INTRODUCTION

Listeria monocytogenes is a Gram positive bacillus, facultative intracellular microbe with a unique life cycle that may enter, persist, grow in phagocytic and non-phagocytic cells, and disseminate across tissues and remain in host cells. It is a foodborne bacteria that cause listeriosis (Farber & Canada, 2015; Lamond & Freitag, 2018). It is transmitted via consumption of contaminated food such as vegetables, cheese, ice-cream, meat and dairy products (FDA, 2019a; CDC, 2016). L. monocytogenes is able to cause aggressive diseases in human with weak immunity (such as elderly, neonates, and pregnant-women) resulting in gastroenteritis, meningitis, septicemia, stillbirth, miscarriage, or serious contagions to baby with a 20%–60% mortality rate (Lamond & Freitag, 2018; Schlech III, 2019; Ireton et al., 2021). It is worth mentioning that antimicrobial resistance is a challenge in our community because of the high resistance rates among various bacteria (Assafi et al., 2015; Rasheed & Hussein, 2020 a &b; Hussein et al., 2019) including Listeria (Al-Brefkani & Mammani, 2019). Studying the virulence genes in pathogenic bacteria can give a better insight about the pathogenicity of bacteria and the severity of infections caused by microorganisms (Hussein et al., 2008; Rasheed & Hussein, 2020). Thus, the virulency of L. monocytogenes is mostly controlled by some virulence related genes that are associated with severe human infections (Kose & Yakupogullari, 2015). Such genes play an sentential role in the pathogenicity and evasion from hosts immune responses because they involve in the essential phases of pathogenicity such as adhesion, invasion, multiplication, and distribute intracellularly (Campuzano-Maya, 2014). The most common virulence-associated genes of L. monocytogenes are internalins that are encoded by (inlA, inIB, inlC and inlJ genes), listeriolysin which is encoded by hlyA gene, actin encoded by actA gene and the invasion associated protein encoded by iap gene (Ward et al., 2004). The expression of these virulent associated factors are involved in the pathogenesis of L. monocytogenes, which is operated and coincide by the regulatory prfA gene (Orsi & Wiedmann, 2016; Vasanthakrishnan et al., 2015). It has been suggested that the shift of L. monocytogenes from a non-pathogen to sever pathogenic bacteria was related to the prevalence of virulence associated genes (Tan et al., 2015; Schlech III, 2019). Therefore, this study was aimed to investigate the presence of five crucial L. monocytogenes virulence genes in human and food product samples by using multiplex Polymerase Chain Reaction (PCR).

MATERIALS AND METHODS

Study isolates

A total of 48 frozen L. monocytogenes isolates that were obtained by Al- Brefkani and Mammani (Al-Brefkani & Mammani, 2019a) were used that were isolated from various foods (41 isolates) and human clinical samples (7 isolates) from June 2021 to July 2022 in Duhok Province, Iraq. Human samples included blood (3), high vaginal swabs (HVS) (3) and urine (1), while, foods were from meat products; frozen chicken meat (27), fresh red meat (10), and dairy products; raw milk (3), white soft cheese (1). Blood samples were collected from septicemia and meningitis cases at Duhok Kidney and Diseases Transplantation Center and Hevi Pediatric Teaching Hospital, while, the urine and HSV samples were collected from women with poor obstetric history such as miscarriage and stillbirth attended the Duhok Obstetrics and Gynecology Teaching Hospital. Meat and dairy products were obtained from the Food Testing Laboratory of the Directorate of Prevention Affairs in Duhok.

Laboratory procedures

All isolates were microbiologically detected by using Gram staining, catalase test, haemolysis test on sheep blood agar and Harlequin™ Listeria Chromogenic Agar (ISO) subculture (Neogen®Company-UK) that was used to discriminate L. monocytogenes from other Listeria spp (Al-Brefkani & Mammani, 2019a). Microgen Listeria-ID microwell test strip (Microgen-UK) was used for biochemical confirmation which includes eleven desiccated carbohydrate components (Dominguez Rodriguez et al., 1986). All phenotypically identified isolates were confirmed by PCR technique using species specific 16s rRNA primers (Macrogen, South Korea). The DNA extraction and PCR conduction were performed according to Al-Brefkani and Mammani (Al-Brefkani & Mammani, 2019a). The molecular work was conducted at Duhok Research Center (DRC) at College of Veterinary Medicine, University of Duhok, Duhok, Iraq.

