapse o time short-er than 4 hours. Recommendations also underlinethe importance o dedicating specic areas to thepreparation, preservation and administration omilk, tasks that should be perormed by specicallytrained and qualied personnel.

Reducing risks connected to E. sakazakiiis manda-tory or all people involved: producers, parents, healthproessionals. This purpose can be ullled ollowingseveral combined steps [6, 31] illustrated in Table 3.

CONCLUSIONS

E. sakazakiiis an emerging pathogen, oten trans-mitted through powdered milk and responsible ora series o inections, some o which with potentialatal outcomes, in a particular segment o the popu-

lation.Factors contributing to increase the risk o inec-

tion include: patients susceptibility, level o con-tamination o ood, tolerance to temperature, speedo growth, inectious dose and the virulence o themicroorganism.

Recently, in order to ace the risks connected tomicrobiologic contamination, producers have beenobliged to adopt new control measures based on theHACCP system (Hazard Analysis Critical ControlPoint).

During production

Monitor raw materials, specifcally ingredients

which do not require urther thermal treatment

beore mixing

At home

Use clean and disinected containers

In hospital/nurseries

Follow practices o good hygiene in preparation

areas

Reduce level o Enterobacteriaceaein areas

used or production in order to prevent

subsequent contamination

Prepare only ood enough or the meal

avoiding the preparation o ollowing meals; i

necessary limit the number o meals prepared

in advance to 1-2

Produce guidelines related to preparation,

handling, preservation and control procedures

or the product, and make them available to

trained personnel

Increase requency o inspections on ood

production environments and on the end

product

Avoid leaving unused reconstituted milk at

room temperature

Have access to a room reserved or

preparation, which has an area or stocking the

product and to which only authorized personnelhave access

Identiy the sources o possible contamination

and take corrective measures

Store in a rerigerator the reconstituted product I a dedicated room is not available, select an

area reserved or this purpose

Revise the instructions or milk preparation

suggesting a higher water temperature, but

not higher than 80 C, or solubility (> 70 C).

A temperature too high would damage the

nutritional characteristics o the product

Reduce as much as possible the lapse o time

between the reconstitution o the ormula and

its use

Have access to utensils and equipment

manuactured in order to be easily sanitized

Sterilize all utensils used or preparation

with thermal treatment (e.g., washing in

dishwashers) or with autoclave

Whenever possible use disposable utensils

Employ qualifed and specialized personnel

(i.e., dieticians)

Store the reconstituted product in a rerigerator

Reduce as much as possible the lapse o time

between reconstitution o the product and its use

Avoid leaving reconstituted milk at room

temperature i unused

Appropriately seal all containers with

remaining milk, place them in the rerigerator,

noting down an expiration date

Whenever possible, use milk in liquid orm

Enorce appropriate control measures which can

assess potential hazards, identiy critical controlpoints (CCP), monitor non-conormities and

necessary corrective actions, and register results

Table 3 | Combined steps to reduce risks connected to E. sakazakii

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279EntErobactErsakazakii

Nowadays in Italy, as well as in other Europeancountries, new microbiological criteria that can beapplied to oods, have been enorced in compliancewith EEC Regulation n. 1441/2007 dated December5, 2007 [32]. In particular, with respect to E. sakaza-

kii, there must not be any trace o the microorganismin 10 grams o tested product taken rom 30 samples.The products taken into consideration are powderedoods or inants and powdered dietetic oods or un-der 6 months inants having special medical needs.

An investigation on marketed inant powderedmilk samples showed the presence o levels o con-tamination ranging rom 0.36cu/100g to 66.0cu/100g [33]. These samples, even i the levels o con-tamination were low, were nevertheless consideredat risk. This is due to the microorganism ability togrow in the reconstituted product ater preparation,during handling beore use, and during preservation

i at room temperature. Such data are supportedby the opinion recently expressed by EFSA (2004meeting) on microbiological risks regarding oodsor newborns and inants. Experts reported that E.sakazakii, in the same way as Salmonella, is the mi-croorganism raising the highest concern in respectto the above mentioned oods. It has been pointedout that the presence o this pathogen represents aserious risk i conditions ater reconstitution allowits replication [4].

It should be noted that the reduction in the re-quency oE. sakazakiicontamination o inant or-mula powder might reduce our to ve times the risko inection, while minimising the lapse o time be-tween preparation and consumption might reduce

thirty times the risk [4].Since E. sakazakiidoes not withstand the tempera-

tures at which milk is pasteurized, but is easily oundin the environment, post-pasteurization, as well aspreparation and handling beore consumption, rep-resent critical points. That is why these are the stepsduring which a preventive action is needed to elimi-nate or avoid risks o contamination and replicationo the microorganism.

It is worth pointing out that breasteeding shouldalways be supported and encouraged since a moth-ers milk constitutes the preerred ood or newborninants especially in their early months. When this

is not possible, a mother should be well inormedand trained on the importance o hygiene while han-dling, preparing and storing powdered milk.

Acknowledgment

The authors wish to thank Eleonora Lacorte or review o theEnglish text.

Receivedon 16 January 2008.

Acceptedon 11 June 2008.

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