IDENTIFICATION OF CAMPYLOBACTER SPP . ISOLATED FROM POWDERED INFANT MILK FORMULA

Campylobacter species are Gram-negative bacilli that are characterized by being catalase-positive, oxidase-positive, motile, microaerophilic bacteria, and non-spore-forming. Campylobacter is often isolated from animal sources, as it inhabits the gastrointestinal tract of both wild and domestic animals and birds, especially poultry. Milk may be possibly contaminated by the direct discharge of a mastitis-affected cow or by excrement from diseased or colonized cattle during milking. Powdered infant formula (PIF) is considered a non-sterile product and may be contaminated intrinsically or extrinsically with various bacteria that can cause critical illness in infants. This study was performed to detect and identify Campylobacter spp. in powdered infant milk formula by phenotypic and genotypic methods. Ten isolates of Campylobacter from eighty-six samples were phenotypically identified and confirmed genotypically by PCR, with a pattern of 11.6%. This Campylobacter could potentially be transmitted to children by PIF consumption that has not been adequately handled, prepared or processed.


INTRODUCTION
One of the most frequent causes of bacterial gastroenteritis in people is Campylobacter spp.
The genus Campylobacter includes thirty-nine species, and this number is constantly increasing as new species are discovered (Zhong et al., 2022).
Campylobacter is a spiral or curved "gull-winged" Gram-negative, nonspore-forming, motile rod and microaerophilic bacteria.It ranges between 0.2 and 0.5μm in width and between 0.5 and 0.8 μm in length.Their unique corkscrew-like motion can be explained by the presence of a single flagellum (sometimes multiple flagella) at one or both cell poles.The alteration from spiral to coccoid morphology is influenced by nutrient limitation, medium aeration, and the level of free radicals (Barros-Velázquez Jorge et al., 1999).
Campylobacter jejuni, C. coli, C. lari, and C. upsaliensis all grow easily at 37°C in microaerophilic circumstances (5% O2, 10% CO2 and 85% N2), and the majority of these species' strains will also grow at 42°C, demonstrating that all of the clinically relevant Campylobacter spp.are thermotolerant in nature (Fouts et al., 2005).
A few hundred bacteria are sufficient to cause intestinal colonization in humans, which leads to campylobacteriosis (Backert, 2020).According to the French National Reference Center for Campylobacters and Helicobacters, the majority of these that cause human illnesses include the thermotolerant species Campylobacter jejuni (C.jejuni) and Campylobacter coli (C.coli) (Sifré et al., 2015), which is followed by a variety of other species, including C. upsaliensis, C. hyointestinalis and C. lari (Ketley, 1997 andTang et al., 2020).
Campylobacter was proven to be the cause of outbreaks related to the consumption of milk, cheese, and other dairy milk products, with a pattern of 22.2% (EFSA, 2017).Milk may be possibly contaminated by the direct discharge from a mastitis-affected cow or by excrement from diseased or colonized cattle during milking (El-Shaboury et al., 2003).
For young children who are more vulnerable to dehydration and loss of nutrients, such as sodium and protein, as a result of the diarrheal disease, Campylobacter infection is considered hazardous (Ahs et al., 2010 andAsuming-Bediako et al., 2019).
Cross contamination and insufficient heat treatment were the most often reported outbreak causes, attributed to pasteurized milk (Mahmood et al., 2009).
Actually, to reduce the danger of developing Campylobacter, meals must be heated to a temperature of 70° C. (A. Facciolà et al., 2017).Additionally, after handling contaminated items, appropriate hand washing is important, because a quick rinse or wash may not be enough to entirely eradicate pathogens such as C. jejuni (Acuff et al., 1986),

MATERIALS AND METHODS
Ethical statement.According to the World Medical Association's code of ethics (Declaration of Helsinki), the study was approved by the ethical committee of Assiut University's faculty of medicine.The number is 17101914.
Collection of samples: seventy-six PIF samples were collected from Assiut University Children's Hospitals.Samples were collected under a septic condition.

Preparation of PIF samples:
For the isolation of Campylobacter from PIF, 5 g of PIF sample was mixed with 30 ml of sterile distilled water, as per the general dissolving instructions on milk bottles.The tubes were mixed with vortex and labeled.The PH determination of PIF was done using pH test paper (pH 6-8 range) and sterile NaOH 2N to adjust PH to 7.5 ± 0.2 if necessary.Centrifuge a 50 g portion at 20,000 × g for 40 minutes.The supernatant was discarded (the fat layer), while the pellet was dissolved and inoculated (Jan M. Hunt et al., 2001).

Culture on solid media:
• mCCDA: C. jejuni showed a gray, small, sheen appearance, moist, and have a special odor, while C. coli typically had a wet, creamy gray appearance and a slightly raised shiny surface (Sallam, 2007 andAli A. AL-Edany et al., 2015).