Multiplex PCR for the identification of virulence-associated genes

For amplification of inIA, inIB, inIC, inIJ and prfA genes, specific primers were used as shown in Table 1 (Klein & Juneja, 1997; Liu et al., 2007) with minor modifications using thermocycler machine (Applied Biosystems 9700, USA). The final volume of master mix reaction was 25 with following contents: 12.5 µL of ready-to-use Prime Taq Premix (GeNet Bio- South Korea), 2 µL of DNA (10 ng) template from each L. monocytogenes isolate and 1.0 µL of each primer (GeNet Bio- South Korea), (stock concentration, 10 µM), together with 5.5 µL of ddH₂O (Ambion-USA) was prepared for each reaction. For L. monocytogenes internalin (InIA, InIB, InIC and InIJ) genes, the cyclic program was adjusted as following: initial heat denaturation at 94˚C for 2 min, 30 cycles of denaturation at 94˚C for 20 sec, annealing at 55°C for 20 sec, and extension at 72°C for 50 sec. The final extension set upped at 72˚C for 2 min, then incubated at 4°C (Liu et al., 2007). The PCR settings were made up for prfA gene as follows: initial denaturation at 95°C for 4 min, 35 cycles at denaturation at 94°C for 30 sec, annealing at 63°C for 60 sec and extension at 72°C for 60 sec. Final extension was at 72°C for 5 min (Klein & Juneja, 1997). The gel electrophoresis conditions were conducted following the instructions defined by Liu et al (Liu et al., 2007). The DNA ladder (100 bp) (Promega) was used as a marker for measuring the amplicon size of amplified genes. A previously sequenced L. monocytogenes strain (access number: MK968366) was used as a positive control (Al-Brefkani & Mammani, 2019b).

Table 1: Oligonucleotide primers sequences

		Virulence associated genes
Target gene	Primer	Primer sequence (5' →3')	bp	Reference
prf A	ELMPRFF	5′-CGGGATAAAACCAAAACAATTT-3′	508	(Klein & Juneja, 1997)
	ELMPRFR	5′-TGAGCTATGTGCGATGCCACTT-3′
inlA	Internalin AF	5'-ACTATCTAGTAACACGATTAGTGA-3'	250	(Hudson et al., 2001)
	Internalin AR	5'-CAAATTTGTTAAAATCCCAAG TGG-3'
inlB	Internalin BF	5'-AAGCACAACCCAAGAAGGAA-3'	1107	(Johnson et al., 2004)
	Internalin BR	5'-AAAATTCCACTCATGCCCAC-3'
InlC	Internalin CF	5'-AATTCCCACAGGACACAACC-3'	517	(Liu et al., 2007)
	Internalin CR	5'-CGGGAATGCAATTTTTCACTA-3'
inlJ	Internalin JF	5'-TGTAACCCCGCTTACACAGTT-3'	238	(Liu et al., 2007)
	Internalin JR	5'-AGCGGCTTGGCAGTCTAATA-3'

Table 2: Relationship between Virulence factors and various sample sources.

Sample Source	Positivity of virulence associated genes (no=48)
Sample Source	prfA no. (%)	InIA no. (%)	InIB no. (%)	InIC no. (%)	InIJ no. (%)	Total no.
HVS	2(66.7%)	2(66.7%)	2(66.7%)	3(100%)	3(100%)	3
Blood	3(100%)	2(66.7%)	3(100%)	3(100%)	3(100%)	3
Urine	0(00.0%)	1(100%)	1(100%)	1(100%)	1(100%)	1
Frozen chicken meat	22(81.5%)	22(81.5%)	26(96.3%)	24(88.9%)	22(81.5%)	27
Fresh red meat	10(100%)	8(80%)	9(90%)	10(100%)	8(80%)	10
White soft cheese	1(100%)	1(100%)	1(100%)	1(100%)	1(100%)	1
Raw milk	1(33/3%	2(66.7%)	1(100%)	3(100%)	2(66.7%)	3
Total	39(81.2%)	38(79.2%)	44(91.7%)	45(93.8%)	40(83.3%)	48
Chi-Square	12.505	1.471	6.158	2.489	2.347
P value	0.052	0.961	0.406	0.870	0.885

