IDENTIFICATION OF CRONOBACTER SAKAZAKII ISOLATED FROM POWDERED INFANT FORMULA AND STOOL OF INFANTS

Cronobacter sakazakii is an emerging opportunistic pathogen contaminating powdered infant formulas causing lethal threats to neonates and immune-deficient infants. It causes life-threatening infections, septicemia, neonatal meningitis, and necrotizing enterocolitis. The aim of this study was to test the commercially available formulas that are intended for consumption by 0-6 months old infants (neonates and immune-compromised infants), for the presence of Cronobacter spp., and to determine the presence of C.sakazakii in the stool of these infants who consumed these formulas through conventional methods as culturing, biochemical tests and PCR. Fifty PIF samples (different brands) retailed in upper Egypt were collected from Assiut University Children Hospital at the Gastroenterology and Hepatology and Preterm Units, and we checked the presence of C.sakazakii in them. Fifty Stool samples were also collected from the infants who were fed the studied PIF samples, to study the presence of C.sakazakii in the stool of these infants. The samples underwent three steps of pre-enrichment, enrichment procedures, and subculture onto chromogenic Enterobacter sakazakii agar plates. Biochemical tests were afterwards carried out. Finally, molecular characterization using specific PCR was done to detect Cronobacter sakazakii, targeting the ESA_02797 gene which is found in all C.sakazakii strains . The results of this study shed light on the immense need for applying effective prevention and control measures and taking all the precautions needed during the production and preparation of PIF to hinder its contamination with C.sakazakii and to prevent the spreading of such fatal infections to infants with low immunity and neonates.


INTRODUCTION
Cronobacter sakazakii (C.sakazakii) is attracting considerable attention as a fatal emerging neonatal pathogen, that is associated with many outbreaks of serious life-threatening septicemia, necrotizing enterocolitis, as well as meningitis in infants and neonates globally (Elkhawaga et al., 2020).
C.sakazakii, formerly named Enterobacter sakazakii, a non-spore-forming food-borne pathogen that is a peritrichous rod belonging to the family Enterobacteriaceae (Feeney et al., 2015 andPakbin et al., 2022), in 1980 it was classified as a new species.The generic assignment for E. sakazakii given by DNA-DNA hybridization was not clear, it was 53-54% related to the species in two genera, Citrobacter & Enterobacter.The species was classified in Enterobacter due to being closer to E. cloacae phenotypically and genotypically than C. freundii (the genera type species) (Iversen et al., 2007).
The genus now includes 9 species, C. sakazakii, C. turicensis, C. malonaticus, C. dublinesis, C. muytjensii, C. condimenti and C. universalis.All of them were linked to clinical infections in immune-compromised adults and infants except for C. condimenti (Saad and Amin, 2014).
The pathogen was called the yellow pigmented Enterobacter cloacae due to its characteristic yellow pigment production as well as its biochemical reactions.Later, its name was changed to Enterobacter sakazakii following the DNA hybridization studies and the antibiotic sensitivity tests.Once more in 2008, it was reclassified by Iversen on the basis of sequencing the 16S rRNA gene, ribotyping, and fluorescence labeledamplified fragment length polymorphism fingerprinting (Gan et al., 2021).
Powdered infant formula, a substitute for the milk of mothers and a major nutrition source for infants globally, is industrially produced and formulated using specific amounts of fats, proteins, carbohydrates, minerals, and vitamins.High chances of contamination with pathogens resemble a risk, as it could lead to serious neonatal illnesses (Song et al., 2018).The contamination of the reconstituted PIF might occur intrinsically or extrinsically (Jang et al., 2020).
Powdered infant formula is not a sterile product, being a medium that is nutrientrich, when reconstituted it can support the growth of bacteria, when some favorable growth conditions are available like water, time, and suitable temperature.Thus, once it is rehydrated, the only conditions that can limit bacterial growth and progressing infections are the time of storage and the temperature.C. sakazakii possesses the characteristics of high tolerance to desiccation and osmotic stress and can grow at low temperatures as 5.5 °C, which is the temperature of refrigerators at houses (Deeb, 2010).
In March 2020, neonatal sepsis cases caused by the emerging lethal pathogen C. sakazakii were reported for the first time in Egypt.C. sakazakii was detected in the water, herbs, and the contaminated PIF (Elkhawaga et al., 2020).
Infants that were able to survive the infection with C. sakazakii often suffer delayed neurological symptoms like brain abscesses, delayed development of the brain, or hydrocephalus.For that reason, the International Commission on Microbiological Specification for Foods (ICMSF) decided to classify C. sakazakii as one of the severe hazards for restricted populations and has considered it lifethreatening having substantial chronic sequelae throughout long durations (Feeney et al., 2015 andGan et al., 2021).

