ISOLATION AND CHARACTERIZATION OF LISTERIA MONOCYTOGENES FROM SOME IRAQI MISCARRIAGE WOMEN

This study was aimed to find out how common Listeria monocytogenes in a group of Iraqi women who had miscarriages. Also, investigates the dependability of molecular diagnosis, specifically evaluating the target hlyA gene as an alternate solution tool for identification . A total of 200 specimens comprising cervical swabs were collected from 176 women suffered from miscarriage and 24 women as control. The detection of L. monocytogenes in miscarriages women by using HiCrome™ Listeria Agar Base as both a selective and differential agar medium for quick and direct recognition of Listeria species. The biochemical tests were achieved for confirmation identification. In addition, virulence-associated genes ( hlyA gene) isolates were identified using a Real-time PCR approach. Results show out of the 200 (patients and control) specimens, 176 women with miscarriages were analyzed and it was found that 117 (66.5%) specimens from abortion cases were negative for L. monocytogenes , while 59 (33.5%) specimens were positive for Listeria species. All 24 women of control give negative result. Seventy-five (32.4%) isolates from the 59 positive women showed the presence of the hlyA gene in Real-time PCR. We found that molecular diagnostic through Real-Time PCR is a quick and accurate diagnostic method that can help minimize the time it takes to start a specific treatment and track its success.

. L. monocytogenes is an animal pathogen causing infectious diseases.It was found that this species was one of the agents responsible for abortions in infected cows (15).Also, Listeria monocyogenes were identified and isolated from contaminated food and contribute to food poisoning (8).This is a foodborne disease that can cause complicated infections in immunocompromised individuals, embryos, and neonates (5).Pregnant mothers account for 20-30% of listeriosis occurrences, according to studies.Listeriosis can cause bacteremia, amnionitis, and infections of the embryo leading to premature birth, miscarriage, stillbirth, and other serious health concerns for newborns (13).The molecular identification of L. monocytogenes was conducted by several virulence genes.These genes include the internalins, listeriolysin O, phosphatidylinositol-phospholipase C, actin (act A), iap (invasion associated protein), and virulence regulators (encoded by prf A).Infections and pathogenicity are aided by several virulence factors (11).L. monocytogenes, Toxoplasma gondii, rubella, herpes simplex virus (HSV), measles, and cytomegalovirus are among the diseases that have been linked to sporadic spontaneous pregnancy loss.The following mechanisms have been proposed for infectious causes of pregnancy loss: a) direct infection of the fetus, uterus, or placenta, b) amnionitis, c) placental insufficiency, d) an infected intrauterine device, or e) chronic endometritis or endocervicitis (7).A meta-analysis study showed there is a significant relationship between the infection with L. monocytogenes and spontaneous abortion (20).In Iraq there is a little prevalence or incidence data about the listeriosis infections and their correlation with recurrent abortion in Iraqi pregnant women, therefore this study was aimed to investigate the prevalence of L. monocytogenes in a group of Iraqi women with recurrent abortion and identification of this bacterium by a molecular assay using the virulence gene (hly).

MATERIALS AND METHODS Samples collection:
The cervical Swab specimens were collected using sterile transport swab medium as follows: 1-From patients with miscarriage, the swab specimens were collected after two to four weeks from the last abortion.2-Healthy (control) women, the swab specimens were collected before or after the menstrual period.These clinical specimens of patients and control groups were collected from public maternity hospitals in Baghdad city during the period from November 2019 to December 2020.

Bacterial culture media
The culture media used in this study included Brain Heart Infusion Broth M210 (BHI, Himedia), HiCrome Listeria Selective Supplement FD181, and HiCrome™ Listeria Agar Base, Modified M1417 (HiMedia, India).All media were prepared according to manufacturing instructions.The pH was adjusted to 7.3 (± 0.2) and sterilized in an autoclave at 121 ᵒC for 15 minutes at 15 pounds per square inch (PSI).Listeria monocytogenes isolation L. monocytogenes was isolated from clinical specimens.The specimen swab was firstly inoculated in an autoclaved and sterilized universal containing 10 ml of BHIB and incubated at 37 °C for 24-48 hours.The broth culture was then streaked on the surface HiCrome™ Listeria Agar Base, with selective supplement, a selective and differential agar medium suggested for quick and direct identification of Listeria species, and then incubated at 36°C ± 2°C for 24-48 hours.Then isolates colonies were detected.