DISCUSSION

The high virulence of L. monocytogenes lies in its ability to penetrate intestinal wall, placenta and blood–brain barriers to cause severe CNS infections (Disson & Lecuit, 2013; Vimentin et al., 2018; Schlech III, 2019). Furthermore, the capability of L. monocytogenes to live in harsh circumstances such as growing in up to 45 °C, resistance to cold, resistance to various disinfectants and could stay at surfaces for long time, increases its ability to cause infections (CDC, 2016). Listeria is considered one of the bacteria of interest for the health sector due to the severity of its infections and its complications among infected individuals especially individuals with weak immunity (Lamond & Freitag, 2018; Schlech III, 2019; Ireton et al., 2021). The severity of these infections is controlled by various virulence genes. Several surface related proteins of the family “internalins” (InIA, InIB and InIC) of L. monocytogenes bacterium interact with specific host receptors to facilitate entry and cross human cells (Ireton et al., 2021). The present study investigated the presence of some of L. monocytogenes virulence genes (InIA, InIB, InIC, InIJ and prfA), that enhance the bacteria to transit between human cells and increase its pathogenicity.

The regulatory prfA gene is considered essential in pathogenicity as it controls and regulates the expression of some virulence genes and it is thought hypothetically that its absence lead the bacteria to lose its ability to cause infections (Osman et al., 2019). Our results revealed that 81.2% of L. monocytogenes isolates carried prfA gene. In consistent with the present study, the prfA gene was detected in 77.7% of L. monocytogenes isolates from different clinical and food samples in Egypt (Osman et al., 2019). On the other hand, the prfA gene was not found in any of the tested isolates in a study conducted in Egypt (Abdeen et al., 2021). However, in Northern region of Iran was found that prfA gene was in almost all of the investigated isolates (Jamali et al., 2015). This diversity in results need further studies for more comprehensive explanation.

Furthermore, the total prevalence of InIA, InIB, InIC and InIJ genes were 79.2%, 91.7%, 93.8% and 83.3%, respectively, in both human and food product samples. In contrast, the prevalence rate of the internalins genes in Egypt were somehow less than that of our finding rates that revealed the following results, inIA (74.1%), inIB (81.5), inIC (70.4%), and InIJ (66.7%) (Osman et al., 2019). In disagreement with our results, the inIA gene was not found in any of the tested isolates in a study performed in Egypt (Abdeen et al., 2021). However, in a study conducted in Iran that found the rates of inIA, inIB, inIC, and inIJ genes were almost in all of the investigated isolates (Jamali et al., 2015). The differences in the virulence genes rate might be due to the differences in the sample type or the presence of different strains in different regions.

Various studies showed that the presence of internalins (InIA, InIB, InIC, and InIJ) genes could raise the mortality rate as these genes have a significant potential to initiate human infections (Liu et al., 2007; Ireton et al., 2021). The incidence of multiple virulence factors has been reported in this study in which all tested isolates (100%) showed the presence of multi-virulence genes. Three virulence factors were noticed in 20.8%, four genes were detected in 33.3% and all five tested virulence genes were identified in 45.8% of L. monocytogenes isolates. In agreement with our results, Tahoun et al. (2017) reported that 60.8% L. monocytogenes isolates showed the presence of multiple virulence associated genes (Tahoun et al., 2017). The occurrence of multiple virulence genes in single L. monocytogenes isolate is also supported by several recent studies (Du et al., 2017; Kaur et al., 2018; El-Demerdash & Raslan, 2019). The presence of multiple virulent genes in the same isolate will increase its pathogenicity and its ability to cause life threating infections. Thus, further studies are needed to investigate the virulogentic profile by recruiting a bigger sample size and following up the outcomes of these infections.

In conclusion, this study showed the prevalence of major virulence-associated genes in tested L. monocyogenes isolates. It also showed the occurrence of multiple virulence related genes in single isolates. Therefore, infection with such a microorganism may cause life-threatening infections. Additionally, application of multiplex PCR for detection virulence genes provides rapid and immediate confirmation of L. monocytogenes virulence-associated genes. The association between those genes and clinical outcomes has to be investigated further.

REFERENCES

Abdeen, E. E., Mousa, W. S., Harb, O. H., Fath-Elbab, G. A., Nooruzzaman, M., Gaber, A., Alsanie, W. F., & Abdeen, A. (2021). Prevalence, antibiogram and genetic characterization of listeria monocytogenes from food products in Egypt. Foods, 10(6), 1–13. https://doi.org/10.3390/foods10061381

Al-Brefkani, A. M. T., & Mammani, I. M. A. (2019a). SEASONAL CHANGES IN THE OCCURRENCE OF LISTERIA MONOCYTOGENES IN DUHOK PROVINCE. 7(1), 5–9. https://doi.org/10.25271/sjuoz.2019.7.1.549

Al-Brefkani, A. M. T., & Mammani, I. M. A. (2019b). Characterisation of Listeria monocytogenes from Food and Human Clinical Samples at Duhok, Kurdistan Region of Iraq. Journal of Pure and Applied Microbiology, 13(4), 2215–2226. https://doi.org/10.22207/JPAM.13.4.35

Assafi, M. S. A., Ibrahim, N. M. R., Hussein, N. R., Taha, A. A., & Balatay, A. A. (2015). Urinary Bacterial Profile and Antibiotic Susceptibility Pattern among Patients with Urinary Tract Infection in Duhok City, Kurdistan Region, Iraq. International Journal of Pure & Applied Sciences & Technology, 30(2), 54–63.

Campuzano-Maya, G. (2014). Hematologic manifestations of Helicobacter pylori infection. World Journal of Gastroenterology: WJG, 20(36), 12818.

CDC. (2016). People at risk. Retrieved August,8, 2022, from

https://www.cdc.gov/Listeria/risk.html#:~:text=Listeria%20is%20most%20likely%20 to,they%20rarely%20become%20seriously%20ill.

Disson, O., & Lecuit, M. (2013). Invitro and invivo models to study human listeriosis: Mind the gap. Microbes and Infection, 15(14–15), 971–980. https://doi.org/10.1016/j.micinf.2013.09.012

Dominguez Rodriguez, L., Vazquez Boland, J. A., Fernandez Garayzabal, J. F., Echalecu Tranchant, P., Gomez-Lucia, E., Rodriguez Ferri, E. F., & Suarez Fernandez, G. (1986). Microplate technique to determine hemolytic activity for routine typing of Listeria strains. Journal of Clinical Microbiology, 24(1), 99–103.

Du, X., Zhang, X., Wang, X., Su, Y., Li, P., & Wang, S. (2017). Isolation and characterization of Listeria monocytogenes in Chinese food obtained from the central area of China. Food Control, 74, 9–16.

El-Demerdash, A. S., & Raslan, M. T. (2019). Molecular characterization of listeria monocytogenes isolated from different animal-origin food items from urban and rural areas. Advances in Animal and Veterinary Sciences, 7(Special Issue 2), 51–56. https://doi.org/10.17582/journal.aavs/2019/7.s2.51.56

Farber, J. M., & Canada, H. (2015). Listeria monocytogenes , a Food-Borne Pathogen. October 1991.

FDA. (2019a). Listeria (Listeriosis). Retrieved August 10, 2022 from” https://www.fda.gov/food/foodborne-pathogens/listeria-listeriosis

Hudson, J. A., Lake, R. J., Savill, M. G., Scholes, P., & McCormick, R. E. (2001). Rapid detection of Listeria monocytogenes in ham samples using immunomagnetic separation followed by polymerase chain reaction. Journal of Applied Microbiology, 90(4), 614–621.

Hussein, N. R., Mohammadi, M., Talebkhan, Y., Doraghi, M., Letley, D. P., Muhammad, M. K. & Atherton, J. C. (2008). Differences in virulence markers between Helicobacter pylori strains from Iraq and those from Iran: potential importance of regional differences in H. pylori-associated disease. Journal of clinical microbiology, 46(5), 1774-1779.

Hussein, N., Salih, R. S., & Rasheed, N. A. (2019). Prevalence of methicillin-resistant Staphylococcus aureus in hospitals and community in Duhok, Kurdistan region of Iraq. International Journal of Infection, 6(2).

Ireton, K., Mortuza, R., Gyanwali, G. C., Gianfelice, A., & Hussain, M. (2021). Role of internalin proteins in the pathogenesis of Listeria monocytogenes. Molecular Microbiology, 116(6), 1407–1419. https://doi.org/10.1111/mmi.14836

Jamali, H., Paydar, M., Ismail, S., Looi, C. Y., Wong, W. F., Radmehr, B., & Abedini, A. (2015). Prevalence, antimicrobial susceptibility and virulotyping of Listeria species and Listeria monocytogenes isolated from open-air fish markets. BMC Microbiology, 15(1), 1–7. https://doi.org/10.1186/s12866-015-0476-7

Johnson, J., Jinneman, K., Stelma, G., Smith, B. G., Lye, D., Messer, J., Ulaszek, J., Evsen, L., Gendel, S., & Bennett, R. W. (2004). Natural atypical Listeria innocua strains with Listeria monocytogenes pathogenicity island 1 genes. Applied and Environmental Microbiology, 70(7), 4256–4266.