Identification of isolated colonies: 1-Gram-stained smear: (Smith Ann C. et al., 2005).
Films were made from the suspected colonies grown on mCCDA by taking a loopful from the colonies with a drop of distilled water on a clean, properly dry glass slide, then stained with Gram's stain and examined microscopically.Campylobacter spp. is Gram-negative, so the suspected result was pink-colored rods that were very slender with curved or spiral-shaped rods with a "gullwinged" appearance.

2-Motility test: (American Society for Microbiology, 2016).
Semisolid media was used to perform this test by using an inoculating needle and the stab technique.Each organism was aseptically transferred to a tube of the motility medium and incubated for 24-48 h by using AnaeroPack-Anaero sachets (MGC, japan) and an anaerobic jar.Positive results when widespread, hazy growth diffused in the medium, making it a little opaque.

3-Biochemical tests:
a. Catalase test: (Reiner Karen, 2010) A colony was added to a small spot on a slide.One drop of 3% H2O2 was put on it.The evolution of gas indicates catalase activity as a positive result, which is presumptive for Campylobacter spp.

b. Oxidase test: (Gupta et al., 2022)
Oxidase activity was tested on oxidase disks.By using a glass loop, a wellgrown colony from a fresh culture medium was applied to the reaction on the disk.Violet coloration indicates a positive reaction within 20-60 seconds after getting contact, which is presumptive for Campylobacter spp.

c. Hippurate hydrolysis test: (OIE Terrestrial Manual, 2008)
This test was used to differentiate between C. jejuni and C. coli.A heavy loopful of fresh growth from a suspected colony was suspended in 400 µl of a 1% sodium hippurate solution.The culture was incubated at 37°C for 2 h.Then 200 µl of 3.5% ninhydrin solution was added to the tube's side to make an overlay.The culture was re-incubated at 37°C for 10 minutes.Positive reactions gave a dark purple color, but negative reactions showed a clear or gray color.The suspected result with C. jejuni was positive, but C. coli was negative.

Detection of the amplified product
An agarose gel with a 1.5% concentration was used to test the PCR amplicons.Then stained with ethidium bromide, and observed for two hours under ultraviolet (UV) radiation at 80 volts.

Statistical analysis
All results were carried out and analyzed using IBM SPSS 26.0, and categorical variables were described using numbers and percentages (N, %).

RESULTS
Campylobacter spp. was isolated from PIF samples in a percentage of 11.6 % (10 /86).C. jejuni and non-C.jejuni had a frequency pattern of 50% for each one.
According to age groups of children, the frequency of positive Campylobacter spp.isolates from PIF with the group ages from zero to 3 months was 10.5%, while the group ages more than 3 to 6 months was 15.6%, but the age groups >6-9 and >9-12 were zero and 11.1%, respectively, as shown in table (4).

On Culture media
As shown in figure (1), Campylobacter spp.were isolated on mCCDA as creamy to grayish-colored, moistened, with or without a metallic luster, with a slightly raised, shiny surface, and having the tendency to spread across the plate

DISCUSSION
According to the current investigation, 11.6% of Campylobacter spp.isolates were found in PIF.About 50% of PIF-positive samples were C. jejuni, and 50% were non-C.jejuni.Mahmood et al. (2009)

CONCLUSION
Powdered infant formula (PIF) was considered a source of Campylobacter infection as it can survive osmotic and high-temperature conditions up to 70 °C, so we recommend reconstitution of PIF with hot water to avoid any possible Campylobacter contamination.
Even a quick hand wash or rinse may not remove C. jejuni absolutely after handling contaminated foods, so we recommend washing hands properly by mothers during handling and preparation of PIF, as it is very important to prevent Campylobacter infection in their children.

Figure
Figure (1): Campylobacter spp. on mCCDA Identification of the isolated Campylobacter colonies by Gram stain, motility test, biochemical tests and genotypic identification by PCR 1-Gram stain: Film from colonies under the microscope revealed Gram-negative, very slender, curved, or spiral-shaped rods with a "gullwinged" appearance.Tendency to form coccoid and elongated shapes as shown in figure (2) 2-motility test: Campylobacter spp.showed widespread, hazy growth diffused in the medium, making it a little opaque, indicating positive

Table 1 :
Types and numbers of PIF used in this study

Table 2 :
Oligonucleotide sequences used to identify Campylobacter spp.according to(Huang  et al., 2009)

Table 3 :
Frequency of Campylobacter spp.isolated from different types of PIF.

Table 4 :
Frequency of Campylobacter spp. in examined PIF isolates according to children's age groups.
Elmalı and Can, (2019), which all were C. jejuni in raw milk and 9% in pasteurized milk byAdmasie et al. (2023).On the other hand, in Eastern Cape Province, South Africa, it was higher than this study by 26.38% in raw milk samples by Igwaran and Okoh, (2020).In Hatay, Turkey, byElmalı and Can, (2019), Campylobacter spp.frequency was lower by 5.2% in raw milk samples.This variety in results may be due to environmental factors, the year that samples were collected, and the geographical location.PIF couldn't be sterile, according toLosio et al. (2018).2-Mother cross-contamination during handling and preparation of PIF.