Cronobacter spp., foodborne pathogens threatening neonates and infants

Abstract

Cronobacter spp. (formerly Enterobacter sakazakii) are special foodborne pathogens. Cronobacter infection can cause necrotizing enterocolitis, sepsis and meningitis in all age groups, especially neonates and infants, with a high fatality of up to 80%, although the infection is rare. Outbreaks of Cronobacter infection are epidemiologically proven to be associated with contaminated powdered infant formula (PIF). Cronobacter spp. can resist dry environments and survive for a long period in food with low water activity. Therefore, Cronobacter spp. have become serious pathogens of neonates and infants, as well as in the dairy industry. In this review, we present the taxonomy, pathogenesis, resistance, detection and control of Cronobacter spp.

Keywords

Cronobacter spp. / desiccation resistance / pathogen control / pathogen detection / powdered infant formula

Introduction

Cronobacter spp. (formerly Enterobacter sakazakii) are Gram-negative foodborne pathogens that can cause necrotizing enterocolitis, sepsis and meningitis in all age groups, especially neonates and infants^[1,^2]. Fatality resulting from infections in neonates and infants is up to 80%^[3,^4]. Thus, the International Commission for Microbiological Specifications for Foods listed Cronobacter spp. as “severe hazard for restricted populations, life threatening or substantial chronic sequelae or long duration”^[5]. In 2004 and 2006, FAO-WHO listed Cronobacter spp. and Salmonella spp. as Category A pathogens after reviewing the organisms found in powdered infant formula (PIF) and epidemiologically linked with neonatal infections^[6].

Taxonomy of Cronobacter

In 1961, two doctors reported the first cases of Cronobacter spp. infection in the UK^[7]. In 1965, Cronobacter spp. were called “yellow-pigmented Enterobacter cloacae” because of their similar characteristics to Enterobacter cloacae and their yellow pigment production^[8]. The differences between Cronobacter spp. and other Enterobacteriaceae were revealed with the development of technology. DNA-DNA hybridization distinguished Cronobacter spp. from Enterobacter cloacae (difference between Cronobacter and Enterobacter cloacae was over 50%) and Cronobacter spp. were named as Enterobacter sakazakii in 1980. 16S rDNA and hsp60 sequencing justified Cronobacter being erected as a new genus^[9]. Currently there are seven described Cronobacter spp.: C. sakazakii, C. malonaticus, C. turicensis, C. universalis, C. muytjensii, C. condimenti and C. dublinensis (with three subspecies dublinensis, lactaridi and lausannensis)^[10].

Analysis of 16S rRNA gene sequence is widely used in phylogenetic studies of bacteria. It can identify strains to genus, and often to species^[11]. However, 16S rRNA gene sequence analysis is not accurate enough to distinguish C. sakazakii from C. malonaticus^[9]. Multilocus sequence typing is a better option for Cronobacter species identification. It characterizes bacterial isolates by multiple housekeeping gene sequence analysis (usually seven genes). A multilocus sequence typing scheme has been established for Cronobacter based on the genes atpD, fusA, glnS, gltB, gyrB, infB and ppsA. All seven species of Cronobacter spp. can be identified and distinguished^[12] and the results also show that C. sakazakii strain ST4 has caused the major meningitis cases investigated^[6]. The protocol and related data are publicly accessible online (PubMLST website: Cronobacter MLST Database), providing profiles and details of 2233 strains (accessed: 28 October 2017).

Disease caused by Cronobacter infection

All Cronobacter spp. are associated with human infections except for C. condiment^[10]. In the US, the infection rate of Cronobacter is about 1:100000^[13]. The US Centers for Disease Control and Prevention (CDC) usually reports four to six infection cases every year (data source: CDC website/CDC features/Disease and Conditions/Learn about Cronobacter infection, accessed: 10 April 2017). However, the CDC also said they did not have a full count. Developing countries reported fewer infection cases, perhaps due to the absence of well-established microbial analysis systems^[14]. Therefore, the infection incidence is probably underestimated. Nevertheless, it is certain that Cronobacter infection is rare. However, the high mortality, serious neurological sequelae and susceptible population of the infection still make it a serious health threat. Cronobacter infection is also the most costly food-associated infection in the US due to loss of life and complicated treatment after acute infection, with an estimated cost of 1 million USD per case^[15].