Kaur, S., Singh, R., Sran, M. K., & Gill, J. P. S. (2018). Molecular characterization of Listeria monocytogenes in white meat samples from Punjab, India. Indian Journal of Animal Research, 52(11), 1635–1641. https://doi.org/10.18805/ijar.B-3414

Klein, P. G., & Juneja, V. K. (1997). Sensitive detection of viable Listeria monocytogenes by reverse transcription-PCR. Applied and Environmental Microbiology, 63(11), 4441–4448.

Kose, A., & Yakupogullari, Y. (2015). A Rapidly Fatal Sepsis Caused by Listeria Monocytogenes Type-4b in A Patient with Chronic Renal Failure. Jundishapur Journal of Microbiology, 8(3).

Lamond, N. M., & Freitag, N. E. (2018). Vertical transmission of listeria monocytogenes: Probing the balance between protection from pathogens and fetal tolerance. Pathogens, 7(2). https://doi.org/10.3390/pathogens7020052

Liu, D., Lawrence, M. L., Austin, F. W., & Ainsworth, A. J. (2007). A multiplex PCR for species- and virulence-specific determination of Listeria monocytogenes. Journal of Microbiological Methods, 71(2), 133–140. https://doi.org/10.1016/j.mimet.2007.08.007

Orsi, R. H., & Wiedmann, M. (2016). Characteristics and distribution of Listeria spp., including Listeria species newly described since 2009. Applied Microbiology and Biotechnology, 100(12), 5273–5287.

Osman, K. M., Kappell, A. D., Fox, E. M., Orabi, A., & Samir, A. (2019). Resistance , and Phylogenetic Analysis of Biofilm-Producing Listeria monocytogenes Isolated from Di ff erent Ecological Niches in Egypt : Food , Humans , Animals , and Environment. Pathogens, 9(5).

Rasheed, N. A., & Hussein, N. R. (2020a). Characterization of different virulent factors in methicillin-resistant Staphylococcus aureus isolates recovered from Iraqis and Syrian refugees in Duhok city, Iraq. PloS One, 15(8), e0237714.

Rasheed, N., & Hussein, N. R. (2020b). The nasal carriage of Staphylococcus aureus and its antimicrobial susceptibility pattern in secondary school students in Kurdistan region, Iraq. Journal of Kermanshah University of Medical Sciences, 24(1).

Schlech III, W. F. (2019). Epidemiology and clinical manifestations of Listeria monocytogenes infection. Microbiology Spectrum, 7(3), 3–7.

Tahoun, A. B. M. B., Abou Elez, R. M. M., Abdelfatah, E. N., Elsohaby, I., El-Gedawy, A. A., & Elmoslemany, A. M. (2017). Listeria monocytogenes in raw milk, milking equipment and dairy workers: Molecular characterization and antimicrobial resistance patterns. Journal of Global Antimicrobial Resistance, 10(April 2019), 264–270. https://doi.org/10.1016/j.jgar.2017.07.008

Tan, M. F., Siow, C. C., Dutta, A., Mutha, N. V. R., Wee, W. Y., Heydari, H., Tan, S. Y., Ang, M. Y., Wong, G. J., & Choo, S. W. (2015). Development of ListeriaBase and comparative analysis of Listeria monocytogenes. BMC Genomics, 16(1), 1–19.

Vasanthakrishnan, R. B., de las Heras, A., Scortti, M., Deshayes, C., Colegrave, N., & Vázquez-Boland, J. A. (2015). PrfA regulation offsets the cost of Listeria virulence outside the host. Environmental Microbiology, 17(11), 4566–4579. https://doi.org/10.1111/1462-2920.12980

Vimentin, H. C., Ghosh, P., Halvorsen, E. M., Ammendolia, D. A., Mor-vaknin, N., & Riordan, M. X. D. O. (2018). Invasion of the Brain by Listeria monocytogenes Is Mediated by InIF and host cell vimentin. MBio, 9(1), 1–11.

Ward, T. J., Gorski, L., Borucki, M. K., Mandrell, R. E., Hutchins, J., & Pupedis, K. (2004). Intraspecific phylogeny and lineage group identification based on the prfA virulence gene cluster of Listeria monocytogenes. Journal of Bacteriology, 186(15), 4994–5002. https://doi.org/10.1128/JB.186.15.4994-5002.2004