Detection and pathogenicity of Listeria monocytogenes in common carp (Cyprinus carpio) fish in Baghdad, Iraq

A few reports are available for detection of L. monocytogenes in fish in Iraq, however, the current study was undertaken to investigate the potential role of Listeria spp. in common carp fish in Baghdad province, Iraq. A total of fresh thirty raw common carp (Cyprinus carpio) were purchased from fish sellers of various local markets in Baghdad city from (December 2017 to March 2018) The viscera was removed aseptically, the bacterial isolation and identification was conducted by a conventional culture method using Listeria selective media, biochemical tests and Vitek 2 for gram-positive. Pathogenicity of isolates was studied in vivo by inoculating mice with bacterium. Targeting virulence associated genes was used to detect the virulence and to confirm the L. monocytogenes isolates. The isolates were tested for antimicrobial susceptibility by disk diffusion method for 12 antibiotics. The results revealed that 6.66% of L. monocytogenes were identified from common carp fish viscera and the isolates were pathogenic in mice. L. monocytogenes virulence associated genes were detected in both isolates, while L. innocua virulence associated gene (Lin0372) was detected in one of the two isolates. The isolates were resistant to 7 out of 12 antibacterial drugs including tetracycline, ampicillin, methicillin, cefixime, oxacillin, cefotaxime and penicillin G. The results suggest that presence of L. monocytogenes in fish may have a serious role in public health hygienic in humans.


Introduction
Listeria spp. are distributed in nature, soil, sewage, dust and water, and isolated from poultry, dairy products and domestic and wild animals. L. monocytogenes is considered emerged and an important foodborne pathogen causing severe diseases in humans especially in low immune system patients, including the pregnant women, elderly and newborns (1,2). Listeriosis infection has increased with increasing of ready-to-eat, heat-and-eat food product consumption. Also, Listeria spp. were isolated from food products and several studies have shown that the seafood such as fish and fish products could be contaminated with L. monocytogenes. Thus, it is considered as the important cause of a number of sporadic listeriosis caused by consumption of fish and its products (1)(2)(3)(4)(5)(6). Many virulence factors have been used to detect the pathogenicity of L. monocytogenes, these include listeriolysin O (hlyA), intenrnalins (inlA, inlB), phosphatidyl-inositolphospholipase C (PI-PLC, plcA), actin (actA), virulence regulator (prfA) and Iap (invasion associated protein) (7,8). L. monocytogenes infections in human are caused by serotypes 1/2a, 1/2b, 1/2c and 4b strains, and 4b serovar is responsible for all major outbreaks of invasive listeriosis (7,9). Differentiation of L. monocytogenes from other Listeria spp. may not be distinguished using conventional techniques such as selective and differential media and standard biochemical tests for differentiation of L. monocytogenes, L. innocua, L. ivanovii and L. seeligeri strains that show similar results to L. monocytogenes in these tests (10,11). It was found that 1.4% of L. monocytogenes isolates were misidentified as L. innocua using Vitek 2 (12). Therefore, Listeria spp. confirmation must be performed by molecular methods such as polymerase chain reaction (PCR) and sequencing (12,13).
Although L. monocytogenes has been reported sensitive to many antibiotics, on the other hand, it has been reported as multi-drug resistant in other studies (7,14). Accordingly, this study aimed to detect and pathogenicity of L. monocytogenes in fish in Baghdad city, Iraq.

Ethical approval
The study was carried out in the laboratory of Zoonotic Diseases unit, College of Veterinary Medicine, University of Baghdad, from Dec 2017-March 2018, and approved by the Animal Care and Use Committee (Approval No. 1788/25 Nov 2017).

Isolation of Listeria
A total of 30 fresh raw common carp (Cyprinus carpio) fish purchased from fish sellers of various local markets in Baghdad city from December 2017-March 2018were subjected to cultural and PCR methods to detect of L. monocytogenes. Isolation was carried out according to (15,16). By using 70% ethanol and povidone-iodine fish skins were cleaned and disinfected, after that fish was opened with a sterile scalpel blade and viscera was removed to a sterile mortar and pestle containing sterile normal saline and well ground. Five ml of suspension were inoculated into 15ml Listeria enrichment broth (LEB) (Himedia) and incubated at 37ºC for 24 hrs. Then a loopful from LEB was streaked on to PALCAM agar (Himedia and incubated at 37ºC for 24 hrs. Colonies surrounded by a black zone on PALCAM agar plates were picked and inoculated onto HiChrome Listeria agar (Himedia) then a bluish green colony were selected and stained with Grams' stain. Further confirmation was applied by Vitek 2 Grampositive identification card.

Pathogenicity in mice
The pathogenicity of Listeria isolates in mice was investigated according to Menudier and his co-worker method (17). Briefly, the isolates were grown on trypticase soya agar plate at 37°C for 24 hrs. Then, 3-5 colonies were picked and homogenized with sterile normal saline and the of bacterial suspension was adjusted to McFarland tube No. 0.5 that's equal to 10 8 CFU/ml . To each isolate, three Balb/c mice weighing 18-20g were injected intraperitoneally (I/p) with approximately 5×10 7 CFU in 0.5 ml. Control animals were injected with 0.5 ml (I/p) with normal saline. The animals were observed for 7days.

Molecular identification
The genomic DNA of the isolates was extracted according to the protocol of a commercial Wizard Genomic DNA purification Kit, Promega. PCR on 16S rRNA was applied using 27F primer (F-GAG TTT GAT CTT GGC TCA G) and 1492R primer R-TAC GGT TAC CTT GTT ACG ACT T) yielding about 1,300 bp (18).
The PCR reaction mixture volume of 25 µl included: (PCRpremix12.5µl, forward primer 1 µl, revers primer1 µl, nuclease free water 8.5 µl and finally DNA 2 µl). After that the amplification was done inside thermo-cycler (BioRad, USA) using the following program: initial denaturation at 95ºC for 5 min and1cyle; denaturation at 95ºC for 30 sec and 30 cycles; annealing at 60ºC for 45 Sec and 30 cycles and extension at 72ºC for 1 min and 30 cycles and final extension at 72ºC for 7 min and 1 cycle hold at 4 for 10 min and 1 cycle. To confirm the presence of the amplified DNA fragments, 1% agarose gel electrophoresis was performed. For sequencing, the PCR products were sent for Sanger sequencing using ABI3730XL, automated DNA sequencer by Macrogen Corporation, Korea. The similarity was applied using the Basic Local Alignment Search Tool (BLAST) in the National Center for Biotechnology Information (NCBI) for analyzing the results.

Isolation and identification
The results revealed the isolation of only two isolate of Listeria spp. 6.66%. On HiCrome Listeria agar, a bluish green colony were observed with ß hemolysis on blood agar. Gram positive rods, catalase positive, ferment lactose and L-rhamnose and showed CAMP test with Staphylococcus aureus giving 97% L. monocytogenes by Vitek 2.

Isolates pathogenicity in mice
The results of pathogenicity indicated that the two isolates were found to be pathogenic to mice, as all mice were dead within 72 hrs. L. monocytogenes was isolated from liver and spleen of dead animals, while control animals remained alive.

Molecular identification
The results obtained using 16S rRNA partial sequencing and analyzed using GenBank databases, showed that the first isolate had100% homology with L. monocytogenes. While the second isolate had 99.3% homology with the corresponding genes of L. innocua strain ATCC 33090 in (NCBI).

Targeting virulence associated genes
Targeting virulence associated genes were used to detect the virulence of the isolates and to confirm isolates of L. monocytogenes. The first L. monocytogenes isolate had L. monocytogenes virulence-associated genes and lacked L. innocua virulence-associated genes that confirmed it was L. monocytogenes. While second isolate also had L. monocytogenes virulence genes and lacked L. innocua virulence genes except harboring to Lin0372 gene as shown in (Figure 1), so this confirms that the second isolate was also L. monocytogenes.

Discussion
L. monocytogenes has been reported from food products in Iraq, including meat and meat products, milk and dairy products (20)(21)(22). In fish, few reports, particularly on the molecular level are available. Our results showed that the detection of L. monocytogenes in carp fish was 6.66%. Compared with other reports, Listeria spp. was isolated from 13% of frozen fish, and L. monocytogenes represented 5% in Basra city of Iraq (23). In Iran, L. monocytogenes isolate was represented 7.6% and 7.72% reported by Jamali et al and Momtaz and Yadollahi (4,24). A higher percentage reported 37.65% in fresh fish in Nigeria by Lennox et al. (25). L. monocytogenes is considered as an important human pathogen, and our results revealed that the fresh common carp contain L. monocytogenes in their viscera due to infection or normally found. Therefore, the present of L. monocytogenes in viscera of fish may lead to contaminate fish meat and then might be a source of foodborne pathogens during the processing procedure. According to culture, biochemical tests, ß hemolysis on blood agar, catalase positive, fermentation of both Lrhamnose and lactose and vitek 2, in addition to pathogenicity study in mice indicated that isolates were L. monocytogenes.
The results of 16S rRNA partial sequencing showed that the second isolate had a similarity of 99.3% with L. innocua ATCC 33090.After detecting the specific genes, this isolate had L. monocytogenes genes and lacked L. innocua genes Lin0454-lin0455 but had Lin0372 gene. High similarities of 99.5% and 99.9% exist between L. monocytegenes, L. innocua, L. ivanovii, L. seeligeri, and L. welshimeri. Genetic similarity is found between L. monocyto-genes serovar 4a strains and L. innocua that possesses L. innocuaspecific genes lin0372 and lin1073. In contrast, L. innocua ATCC 33090 has no mortality in the murine intraperitoneal infection (26). Thus, this isolate is not L. innocua ATCC 33090 but L. monocytogenes and may be serovar 4a. In addition, these isolates are pathogenic due to the presence of virulence genes and causing mortality in mice. Similar results have been reported, L. monocytogenes harboring virulence genes actA, hly, prfA and plcA and generally pathogenic in vivo model of infections (16,27,28).
Other studies reported L. monocytogenes sensitivity to ampicillin, chloramphenicol, rifampin, tetracycline, gentamicin, erythromycin, vancomycin, penicillin and imipenem (31,32). Ampicillin is commonly used in the treatment of Listeria infections, and a high antibiotics resistance of L. monocytogenes strain, with broad used of antibiotics, thus multidrug resistant L. monocytogenes have been isolated from various food samples. The presence of multidrug resistant L. monocytogenes will have a negative impact on the public health and safety of food (18). This study indicated the presence of L. monocytogenes in viscera of raw common carp fish. Therefore, for public health hygiene, viscera should be removed immediately from the fish after harvesting to prevent contamination other tissues.
Hygienic methods should be carried out during transportation and handling processing of fish and its products. In addition to conventional methods, it is necessary to use the virulence associated genes to distinguish between the Listeria spp. and to detect the pathogenic strains.

Conclusion
Our results suggest that L. monocytogenes isolated from fish have the potential role in public health hygienic in humans.