Cronobacter usually cause meningitis, septicemia bacteremia, and necrotizing enterocolitis^[3]. Meningitis is the most common symptom of Cronobacter infection, accounting for about 42% of all cases. Neonate and infant patients may have symptoms of fever, irritability and high-pitched crying. Meningitis may further develop into vasculitis, cerebritis, ventriculitis, hydrocephalus and brain abscesses at a surprising rate^[16]. The death rate is about 50%^[17] and progression to death is usually rapid in infant patients. Survivors often suffer from serious neurological sequelae such as quadriplegia and mental retardation^[14,^18]. Necrotising enterocolitis is another important symptom of Cronobacter infection. Necrotising enterocolitis induced by Cronobacter spp. has a mortality rate of 19.0%^[19]. Factors such as an incomplete immune system, hypothermia and hypoxia contribute to the necrotising enterocolitis development^[20]. Table 1 shows Cronobacter spp. infection cases over the past 10 years.

Pathogenesis of Cronobacter

Cronobacter spp. generally infect the human body through the digestive system. Intestinal epithelium invasion is the first step of Cronobacter infection. Then, neonates and infants may suffer from necrotizing enterocolitis. After that, Cronobacter may enter and multiply in the blood stream to survive and proliferate in blood macrophages. Thus, Cronobacter spp. can spread throughout the body via blood circulation and patients develop symptoms of bacteremia. Meningitis occurs after Cronobacter spp. invade brain endothelium and cross the blood brain barrier. In conclusion, attachment, invasion and host cell injury by Cronobacter infection are the main pathogenic route, and this involves various virulence factors.

Protein ompA is an outer membrane protein of Cronobacter spp. It is important in Cronobacter invasion of cells. Cronobacter strains lacking gene ompA show reduced invasion, 87% less in INT-407 cells^[26] and 83% less in human brain microvascular endothelial cells^[27]. Protein ompA can also promote the attachment of Cronobacter spp. to INT-407 epithelial cells but cannot improve its attachment to human brain microvascular endothelial cells^[17]. The attachment mechanism is likely host cell-type specific.

Lipopolysaccharide is a major cell wall component of Gram-negative bacteria. It is stable even at 100°C and can persist in PIF for a long time after spray drying. It helps Cronobacter spp. translocate through the body and cross the blood-brain barrier to cause meningitis^[28,^29]. Lipopolysaccharide can also cause inflammatory response and inhibit tissue repair^[30,^31]. This contributes to the development of necrotising enterocolitis and meningitis.

The Type VI secretion system was found recently. About 25% of Gram-negative bacteria including Cronobacter have this system^[32] and it has important roles in adhesion, virulence, invasion and proliferation of Cronobacter in cells. This system also produces proteinaceous toxins that are potential virulence factors^[17]. Five related genes were identified from the Cronobacter genome sequence and their contributory roles in meningitis are under investigation^[33–^36].

Other factors such as flagella, zinc metalloprotease, pmrA gene and Cronobacter plasminogen activator also participate in the invasion of Cronobacter spp^[37–^40]. However, further studies are still needed to confirm their function.

Studies on Cronobacter spp. have increased exponentially in recent decades. However, the precise pathogenic mechanism of Cronobacter spp. is still unclear. A thorough understanding this mechanism should provide better strategies to treat and control infection. Consequently, the fatality of Cronobacter infection could be reduced and patients with neurological sequelae will have better quality of life.

Source of Cronobacter

Cronobacter spp. are ubiquitous in the nature. They have been isolated from PIF, herbs, fruits, water, meat and food processing equipment^[41,^42]. Table 2 shows Cronobacter spp. isolated from different sources over the past 5 years. Of all sources, PIF is epidemiologically linked with neonates and infants infections^[21,⁵⁵,⁶⁷–^69]. Although water activity of PIF is low and so numbers of Cronobacter spp. are low, they can proliferate with inappropriate storage and reconstitution.

