Cronobacter: an Emerging Opportunistic Pathogen Associated with Neonatal Meningitis, Sepsis and Necrotizing Enterocolitis

Home , Cronobacter, Cronobacter dublinensis, Cronobacter muytjensii, Cronobacter sakazakii, Cronobacter turicensis

STATE-OF-THE-ART Cronobacter: an emerging opportunistic pathogen associated with neonatal meningitis, sepsis and necrotizing enterocolitis

CJ Hunter1,2 and JF Bean1

Members of the genus Cronobacter are an emerging group of opportunist Gram-negative pathogens. This genus was previously thought to be a single species, called Enterobacter sakazakii. Cronobacter spp. typically affect low-birth-weight neonates, causing life-threatening meningitis, sepsis and necrotizing enterocolitis. Outbreaks of disease have been associated with contaminated infant formula, although the primary environmental source remains elusive. Advanced understanding of these bacteria and better classiﬁcation has been obtained by improved detection techniques and genomic analysis. Research has begun to characterize the virulence factors and pathogenic potential of Cronobacter. Investigations into sterilization techniques and protocols for minimizing the risk of contamination have been reviewed at national and international forums. In this review, we explore the clinical impact of Cronobacter neonatal and pediatric infections, discuss virulence and pathogenesis, and review prevention and treatment strategies.

Journal of Perinatology (2013) 33, 581–585; doi:10.1038/jp.2013.26; published online 28 March 2013 Keywords: Cronobacter sakazakii; meningitis; necrotizing enterocolitis; neonatal infections; infant formula

INTRODUCTION single clinical setting.10 Cronobacter neonatal infection rates Cronobacter is an emerging genus of opportunistic Gram-negative remain low and are reported in the USA as one Cronobacter pathogens associated with potentially fatal neonatal infections, infection per 100 000 infants, 8.7 per 100 000 low-birth-weight including meningitis, sepsis and necrotizing enterocolitis (NEC).1 neonates, and one Cronobacter infection per 10 660 very low- 13 These infections typically affect our smallest and most vulnerable birth-weight neonates. It is suggested that the incidence of patients. Historically, Cronobacter spp. were thought to be a single infection may be under-reported in developing and less species known as Enterobacter sakazakii.2 With improved culture developed countries, perhaps due to a lack of medical 8 and identification techniques, including partial 16S ribosomal microbiology analysis facilities. Figure 1 demonstrates the DNA, hsp60 sequencing and polyphasic analysis, Cronobacter was worldwide distribution of reported cases, the majority are within recognized as its own genus within the Enterobacteriaceae the United States; however, widespread locations are noted. These 8,14–17 family.3 Subsequent genomic sequencing data confirmed the data were generated from multiple reports worldwide. It is change in taxonomy.4 The first Cronobacter genome to be not the frequency of these infections, but rather their impact, sequenced was that of an isolate recovered from a neonatal lethality and vulnerable population affected, which makes this a unit in Tennessee after an outbreak in 2001.5 Sequencing revealed serious health issue. Cronobacter-induced neonatal disease had 18 a single chromosome of 4.4 Mb and 2 plasmids.4 Currently been previously cited to have a mortality of 80%. Friedmann 8 the genus contains eight species that include C. sakazakii, et al. analyzed the microbiologically confirmed cases and C. malonaticus, C. turicensis, C. muytjensii, C. dublinensis, reported an overall lethality of Cronobacter infection as 26.9%. C. condiment and C. universalis.3 In this review, we will explore Cronobacter-associated meningitis is fatal in almost 42% of the clinical impact of Cronobacter neonatal and pediatric patients and Cronobacter-associated sepsis and NEC lethality 8 infections, discuss virulence and pathogenesis and review rates are cited at B10 and 20%, respectively. Meningeal prevention and treatment strategies. Cronobacter infection is established between the fourth and fifth post partum days, and can be fatal within hours to days following the initial clinical symptom.9 Even when infants survive the acute CLINICAL IMPACT insult, long-term neurological delay and poor lifelong outcomes 19 The sentinel case of Cronobacter infection was reported in 1961 by may occur in more than 74% of survivors. Although the majority Urmenyi and Franklyn.6 In that description of terminal neonatal of affected patients are less than 3 months of age; a study from the United Kingdom found infants up to 11 months with meningitis, the bacterial culprit was described as a ‘yellow 20 pigmented Enterobacter cloacae’. This infectious entity was given Cronobacter-associated meningitis. Although older populations 2 may be infected including the elderly,21 premature infants and species-status in 1980 (E. sakazakii), and subsequently redefined 8 as the genus Cronobacter in 2008.7 As this initial description there those weighing less than 2.5 kg remain the most susceptible. are now in excess of 150 reported cases of Cronobacter-related infections.8 The most commonly reported consequences of infection are meningitis, NEC and sepsis.9,10 Other resultant PATHOGENESIS AND VIRULENCE diseases have been reported, including conjunctivis, urinary tract Virulence is the degree of pathogenesis of a species or its ability to infections, diarrhea, tonsillitis and osteomyelitis.11,12 Outbreaks are induce disease in a host. A variety of in vitro and in vivo models most commonly sporadic, but have occurred within clusters in a have been used to investigate the mechanisms by which