Researchers
focused on isolating Cronobacter sakazakii from dairy products as PIF, and from baby foods as herbs and cereals.PIF was epidemiologically linked to infections in infants caused by C. sakazakii.Hence the immune system of infants is immature; researchers tried preventing the contamination of baby foods by irradiation and the addition of probiotic bacteria and tried to control the existing infection in foods by plant essential oil (Abdelhameed, 2017).
The whole genome of all six C. sakazakii strains was analyzed revealing twenty eight different virulence genes present (Holý et al., 2020).
Cronobacter species, like most of the enteric pathogens that interact with humans, has a preferable contact site that it targets which is the mucous membranes or the human mucosa, to easily follow a well-known bacterial infection stratagem that comprises of: (A) Colonizing the mucosal site (intestinal, urinary tract, or respiratory epithelia).(B) Circumvention, subversion, and host defenses exploitation (invasion of the epithelial cells, then internalization and survival within the phagocytic cells, providing a niche for the pathogen with less competition from other organisms, and new rich nutrients).(C) Spreading systemically & multiplying.(D) Host damage (due to host immune system pro-inflammatory modulation, or through expressing exoproteins like toxins,).The flagellum of Cronobacter species induces inflammatory cytokines like IL-8, IL-10, and TNF-α (Jang, Gopinath, et al., 2020).
The general infection's fatality rates range from 42: 80%, and 15: 25% for the neonatal meningitis and septicemia cases, respectively.The highest incidence and severity is in infants, and in outbreaks in intensive care units of neonates (NICU) (Holý et al., 2020).

Aim of the work:
1) Determination of the prevalence of Cronobacter sakazakii in PIF commercially available in the city of Assiut & its prevalence in stool of infants suffering from gastroenteritis fed on these rehydrated PIFs collected from Assiut University Infant Hospital.
2) Determining if there is a link between the presence of C. sakazakii in contaminated PIF & its presence in the stool of infants fed on it.

Samples:
 A total number of 50 powdered infant formula (PIF) samples were collected from formulas used for feeding infants.
 Stool samples were collected from infants suffering from acute diarrhea, and from non-diarrheic infants at infant incubators and at the Gastroenterology and Hepatology unit of Assiut University Children Hospital.A total of 50 samples were collected (40 samples from diarrheic infants & 10 samples from non-diarrheic infants).