Desiccation resistance of Cronobacter

Contaminated PIF is the critical infection source of Cronobacter spp. PIF has a low water activity (a_w: 0.2–0.5) and most common pathogens cannot survive for a long time in such a dry environment, however, Cronobacter is an exception. Edelson et al.^[70] studied the long-term survival of Cronobacter spp. in milk powder. The initial inoculum dosage was about 10⁶ CFU/mL and there were still 300 CFU/mL in the milk powder after 687 days’ storage. Furthermore, Barron and Forsythe^[71] prepared milk powder samples inoculated with Enterobacteriaceae including Cronobacter spp. These samples were kept at room temperature for 30 months. All non-Cronobacter strains had died by 15 months but some Cronobacter strains survived for 30 months. Fei et al.^[72] studied the desiccation resistance of six C. sakazakii strains and two C. malonaticus strains isolated from PIF and processing environments. The results showed that C. sakazakii ST4, ST8 and ST12 had the greatest survival after 1 year. Among these strains, C. sakazakii ST4 has caused the majority of Cronobacter meningitis cases. Therefore, the pathogenesis of Cronobacter spp. is likely to be associated with their resistance to drying. Their desiccation resistance also improves the resistance of Cronobacter to other environment stress such as ionizing radiation^[73], heat^[74] and antimicrobial substances^[75–^78].

The mechanism of desiccation resistance in Cronobacter spp. is still under investigation. Trehalose is a disaccharide formed by two a-glucose units through an a,a-1,1-glucoside bond. It participates in the responses of many organisms to environment stresses, such as Escherichia coli^[79], Gossypium^[80] and Saccharomyces cerevisiae^[81]. Cronobacter spp. mainly produce trehalose in the stationary phase after a drying treatment^[82]. The resistance of Cronobacter spp. to drying is also improved when trehalose is added in the medium^[83]. Transposon mutagenesis has been applied to analyze gene expression in Cronobacter spp. under osmotic and drying stress^[84]. The osmotic and desiccation resistance mechanisms of Cronobacter spp. were quite different. Genes dnaK and dnaJ, encoding two molecular chaperones are responsible for resisting drying. RpoS (RNA polymerase sigma factor) that is a central regulator of stress response also regulates the process of desiccation resistance because C. sakazakii strains deficient in rpoS proved to be more sensitive to drying^[85]. However, none of these genes is related to trehalose and the role of trehalose in the desiccation resistance of Cronobacter spp. needs further examination. Recently, a comparative proteomic analysis of C. sakazakii by iTRAQ was carried out. Results showed that expression level of genes involved in unnecessary survival functions, such as virulence, adhesion and invasion, decreased, while expression level of genes involved in trehalose and betaine uptake such as ABC transporter and secretion system increased during the response of C. sakazakii to desiccation^[86]. As with the pathogenic mechanism of Cronobacter spp., current studies have not completely described the mechanism of desiccation resistance but it seems that multiple metabolic pathways are necessary. Further studies concerning desiccation resistance of Cronobacter spp. are encouraged, to figure out better hazard control strategies in the dairy industry and consuming process.

Detection of Cronobacter

Culture methods were first used to isolate and identify Cronobacter spp. The core of these methods was to determine the special physiologic properties of Cronobacter spp. They can produce a-glucosidase and this distinguishes them from other Enterobacteriaceae. a-Glucosidase can hydrolyze 4-nitrophenyl-a-D-glucopyranoside^[87], 4-methylumbelliferyl-a-D-glucopyranoside^[88] and other substrate to produce a color change. In conjunction with other characteristics such as yellow pigment production and insensitivity to vancomycin, Cronobacter spp. can be isolated from food samples and distinguished from other pathogens. The International Organization for Standardization then adopted this detection method as ISO 22964:2017 Microbiology of the food chain. The evaluation process for Cronobacter detection is shown in Fig. 1 Culture methods are important for Cronobacter detection. However, they are time consuming and these steps are complex. Therefore, simpler and more rapid detection methods have been developed.

Immunological methods have been developed for Cronobacter detection and enzyme linked immunosorbent assay (ELISA) has been widely used^[86,^87]. Nevertheless, ELISA still takes a long time. Therefore, a colloidal gold test strip for Cronobacter detection have been developed to shorten the time and simplify the steps^[89]. It is easy to use and can be adapted to various situations. To make antibody preparation easier and faster, a single chain variable fragment specific to Cronobacter spp. was also identified and produced in a prokaryotic expression system^[90]. Compared with polyclonal and monoclonal antibodies, the preparation time and cost of single chain variable fragment is reduced. Immunological methods can also be combined with better detection methods for Cronobacter spp., such as immunomagnetic separation-polymerase chain reaction (IMS-PCR)^[91].

Nucleic acid amplification methods are more widely used in the detection of Cronobacter spp. Mining of specific gene targets is important in nucleic acid amplification detection methods. Here, we summarize these specific target genes used for the detection of Cronobacter spp. and the PCR and real-time PCR detection methods targeting them (Table 3). Isothermal amplification detection methods have also been also developed. These methods can be run at a stationary temperature and thermal cycling equipment is unnecessary, making isothermal amplification detection easier to carry out. Most isothermal amplification detection methods also show higher amplification efficiency than PCR methods^[108] and isothermal amplification is a promising alternative to PCR. Two isothermal amplification detection methods have been developed for Cronobacter, loop-mediated isothermal amplification (LAMP)^[109] and helicase-dependent isothermal DNA amplification (HDA)^[110]. They are simpler, without the need for PCR amplification, and the sensitivity of the LAMP assay is 1000 and 100 times higher than regular PCR and real time quantitative PCR, respectively^[109]. In addition to these main detection methods mentioned above, other valuable additions to the methods for Cronobacter detection have been developed, such as MALDI-TOF MS^[111] and a label-free aptasensing platform^[112].

However, due to low Cronobacter numbers in contaminated PIF^[113] and the inhibiting effect of food components, enrichment is still necessary in order to increase cell concentrations to the detection limit. To improve detection efficiency, methods that enrich detection targets, such as immunomagnetic separation^[91] and probe-magnetic separation^[114], should be developed.

Control of Cronobacter in PIF

Cronobacter spp. are ubiquitous in nature and they cannot be completely removed from production environments^[115]. Hence, contamination in PIF can occur easily and PIF is the most common vehicle for Cronobacter infection. However, according to national standards, they should not be detectable in PIF given the severe consequences of infection. Therefore, it is important to develop inactivation methods for PIF. The organoleptic, nutritional and functional properties of PIF change under heat treatment^[116], and drying increases the heat resistance of Cronobacter^[74]. Therefore, there has been a focus on non-thermal inactivation methods for Cronobacter spp.

Hydrostatic pressure processing is a non-thermal sterilization method for food products^[117]. Inactivation levels of five to seven log₁₀ cycles in reconstituted PIF were observed with treatment at 100 to 600 MPa^[118]. Gamma irradiation is another useful sterilization for PIF. C. sakazakii and concentration of 8 to 9 log₁₀ CFU/g in a dehydrated infant formula could be eliminated with irradiation at 5.0 kGy^[119,^120]. Ultrasound waves also inactivate Cronobacter spp. However, this method has a limited lethal effect compared with hydrostatic pressure and gamma irradiation. Researchers have tested the addition of antimicrobials to PIF to control Cronobacter spp., including lactoferrin, nisin^[121], vanillin^[75] and polyphenol-rich cocoa powder^[122]. These all had good inhibitory effect on Cronobacter spp., although the safety of such antimicrobials needs to be fully assessed prior to being recommended or approved for industrial application.

The CDC, WHO and the US Food and Drug Administration have all provided guidelines to avoid contamination and infection of Cronobacter spp.: (1) PIF should be prepared in a clean location with sterilize bottles and plastic nipples; (2) The temperature of water for reconstitution must exceed 70°C; (3) The reconstituted PIF must be stored under 4°C and use within 24 h; (4) Once a feeding has started, the reconstituted PIF should be finished within 2 h^[14].

Conclusions

To promptly identify contaminated food and control infection, reliable, sensitive and rapid detection methods for Cronobacter are necessary and need further development. In addition, we still know little about the transmission and pathogenesis of Cronobacter spp. Infection still occurs, albeit at low prevalence, though with often fatal consequences in neonates and infants. Control methods other than the current heating and drying during processing should be considered as alternatives or supplements, given the heating and desiccation resistance of Cronobacter. To thoroughly control the Cronobacter contamination of PIF, increased multidisciplinary effort is needed, including studies of infection surveillance systems, virulence factor identification, regulatory mechanism of resistant genes, efficient non-thermal inactivation methods and reliable prevention strategies.

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