1Division of Pediatric Surgery, Ann and Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, USA and 2Department of Surgery, Northwestern University’s Feinberg School of Medicine, Chicago, IL, USA. Correspondence: Dr CJ Hunter, Northwestern University’s Feinberg School of Medicine, 303 E Chicago Avenue, Chicago, IL 60611, USA. E-mail: [email protected] Received 26 October 2012; revised 24 January 2013; accepted 11 February 2013; published online 28 March 2013 Cronobacter and neonatal infections CJ Hunter and JF Bean 582 Cronobacter induces disease.22–24 Although the distinct results in the loss of mucosal integrity, intestinal barrier mechanisms are not fully elucidated, promising data from the compromise and permits the passage of bacteria into the host genome sequencing effort have highlighted several potential environment. Studies using an in vitro epithelial cell line virulence factor candidates.25 As described, the genome of demonstrated that some strains may be able to disrupt gap Cronobacter sakazakii has B4392 genes.4 Of these, 223 genes junctions between cells.32 Loss of normal intestinal integrity is a were annotated as relevant to virulence and disease.4 Of the 21 key feature in the development of NEC.33 Figure 2 outlines a genes unique to C. sakazakii, the resultant encoded proteins have possible model for Cronobacter infection after oral inoculation. The been identified to have a role in pilus assembly, photransferase bacteria adhere to the intestinal epithelial barrier, causing barrier systems, acid transport and toxin/antitoxin transport systems.26 degradation and soliciting an inflammatory response in the host. Outer membrane protein A, encoded by the ompA gene, is one of This inflammatory response is characterized by the production of the best characterized virulence factors.27 It is required for binding cytokines, which in turn recruit immune cells to the site. Once the and invasion of brain endothelial cells, which are necessary steps barrier is breached Cronobacter spp. may enter the blood stream, during development of meningitis.28 The ompA-encoded gene is resulting in sepsis and meningitis. thought to be universally present in Cronobacter. OmpA is also of In addition to facilitating host damage, virulence factors may crucial importance in the virulence of another meningitis causing provide protection from the detection and response of the innate bacteria, Escherichia coli K1.29 Although the sequences of ompA and adaptive host immune system. Recently, Franco et al.34 between various strains are different, the mechanism of demonstrated resistance to complement-mediated killing in the attachment and invasion of Cronobacter and E.coli K1 at the presence of a plasmid-encoded protein. Cronobacter may also blood–brain barrier may theoretically be similar. E.coli K1 has not effect alterations in immune cell recruitment in the intestine of been specifically associated with outbreaks of NEC. Cronobacter affected subjects.35 Cronobacter has been shown to trigger an virulence is also associated with the production of an enterotoxin- inflammatory cascade and inducible nitric oxide synthase, and like compound, which may further enhance virulence and its interleukin-6 have been implicated in facilitating the downstream translocation across the gut and blood–brain barrier.30 The role of affects.22,35 Both nitric oxide and interleukin-6 have pro-apoptotic this toxin may act in a similar fashion to lipopolysaccaride, activity, and are found at high levels in animal models of mediating Toll-like receptor 4 activation and activating a host Cronobacter infection. In addition to causing local barrier inflammatory response. The production of an enterotoxin was disruption, these cytokines may have profound effects on established in a suckling mouse assay.30 Intraperitoneal injection systemic immunity and inflammation.36 of Cronobacter and oral administration both result in the clinical manifestations of disease; injection of 108 colony forming units resulted in death regardless of the strain used.30 The ability of ENVIRONMENTAL SOURCES Cronobacter to induce death in a mouse model through the oral Contamination of powder infant formula (PIF) leading to administration appears both strain and dose-dependent. The role Cronobacter-associated disease garnered attention both within of Cronobacter enterotoxin remains an area of active investigation. the scientific literature and popular press.37,38 The natural We have previously demonstrated that Cronobacter is able to environment of Cronobacter remains unclear but soil samples, adhere to intestinal epithelial cells both in cultured cell lines and powdered milk substitutes, processed cheese, meats, herbs and in the gavage-fed neonatal rat pup model of NEC22 This spices, the gut of a Mexican fruit fly and the stable fly have all association results in increased epithelial apoptosis and loss of been identified as containing the bacteria.39,40 Although the stable epithelial barrier integrity.31 Our prior data suggest that the fly was thought to have importance as a potential vector, further normal balance of cell proliferation, restitution and programed cell investigation revealed that the true incidence of Cronobacter death may be altered in the presence of Cronobacter infection. in this species was very low and thus it was an unlikely Although programed cell death (apoptosis) is an important aspect candidate. Typically, Cronobacter is not present within the of the gut health and maintenance, a greater degree of apoptosis normal mammalian intestinal tract, and there is only a single

Figure 1. Worldwide distribution of reported cases of neonatal Cronobacter spp. infection. PubMed was queried for all reports of E. sakazakii or Cronobacter infant and pediatric infection. Each case was grouped based on the country, in which it was recorded. Geographical locations of cases are summarized in pie chart format. The others segment include Canada, India, Korea, the Netherlands, New Zealand and Slovenia. The majority of cases are reported in the USA (27.9%).

Journal of Perinatology (2013), 581 – 585 & 2013 Nature America, Inc. Cronobacter and neonatal infections CJ Hunter and JF Bean 583 report of Cronobacter being isolated from a human vaginal develop strategies to eliminate Cronobacter from PIF when canal,41 thereby making vertical transmission less likely. The present. The FAO/WHO (Food and Agriculture Organization and prospect of contaminated PIF constitutes an intolerable mainline the World Health Organization) recommends that formula be access to the most at risk populations. In 1990 an outbreak of reconstituted with hot water (4 70 1C)51 in an effort to reduce the Cronobacter neonatal infection was clearly linked to contaminated bacterial load and reduce the incidence of Cronobacter-associated PIF.42 Two unopened cans were found to contain Cronobacter spp. infections.52 However, certain nutritional elements including One hundred and forty-one breast milk substitutes from 35 vitamins may be altered or lost after heating to such high countries have identified Cronobacter spp. in over 20 samples.43 temperatures. Inappropriate storage and temperature control after The concentrations ranged from 0.36 colony forming units/100 g reconstitution, and poor bottle and plastic nipple cleanliness are a to 66 colony forming units/100 mg of PIF. More recently, potential reservoir of infection. The ability of Cronobacter to form Cronobacter was isolated from 2 of 82 PIF, 5 of 49 weaning biofilms may also provide it protection to host defenses and the foods, 3 of 72 milk powders, 40 of 122 herbs and spices and 15 of ability to survive regular clinical sterilization methods.53 Other 66 other dry food ingredients.44 A total of seven countries sterilization strategies have been investigated.54 Reports have participated in a study to determine the incidence of Cronobacter shown that essential oils and polyphenols are effective biocides in in desiccated products made for infant consumption.17 These a laboratory environment.55 However, the majority of these countries included Brazil, England, Mexico, Indonesia, Jordan, compounds are toxic to human beings, and elevated levels with Korea, Portugal and Malaysia.45 They reported the isolation of PIF would likely pose their own threat. The addition of ultrasound Cronobacter spp. from 1 of 84 samples of follow-up formula and 30 or ultraviolet radiation to heating protocols may improve of 203 weaning foods. Therefore, it is clear that Cronobacter spp. is sterilization processes.56,57 The efficacy of standard detergents recoverable from the desiccated state in a number of powdered has also been investigated,53 and in general Cronobacter appear food products that are provided to infants. In December of 2011, fairly resistant to routine cleaning techniques. Ionizing radiation the Food and Drug Administration reported a series of neonatal has been investigated as a strategy to reduce the presence of disease outbreaks in Florida, Illinois, Missouri and Oklahoma.46 bacteria in PIF.58 Doses of up to 10 kGy have demonstrated a Although temporally related, the sources of these outbreaks reduction in pathogens without causing disturbance of the appear to be different, and unlike the 1990 cases, factory-sealed quality, safety or composition of the formula.59 These data PIF is not the likely source. Contamination of the product after appear promising; however, desiccated Cronobacter exhibit opening appears more likely. PIF is a clean but not sterile product increased radiation-resistance when compared with non- that must conform to specified international microbiological desiccated cells. Several preventative strategies have been criteria. PIF is manufactured through a process of high investigated in vivo. We were able to demonstrate that temperature heating and desiccation. C. sakazakii has been pretreatment with the probiotic bacteria Lactobacillus decreased demonstrated to have remarkable thermotolerance,47 and can the severity of C. sakazakii-induced NEC in an animal model.60 withstand temperatures up to 60 1C for limited periods of time.48 Improved intestinal barrier health and decreased apoptosis were Additionally, Cronobacter may survive up to 12 months at 4 1C.49 also noted. Lactobacillus is a lactic acid producing bacteria, and the Reconstituted formula is an excellent substrate for Cronobacter. presence of lactic acid in the presence of copper has been shown There are also reports of Cronobacter surviving desiccated states to inhibit Cronobacter spp. in vitro.61 Other investigators have for several weeks and being resistant to osmotic stresses.50 It suggested that prebiotics may inhibit Cronobacter intestinal further appears that clinically relevant strains are the most heat adherence, thereby providing clinical protection from disease.62 and desiccation resistant, perhaps having provided them with a The current recommendations regarding formula preparation are survival benefit and the ability to reach their host. It is important summarized in Table 1. It has not been proved definitively that to both identify potential environmental contamination and hospital staff are themselves not a vector for Cronobacter

Figure 2. Model of Cronobacter infection following ingestion of contaminated PIF. Oral inoculation of Cronobacter into an infant after ingestion of contaminated PIF. The bacteria release enterotoxin ( þ E), which may increase bacterial virulence and disrupt the intestinal barrier causing necrotizing enterocolitis. Cronobacter adheres to the intestinal layer. In the setting of a disrupted epithelial barrier, microbes traverse the mucosal layers, gaining access to lower immune and nonimmune cell populations. Mucosal microvascular endothelial cells, which represent the ﬁnal anatomic border before the host’s microcirculation, sense microbial antigens and respond rapidly by mounting an immune response. Cronobacter may enter to vasculature thereby gaining access to the blood–brain barrier resulting in bacteremia and meningitis.

& 2013 Nature America, Inc. Journal of Perinatology (2013), 581 – 585 Cronobacter and neonatal infections CJ Hunter and JF Bean 584 Although great strides have been made in successfully identifying Table 1. Table summarizing problems and proposed solutions to Cronobacter, we lack full understanding of its environment, minimize the likelihood of Cronobacter contamination of infant transmission, pathogenesis and treatment. Work both in vitro formula. Information modiﬁed from Centers for Disease Control, and in vivo will hopefully continue to elucidate the underpinning World Health Organization and Food and Drug Administration of Cronobacter-related infection. Furthermore, better regulation recommendations66–69 and preventative strategies will continue to aid in the control of Problem Solution(s) the introduction of this potentially fatal neonatal pathogen to human food sources. Cronobacter risk Encourage use of breast milk or liquid milk formula, which is usually sterile CONFLICT OF INTEREST Cronobacter Improved surveillance international and contamination at national criteria strategies and monitoring. The authors declare no conﬂict of interest. factory

Cronobacter in PIF Reconstitute formula with water 470 1C. ACKNOWLEDGEMENTS Rapidly cool before feeding to 37 1C We thank Dr HS Seifert for his insightful suggestions and comments in the Cronobacter in Prepare formula in a clean location. preparation of this manuscript. environment Sterilize bottles and plastic nipples, wash hands REFERENCES Cronobacter in Store reconstituted formula at o4 1C, and reconstituted PIF use within 24 h. Once a feeding has 1 Hunter CJ, Petrosyan M, Ford HR, Prasadarao NV. Enterobacter sakazakii:anemerging (home setting) started finish formula within 2 h and pathogen in infants and neonates. Surg Infect (Larchmt) 2008; 9: 533–539. discard any remaining. 2 Farmer 3rd JJ, Davis BR, Hickman-Brenner FW, McWhorter A, Huntley-Carter GP, Asbury MA et al. Biochemical identification of new species and biogroups of Cronobacter in Avoid powdered infant powders when Enterobacteriaceae isolated from clinical specimens. J Clin Microbiol 1985; 21: reconstituted PIF possible. Limit formula bag hang time 46–76. (hospital setting) too4h 3 Iversen C, Druggan P, Schumacher S, Lehner A, Feer C, Gschwend K et al. Development of a novel screening method for the isolation of ‘Cronobacter’spp. Abbreviation: PIF, powder infant formula. (Enterobacter sakazakii). Appl Environ Microbiol 2008; 74: 2550–2553. 4 Kucerova E, Clifton SW, Xia XQ, Long F, Porwollik S, Fulton L et al. Genome sequence of Cronobacter sakazakii BAA-894 and comparative genomic hybridi- transmission. Unfortunately, it is recognized that hospital person- zation analysis with other Cronobacter species. PLoS One 2010; 5: e9556. nel may ignore appropriate hygiene practices and contribute to 5 Himelright I, Harris E, Lorch V, Anderson M, Jones T, Craig A et al. From the Centers the spread of infection.63 Therefore, adherence to strict hand for Disease Control and Prevention. Enterobacter sakazakii infections associated with washing and contact isolation of affected and susceptible infants the use of powdered infant formula--Tennessee, 2001. Jama 2002; 287: 2204–2205. should be employed. Table 1 outlines preventative strategies that 6 Urmenyi AM, Franklin AW. Neonatal death from pigmented coliform infection. are recommended to combat the risks of Cronobacter contamina- Lancet 1961; 1: 313–315. 7 Iversen C, Mullane N, McCardell B, Tall BD, Lehner A, Fanning S et al. Cronobacter tion of PIF. gen. nov., a new genus to accommodate the biogroups of Enterobacter sakazakii, and proposal of Cronobacter sakazakii gen. nov., comb. nov., Cronobacter malonaticus sp. nov., Cronobacter turicensis sp. nov., Cronobacter muytjensii sp. THERAPEUTIC STRATEGIES nov., Cronobacter dublinensis sp. nov., Cronobacter genomospecies 1, and of three subspecies, Cronobacter dublinensis subsp. dublinensis subsp. nov., Cronobacter Prevention is preferable, however, once Cronobacter infection has dublinensis subsp. lausannensis subsp. nov. and Cronobacter dublinensis subsp. been diagnosed, prompt and appropriate antibiotic therapy lactaridi subsp. nov. Int J Syst Evol Microbiol 2008; 58: 1442–1447. should be initiated. Standard supportive measures typically 8 Friedemann M. Epidemiology of invasive neonatal Cronobacter (Enterobacter employed in the management of neonatal sepsis, meningitis sakazakii) infections. Eur J Clin Microbiol Infect Dis 2009; 28: 1297–1304. and NEC should be maintained. The majority of antibiotic data 9 Muytjens HL, Zanen HC, Sonderkamp HJ, Kollee LA, Wachsmuth IK, Farmer 3rd JJ. pertaining to antibiotic susceptibility and resistance profiles of Analysis of eight cases of neonatal meningitis and sepsis due to Enterobacter Cronobacter were determined on established Enterobacter species sakazakii. J Clin Microbiol 1983; 18: 115–120. such as E. cloacae, and later the specific antibiotic profiles of 10 Block C, Peleg O, Minster N, Bar-Oz B, Simhon A, Arad I et al. Cluster of neonatal various E. sakazakii strains. Interestingly, no natural resistance infections in Jerusalem due to unusual biochemical variant of Enterobacter sakazakii. Eur J Clin Microbiol Infect Dis 2002; 21: 613–616. to cephalosporins was detected in wild-type populations of 64 11 Corti G, Panunzi I, Losco M, Buzzi R. Postsurgical osteomyelitis caused by Cronobacter, and these strains appear to lack b-lactamases. Enterobacter sakazakii in a healthy young man. J Chemother 2007; 19: 94–96. C. sakazakii, typical of most Enterobacteriacae, has natural 12 Lai KK. Enterobacter sakazakii infections among neonates, infants, children, and resistance to lincosamides, glycopeptides, streptogramins and adults. Case reports and a review of the literature. Medicine (Baltimore) 2001; 80: fusidic acids. However, the emergence of antibiotic resistance 113–122. patterns has been reported.65 Currently, broad-spectrum 13 Stoll BJ, Hansen N, Fanaroff AA, Lemons JA. Enterobacter sakazakii is a rare cause of antibiotics and supportive care are recommended. In cases of neonatal septicemia or meningitis in VLBW infants. J Pediatr 2004; 144: 821–823. clinical infection, antibiotic sensitivity profiling should be sought, 14 Simon M, Sabate S, Cristina Osanz A, Bartolome R. Dolores Ferrer M. [Investigation in order to administer the most effective therapy. of a neonatal case of Enterobacter sakazakii infection associated with the use of powdered infant formula]. Enferm Infecc Microbiol Clin 2010; 28: 713–715. 15 Flores JP, Medrano SA, Sanchez JS, Fernandez-Escartin E. Two cases of hemor- rhagic diarrhea caused by Cronobacter sakazakii in hospitalized nursing infants CONCLUSION associated with the consumption of powdered infant formula. J Food Prot 2011; Since the first report of a ‘yellow pigmented’ bacterial infection, 74: 2177–2181. our understanding of Cronobacter has greatly improved. 16 Teramoto S, Tanabe Y, Okano E, Nagashima T, Kobayashi M, Etoh Y. A first fatal neonatal case of Enterobacter sakazakii infection in Japan. Pediatr Int 2010; 52: Cronobacter spp. are especially interesting because no single 312–313. bacterium has been found to meet Koch’s postulates for NEC in 17 Baumbach J, Rooney K, Smelser C, Torres P, Bowen A, Nichols M. Cronobacter most circumstances. Both neonatal meningitis and NEC are species isolation in two infants - New Mexico, 2008. MMWR Morb Mortal Wkly Rep devastating diseases, with poor long-term prognosis in survivors. 2009; 58: 1179–1183.