Preparation of samples:
 Sterilization of the surface covers of PIF cans was done with 70% ethanol, then cans were aseptically opened inside a laminar flow cabinet, and the samples were aseptically taken from each product can.
 We suspended ten milligrams of stool in 90 ml buffered peptone water, then incubated it for 1 hour at 37°C, before inoculating each sample onto plates of chromogenic Brilliance Enterobacter sakazakii agar (Chandrasekaran et al., 2018).The procedure of detection of C. sakazakii and its isolation is done through 3 successive steps of pre-enrichment in buffered peptone water broth (BPW), then enrichment in selective Enterobacteriaceae Enrichment Broth (EEB), and finally plating on selective chromogenic media.The suspected colonies were then picked up and then subcultured for further microscopic and biochemical identification.
 Pre-enrichment, Enrichment and culturing procedures: Following the FDA protocol, a flask containing sterile distilled water was prepared (pre-warmed to 45°C), then PIF was added and mixed till completely dissolved, then the flask was incubated at 35 ± 2°C for 18-24 hours.
Ten-fold serial dilution was done through adding 10 ml of the dissolved PIF to 90 ml of EE broth medium (Enterobacteriaceae enrichment), then the diluted solution was used as the base solution to make an additional dilution through transferring 10 ml of it to 90 ml of EE broth, and we repeated this three times to reach final concentrations of 10 -1 , 10 -2 , and 10 -3 .The diluted solutions were then incubated at 35 ± 2°C for 18-24 hours.
A loop full of each sample that is incubated in EE broth was streaked by plating out on plates of Brilliance Enterobacter sakazakii agar, then incubated for another 18-24 hours at 35 ± 2°C.The green colonies that were presumptive for C.sakazakii (Figure .1)were picked and pure subcultures were performed on MacConkey agar, TSA, and blood agar, and then were incubated overnight at room temperature (25°C) (Table 1).
Gram-stained films were performed from the presumptively positive cultures and the smears were examined.
Biochemical Tests such as Catalase test, Oxidase test, H2S production test, Citrate utilization, and Urease Test were done to further confirm the isolates (Abdeltawab et al., 2019).

 Preservation:
The purified isolates were saved in LB broth supplemented with 20% glycerol at -20°C.DNA extraction (Fayyad and Dwaish, 2016): It was done by rapid boiling, with some modifications: first, the bacterial cultures were centrifuged (5000 rpm/10 min.) to be concentrated to obtain heavy growth, and were placed in 1.5 ml Eppendorf tubes that contained 300 microliters of distilled water, vortex of the samples for a few seconds, and then the tubes were placed in a water bath at 95˚C for 30 minutes, followed by 10 minutes of centrifugation at 5000 rpm, then we transferred the supernatant to a new sterile Eppendorf tube and stored it at -80˚C until used.

Preparation of PCR reactions:
A 20-μL reaction mixture was prepared to carry out the PCR in, consisting of 10μL of the PCR Master Mix (2X).The solutions of the template DNA were added, and the master mix was prepared according to We supplied the submarine gel with a 100 volts direct current for 1 hour through a power supply.
Then we visualized the gel and photographed it using the gel documentation system.

RESULTS
 C. sakazakii was isolated from a total of 24/50 PIF samples with a percentage of 48% positive PIF samples for C. sakazakii.It was isolated in a percentage of 43.75% from PIF samples intended for feeding infants aged 1-3 months, and in a percentage of 55.55% from PIF samples intended for infants aged 4-6 months (Table 3).
 C. sakazakii was isolated from 25/50 of the total stool samples with a percentage of 50% positive stool samples.It was isolated from 53.125% of the stool samples of infants aged 1-3 months, and from 44.44% of the stool samples from infants aged 4-6 months (Table 4).
 The results of the stool samples collected from infants with acute diarrhea were positive in a percentage of 50%.And according to the ages of infants, C.
sakazakii was isolated from 31.58% of the diarrheic stool samples from infants aged 1-3 months, and in a percentage of 18.42% from the diarrheic stool samples from infants aged 4-6 months (Figure 2).
 The study included 12 stool samples from non-diarrheic infants.The samples were positive in a percentage of 50% (Figure 3).

The correlation between the results of PIF and Stool samples:
We found that 62% of the infants had similar results for their PIF and stool samples, as 30% of the infants in the study showed positive results for both their samples, and 32% of the infants in our study showed negative PIF and stool samples.

Percentage of PIF and Stool Samples similarities
Both

DISCUSSION
Our study isolated C. sakazakii from 43.75% of the PIF samples intended for feeding infants aged 1-3 months.And the pathogen's percentage in PIF samples intended for infants aged 4-6 months was 55.55%.Making 48% of the total studied PIF samples positive.
Similarly, Elsheikh et al. (2021) found that the prevalence of Cronobacter sakazakii in preterm showed a significant increase than in full-term.
These results were very different from the range of prevalence reported by many other studies.Enem et al. (2020) declared that the prevalence of C.sakazakii in their study that was isolated from PIF samples ranged between 5.6% and 3.1% between the different locations where their study took place.
Another study conducted by Mardaneh and Soltan Dallal, (2017) showed different prevalence rates of 7.2% of the samples positive for C.sakazakii.While others isolated C.sakazakii strains from the studied PIF samples in a percentage of 6.86% (Pakbin et al., 2022).
The variation of the prevalence rates may be directly proportional to the different levels of perception of personal hygiene, and the educational level of the caregivers and the PIF handlers, however, there are some reports that indicate that the contamination of PIF with C. sakazakii is very common, as the formulas are the most common vehicle for C.sakazakii known in neonatal infections (Strysko et al., 2020), and the contamination by Enterobacteriaceae members is inevitable if poor hygienic practices were used in the manufacturing of PIF (Güner et al., 2011).
In our study, C. sakazakii was found in stool samples collected from infants aged 1-3 months fed on the studied PIF samples in a percentage of 53.125% and was isolated from stool samples of infants aged 4-6 months in a percentage of 44.44%, making the total percentage of positive stool samples in the study 50%.
Results of another study found 1271 stool samples out of 2304 samples (55.2%) containing Cronobacter rRNA gene sequences (Chandrasekaran S et al., 2018).And another previous study showed different results finding a low prevalence in infant stool samples, as low as 4% (Awadallah et al., 2018).
Upon studying the prevalence in the stool of infants with the clinical presentation of acute diarrhea that might be caused by ingesting PIF infected with C. sakazakii, the total percentage of positive stool samples in our study was 31.58% in diarrheic infants aged from 1-3 months, and 18.42% in diarrheic infants aging from 4-6 months.
Our study also included 12 stool samples from non-diarrheic infants, and C. sakazakii was isolated from them at a percentage of 50% (6 stool samples were positive out of 12 samples).
The study held by Elsheikh et al. in (2021) showed close results as the prevalence of C.sakazakii they found in preterm infants was 28%, and in full-term infants it was 32%.
In our study we evaluated the correlation between ingesting contaminated PIF with C. sakazakii and the prevalence of C. sakazakii infections in PIF-fed infants, and found that the percentage of Infants in the study showing similar positive results for both their PIF and stool samples was 30%, and the percentage of Infants who showed similar negative results for both their PIF and stool samples in the study was 32%, while the percentage of infants in the study with different results in their PIF and Stool samples was 38%.Thus, we found that we can't ensure that the PIF samples were the only source that resulted in the presence of the pathogen in the GIT and the stool of the tested infants, as some infants had the pathogen in their stool while their analyzed PIF samples were not contaminated and vice versa.
The presence of C.sakazakii in the stool of these infants proven to have clear uncontaminated PIF samples analyzed might be due to having ingested a previous contaminated PIF can prior to our analysis, or that these infants might have caught the pathogen from the hands of caregivers or other contaminated sources that might have reached the infant's mouth, which is linked to unhygienic practices of the caregivers in uneducated families and in rural areas (Cho et al., 2019).

Figure
Figure (2): The percentage of positive stool samples in infants suffering from acute diarrhea in different age groups.

Figure ( 4 ):
Figure (4):The correlation between the results of PIF and Stool samples of the same infant in the study.

Table 1 :
The characters, microscopic morphology and biochemical reactions of C. sakazakii.

Table 2 :
PCR components and their quantities.

Table 3 :
The percentage of the positive results of PIF samples intended for feeding different age groups:

Table 4 :
The percentage of positive results in Stool samples.
According to our research, we can't ensure that consuming the contaminated PIF samples was the only cause that resulted in the presence of the pathogen in the GIT and the stool of the infected infants.