Listeria monocytogenes Identification
The suspect colonies were identified as L. monocytogenes using a selective and differential agar (as mentioned previously), after which they were submitted to biochemical tests such as Gram staining, oxidase, and catalase tests, carbohydrate fermentation tests (mannitol, rhamnose, glucose).As pathogenic Listeria spp. was distinguished from non-pathogenic spp., all of the described Listeria isolates were tested for hemolysis on (5%) sheep blood agar (SBA).The L. monocytogenes isolates were also confirmed using a Real-time PCR method to determine the presence of a particular virulence feature, hylA gene.Molecular detection of L. monocytogenes hlyA gene: DNA extraction: By using Genomic DNA Extraction Kit, Genome Quick Bacteria (Dongsheng Biotech Co., Korea).This kit provides a straightforward and quick approach for obtaining high-quality genomic DNA from Gram-Positive and Gram-Negative Bacteria, which may then be utilized as the analysis target.

Detection of L. monocytogenes hlyA gene
Quantitative Real-time PCR (Q-PCR) assay is the confirmation process to determine the presence of L. monocytogenes hlyA gene in the clinical specimens that gave positive results in selective and differential HiCrome™ Listeria agar base and biochemical tests previously.The process was conducted by specific detection of hlyA gene in the Purified DNA.Positive results in detection hlyA gene observed as amplification exponential florescent curves (Presence of Taqman Probe) at specific computer screen attached with the thermal cycler system.The design of primers and probe for hlyA gene was conducted using Beacon Designer and used in this study are show in Table (

Q-PCR melting curve with SYBR Green
Moreover, to confirm our specimens that gave positive results in the previous step were highly pure, additionally there were no contaminations in our work or primer-dimer and other non-specific products, so melting curves were done.Program conditions with a total volume of 20μl are mentioned in Table 3 to detect hlyA gene of the same primers above with SYBR Green instead of the probe in this technique.

RESULTS AND DISCUSSION Isolation and identification of Listeria spp
In this work, Listeria spp.were confirmed and identified using HiCrome™ Listeria Agar Base, Modified (HIMEDIA M1417).The selective chromogenic measurement of βglucosidase activity, as well as rhamnose fermentation, is the bases of this medium.The pure chromogenic substrate in the medium is hydrolyzed by Listeria species, resulting in blue-colored colonies.Other organisms cannot use the chromogenic substrate because the β-glucosidase activity is peculiar to Listeria spp., resulting in white colonies.As well as the feature of rhamnose fermentation is used to distinguish Listeria spp (19).L. monocytogenes colonies were bluish-green with a yellow halo (rhamnose positive) Figure ( 1).HiCrome TM Listeria Selective Supplement (FD181) is included in this medium, which inhibits the development of most Grampositive and Gram-negative bacteria, as well as yeasts and molds.

Figure 1. Listeria monocytogenes colonies on HiCrome™ Listeria Agar Base selective and
differential agar (colonies that are bluish-green with a yellow halo).The results of primary identification using biochemical tests were (Gram Staining (+ve), Catalase Positive (+ve), Oxidase Negative (ve), carbohydrate fermentation D-mannitol Negative (-ve), rhamnose and D-glucose Positive (+ve), Hemolysis was β-hemolysis) and for HiCrome™ Listeria Agar Base revealed that out of the 200 (patients and control) specimens, from 176 miscarriages women analyzed, it was found that 117 (66.5%) specimens were Negative for L. monocytogenes, while 59 (33.5%) specimens from abortion cases were positive.all 24 women control gives Negative to this bacterium.Abdalla et al. (1) identified L. monocytogenes from aborted women by using biological and biochemical properties.In addition to molecular diagnosis, the microbes' isolation and identification were validated using staining, culture, morphological, and biochemical testing (2,3).