Journal of Perinatology (2013), 581 – 585 & 2013 Nature America, Inc. Cronobacter and neonatal infections CJ Hunter and JF Bean 585 18 Bowen AB, Braden CR. Invasive Enterobacter sakazakii disease in infants. Emerg 44 Iversen C, Forsythe S. Isolation of Enterobacter sakazakii and other Enterobacter- Infect Dis 2006; 12: 1185–1189. iaceae from powdered infant formula milk and related products. Food Micro- 19 Reij MW, Jongenburger I, Gkogka E, Gorris LG, Zwietering MH. Perspective on the biology 2004; 21: 771–776. risk to infants in the Netherlands associated with Cronobacter spp. occurring in 45 Chap J, Jackson P, Siqueira R, Gaspar N, Quintas C, Park J et al. International survey powdered infant formula. Int J Food Microbiol 2009; 136: 232–237. of Cronobacter sakazakii and other Cronobacter spp. in follow up formulas and 20 WHO. F. Enterobacter sakazakii (Cronobacter spp.) in powdered follow-up for- infant foods. Int J Food Microbiol 2009; 136: 185–188. mulae: meeting report 2008, [cited]Available from http://www.fao.org/ag/agn/ 46 FDA. Investigation of Cronobacter Bacteria Illness in Infants [cited]Available from: agns/jemra/Sakazakii_FUF_report.pdf. http://www.fda.gov/NewsEvents/PublicHealthFocus/ucm285401.htm 2011. 21 Gosney MA, Martin MV, Wright AE, Gallagher M. Enterobacter sakazakii in 47 Alvarez-Ordonez A, Begley M, Hill C. Polymorphisms in rpoS and stress tolerance the mouths of stroke patients and its association with aspiration pneumonia. heterogeneity in natural isolates of Cronobacter sakazakii. Appl Environ Microbiol Eur J Intern Med 2006; 17: 185–188. 2012; 78: 3975–3984. 22 Hunter CJ, Singamsetty VK, Chokshi NK, Camerini BoyleP, Grishin V, Upperman AV 48 Arku B, Fanning S, Jordan K. Heat adaptation and survival of Cronobacter spp. et al. Enterobacter sakazakii enhances epithelial cell injury by inducing apoptosis (formerly Enterobacter sakazakii). Foodborne Pathog Dis 2011; 8: 975–981. in a rat model of necrotizing enterocolitis. J Infect Dis 2008; 198: 586–593. 49 Walsh D, Molloy C, Iversen C, Carroll J, Cagney C, Fanning S et al. Survival 23 Prasadarao NV, Wass CA, Weiser JN, Stins MF, Huang SH, Kim KS. Outer membrane characteristics of environmental and clinically derived strains of Cronobacter protein A of Escherichia coli contributes to invasion of brain microvascular sakazakii in infant milk formula (IMF) and ingredients. J Appl Microbiol 2011; 110: endothelial cells. Infect Immun 1996; 64: 146–153. 697–703. 24 Richardson AN, Lambert S, Smith MA. Neonatal mice as models for Cronobacter 50 Al-Nabulsi AA, Osaili TM, Al-Holy MA, Shaker RR, Ayyash MM, Olaimat AN et al. sakazakii infection in infants. J Food Prot 2009; 72: 2363–2367. Influence of desiccation on the sensitivity of Cronobacter spp. to lactoferrin 25 Stephan R, Lehner A, Tischler P, Rattei T. Complete genome sequence of or nisin in broth and powdered infant formula. Int J Food Microbiol 2009; 136: Cronobacter turicensis LMG 23827, a food-borne pathogen causing deaths in 221–226. neonates. J Bacteriol 2011; 193: 309–310. 51 Edelson-Mammel SG, Buchanan RL. Thermal inactivation of Enterobacter sakazakii 26 Yan QQ, Condell O, Power K, Butler F, Tall BD, Fanning S. Cronobacter species in rehydrated infant formula. J Food Prot 2004; 67: 60–63. (formerly known as Enterobacter sakazakii) in powdered infant formula: a review 52 Chen PC, Zahoor T, Oh SW, Kang DH. Effect of heat treatment on Cronobacter spp. of our current understanding of the biology of this bacterium. J Appl Microbiol in reconstituted, dried infant formula: preparation guidelines for manufacturers. 2012; 113:1–15. Lett Appl Microbiol 2009; 49: 730–737. 27 Nair MK, Venkitanarayanan K, Silbart LK, Kim KS. Outer membrane protein A 53 Kim H, Ryu JH, Beuchat LR. Effectiveness of disinfectants in killing Enterobacter (OmpA) of Cronobacter sakazakii binds fibronectin and contributes to invasion sakazakii in suspension, dried on the surface of stainless steel, and in a biofilm. of human brain microvascular endothelial cells. Foodborne Pathog Dis 2009; 6: Appl Environ Microbiol 2007; 73: 1256–1265. 495–501. 54 Kim H, Ryu JH, Beuchat LR. Survival of Enterobacter sakazakii on fresh produce 28 Singamsetty VK, Wang Y, Shimada H, Prasadarao NV. Outer membrane protein A as affected by temperature, and effectiveness of sanitizers for its elimination. expression in Enterobacter sakazakii is required to induce microtubule con- Int J Food Microbiol 2006; 111: 134–143. densation in human brain microvascular endothelial cells for invasion. Microb 55 Condell O, Iversen C, Cooney S, Power KA, Walsh C, Burgess C et al. Efficacy of Pathog 2008; 45: 181–191. biocides used in the modern food industry to control salmonella enterica, and 29 Prasadarao NV. Identification of Escherichia coli outer membrane protein A links between biocide tolerance and resistance to clinically relevant antimicrobial receptor on human brain microvascular endothelial cells. Infect Immun 2002; 70: compounds. Appl Environ Microbiol 2012; 78: 3087–3097. 4556–4563. 56 Adekunte A, Valdramidis VP, Tiwari BK, Slone N, Cullen PJ, Donnell CP et al. 30 Pagotto FJ, Nazarowec-White M, Bidawid S, Farber JM. Enterobacter sakazakii:infec- Resistance of Cronobacter sakazakii in reconstituted powdered infant formula tivity and enterotoxin production in vitro and in vivo. J Food Prot 2003; 66:370–375. during ultrasound at controlled temperatures: a quantitative approach on 31 Liu Q, Mittal R, Emami CN, Iversen C, Ford HR, Prasadarao NV. Human isolates of microbial responses. Int J Food Microbiol 2010; 142:53–59. Cronobacter sakazakii bind efficiently to intestinal epithelial cells in vitro to induce 57 Arroyo C, Gayan E, Pagan R, Condon S. UV-C inactivation of Cronobacter sakazakii. monolayer permeability and apoptosis. J Surg Res 2012; 176: 437–447. Foodborne Pathog Dis 2012; 9: 907–914. 32 Kim KP, Loessner MJ. Enterobacter sakazakii invasion in human intestinal Caco-2 58 Mahmoud BS. Inactivation effect of X-ray treatments on Cronobacter species cells requires the host cell cytoskeleton and is enhanced by disruption of tight (Enterobacter sakazakii) in tryptic soy broth, skim milk, low-fat milk and whole-fat junction. Infect Immun 2008; 76: 562–570. milk. Lett Appl Microbiol 2009; 49: 562–567. 33 Hunter CJ, Podd B, Ford HR, Camerini V. Evidence vs experience in neonatal 59 Osaili T, Al-Nabulsi A, Shaker R, Ayyash M, Olaimat A, Abu Al-Hasan A et al. Effect practices in necrotizing enterocolitis. J Perinatol 2008; 28(Suppl 1): S9–S13. of environmental stresses on the sensitivity of Enterobacter sakazakii in powdered 34 Franco AA, Kothary MH, Gopinath G, Jarvis KG, Grim CJ, Hu L et al. Cpa, the outer infant milk formula to gamma radiation. Lett Appl Microbiol 2008; 47: 79–84. membrane protease of Cronobacter sakazakii, activates plasminogen and med- 60 Hunter CJ, Williams M, Petrosyan M, Guner Y, Mittal R, Mock D et al. Lactobacillus iates resistance to serum bactericidal activity. Infect Immun 2011; 79: 1578–1587. bulgaricus prevents intestinal epithelial cell injury caused by Enterobacter 35 Emami CN, Mittal R, Wang L, Ford HR, Prasadarao NV. Recruitment of dendritic cells sakazakii-induced nitric oxide both in vitro and in the newborn rat model of is responsible for intestinal epithelial damage in the pathogenesis of necrotizing necrotizing enterocolitis. Infect Immun 2009; 77: 1031–1043. enterocolitis by Cronobacter sakazakii. JImmunol2011; 186: 7067–7079. 61 Al-Holy MA, Castro LF, Al-Qadiri HM. Inactivation of Cronobacter spp. (Enterobacter 36 Emami CN, Petrosyan M, Giuliani S, Williams M, Hunter C, Prasadarao NV et al. Role sakazakii) in infant formula using lactic acid, copper sulfate and monolaurin. of the host defense system and intestinal microbial flora in the pathogenesis of Lett Appl Microbiol 2010; 50: 246–251. necrotizing enterocolitis. Surg Infect (Larchmt) 2009; 10: 407–417. 62 Quintero M, Maldonado M, Perez-Munoz M, Jimenez R, Fangman T, Rupnow J 37 Kandhai MC, Reij MW, Gorris LG, Guillaume-Gentil O, van Schothorst M. et al. Adherence inhibition of Cronobacter sakazakii to intestinal epithelial cells by Occurrence of Enterobacter sakazakii in food production environments and prebiotic oligosaccharides. Curr Microbiol 2011; 62: 1448–1454. households. Lancet 2004; 363: 39–40. 63 Gould DJ, Chudleigh JH, Moralejo D, Drey N. Interventions to improve hand 38 FSNET. Recalled baby formula found in Colorado stores. Colorado Department of hygiene compliance in patient care. Cochrane Database Syst Rev 2007, CD005186. Public Health and Environment 2002. 64 El-Sharoud WM, O’Brien S, Negredo C, Iversen C, Fanning S, Healy B. Character- 39 Baumgartner A, Grand M, Liniger M, Iversen C. Detection and frequency of ization of Cronobacter recovered from dried milk and related products. BMC Cronobacter spp. (Enterobacter sakazakii) in different categories of ready-to-eat Microbiol 2009; 9: 24. foods other than infant formula. Int J Food Microbiol 2009; 136: 189–192. 65 Kilonzo-Nthenge A, Rotich E, Godwin S, Nahashon S, Chen F. Prevalence and 40 Mramba F, Broce AB, Zurek L. Vector competence of stable flies, Stomoxys antimicrobial resistance of Cronobacter sakazakii isolated from domestic kitchens calcitrans L. (Diptera: Muscidae), for Enterobacter sakazakii. J Vector Ecol 2007; 32: in middle Tennessee, United States. J Food Prot 2012; 75: 1512–1517. 134–139. 66 World Health Organization 2007, [cited] Available from: http://www.who.int/ 41 Chenu JW, Cox JM. Cronobacter (’Enterobacter sakazakii’): current status and future foodsafety/publications/micro/pif_guidelines.pdf. prospects. Lett Appl Microbiol 2009; 49: 153–159. 67 Centers for Disease Control. 2011 [cited] Available from: http://www.cdc.gov/ 42 Clark NC, Hill BC, O’Hara CM, Steingrimsson O, Cooksey RC. Epidemiologic typing media/releases/2011/s1230_Cronobacter.html. of Enterobacter sakazakii in two neonatal nosocomial outbreaks. Diagn Microbiol 68 Food and Drug Administration 2007, [cited] Available from: http://www.fda.gov/ Infect Dis 1990; 13: 467–472. ForConsumers/ConsumerUpdates/ucm048694.htm. 43 Muytjens HL, Roelofs-Willemse H, Jaspar GH. Quality of powdered substitutes for 69 Food and Drug Administration 2002, [cited] Available from: http://www.fda.gov/ breast milk with regard to members of the family Enterobacteriaceae. J Clin Safety/MedWatch/SafetyInformation/SafetyAlertsforHumanMedicalProducts/ Microbiol 1988; 26: 743–746. ucm154581.htm.

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