Identification of L. monocytogenes by Q-PCR:
The hlyA gene is one of the important virulence characteristics.Therefore it was chosen as the best target for the detection of this bacterium by real-time PCR.To find genetically common characteristics of newly isolated 57 (32.4%) positive specimens for L. monocytogenes hlyA gene was amplified by Q-PCR and mention in  Furthermore, the quantitative results supplied may be valuable for the follow-up of patients undergoing treatment in specific cases (10).On the other hand, (12) revealed that specificity of molecular detection of hly gene for L. monocytogenes was 100%, in comparison with other Listeria isolates, and under certain settings, this PCR detection method, which is relatively sensitive and more specific, could be used as a quick diagnostic method from contaminated food.Following amplification, a melt curve analysis was performed to verify the specificity of the amplified products by their specific melting temperatures Tm (6).The protein with poreforming activity is the most important factor in vacuole lysis.The 58-kDa protein (Listeriolysin O) is encoded by the (hlyA) gene.The hlyA gene also has been targeted for the development of PCR-based tests for L. monocytogenes detecting (9).According to the findings of a study conducted in Erbil, Iraq, the Q-PCR detection method, which included a set of primers and a hybridization probe, was very effective in monitoring the L. monocytogenes hlyA gene in meat (16).In one study in Italy, the Italian National Reference Laboratory for L. monocytogenes of Istituto Zooprofilattico Sperimentale dell' Abruzzo e del Molise (IZSAM) received a total of 37 clinical strains isolated from patients with listeriosis symptoms and 1374 strains correlated to the outbreak for outbreak investigation.A real-time PCR assay was discovered to be capable of screening thousands of L. monocytogenes strains in a short amount of time, saving time and money (17).The presence of virulence factors associated genes, actA, hlyA, plcA, and prfA in all strains with high antibiotic resistance indicated that L. monocytogenes strains were highly virulent, according to a research in Iraqi pregnant women at Al-Diwaniya hospitals, that included determination of virulence genes as well as antimicrobial susceptibility (4).Listeria monocytogenes can infect asymptomatic pregnant mothers, fetus infections are a dangerous illness that can lead to premature birth, abortion, sepsis, CNS involvement, or even death.If a pregnant woman exhibits symptoms similar to influenzas, such as headache, myalgia, fever, diarrhea, or any other digestive-related symptoms, the performance is practically identical to influenza (18).CONCLUSION The prevalence of L. monocytogenes in cases of spontaneous abortions among Iraqi aborted women was 32.4%.It seems that the hlyA gene has an essential role in the early and accurate identification of the bacteria in aborted women by using Q-PCR.Additional studies are documented to investigate the routine monitoring of pregnant women for L. monocytogenes to reduce the illness load and manage treatment.

Figure 2 .
Figure 2. Real-time PCR for detection hlyA gene of Listeria monocytogenes.(Quantitation Data (Green), Listeria monocytogenes, Green filter -FAM) The results of Q-PCR confirmed the previous results of HiCrome™ Listeria Agar and biochemical tests, where fifty-seven isolates from all positive women (n=59) showed the presence of the hlyA gene by Real-time PCR.Both molecular and traditional methods are effective in identifying L. monocytogenes, the molecular method remains the most accurate.For this small variation in results is attributed to the possibility of weak bacterial growth in these two samples, which resulted in DNA loss during the extraction process.The designed primers specifically amplify the L. monocytogenes hlyA gene, which encodes listeriolysin O and produces reproducible quantitative data over a large dynamic range of concentrations.The real-time PCR-hly assay demonstrated useful as a supplement to traditional bacteriological CSF culture by improving the speed and accuracy of diagnosing L. monocytogenes CNS infection.Furthermore, the quantitative results supplied may be valuable for the follow-up of patients undergoing treatment in specific cases(10).On the other hand,(12) revealed that specificity of molecular detection of hly gene for L. monocytogenes was 100%, in comparison with other Listeria isolates, and under certain settings, this PCR detection method, which is relatively sensitive and more specific, could be used as a quick diagnostic method from contaminated food.Following amplification, a melt curve analysis was performed to verify the specificity of the amplified products by their specific melting temperatures Tm(6).The protein with poreforming activity is the most important factor in vacuole lysis.The 58-kDa protein (Listeriolysin O) is encoded by the (hlyA) gene.The hlyA gene also has been targeted for

Table 1 . Primers of hlyA gene used in this study for species detection Listeria monocytogenes by Real-time PCR
1) as follows: