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

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

N.M. Al-Gburi

Zoonotic Diseases unit, College of Veterinary Medicine, University of Baghdad, Baghdad, Iraq, [email protected]

Abstract

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.

Keywords: L. monocytogenes, Lin0372, Antimicrobial susceptibility, PCR, Fish

DOI: 10.33899/ijvs.2019.125980.1205, © 2020, College of Veterinary Medicine, University of Mosul.

This is an open access article under the CC BY 4.0 license (http://creativecommons.org/licenses/by/4.0/).

کشف وإمراضیة L. monocytogenesفی اسماک الکارب الشائع (Cyprinus carpio)

فی مدینة بغداد، العراق

نغم محمد الجبوری

وحدة الأمراض المشترکة، کلیة الطب البیطری، جامعة بغداد، بغداد، العراق

الخلاصة

لا توجد دراسات وافیه عن وجود جراثیم L. monocytogenesفی الأسماک فی العراق، فقد أجریت الدراسة الحالیة للکشف عن جراثیم Listeria فی أسماک الکارب الشائع فی محافظة بغداد / العراق. تم شراء ثلاثین من اسماک الکارب الشائع Cyprinus carpio من بائعی الأسماک من مختلف الأسواق فی مدینة بغداد للفترة من کانون الأول 2017- أذار 2018. تمت إزالة الأحشاء بطریقة معقمة. اجری العزل الجرثومی باستخدام الوسائط الانتقائیة الخاصة بجراثیم Listeria والاختبارات الکیمیائیة الحیویة و Vitek 2 موجبة لصبغة کرام. تم إجراء فحص الإمراضیة بحقن العزلات فی الفئران، استخدام جینات ضراوة للکشف عن ضراوة وتأکید عزلة L. monocytogenes. تم اختبار عزلات L. monocytogenesلمعرفة مدى حساسیتها للمضادات الحیویة باستخدام اثنی عشر من المضادات الحیویة. کشفت النتائج عن عزل عزلتان من 6.66% L. monocytogenes من سمک الکارب الشائع وکانت العزلات ضاریة فی الفئران. وان کلا العزلتین یحملان جینات الضراوة الخاصة L. monocytogenesإلا أن واحده من العزلات کانت حامله للجین Lin0372 الخاص بال L. innocua. إضافة الى ذلک وجدت أن العزلات کانت مقاومة لسبعة من أصل أثنی عشر الأدویة المضادة للبکتیریا التألیه التتراسیکلین، الأمبیسلین، المیثیسیلین، السیفإکسیم، الأکسنسلین والسیفوتاکسین والبنسلین G. تشیر النتائج إلى أن وجود L. monocytogenesفی الأسماک لها دور على الصحة العامة.

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 (1-6). Many virulence factors have been used to detect the pathogenicity of L. monocytogenes, these include listeriolysin O (hlyA), intenrnalins (inlA, inlB), phosphatidyl-inositol-phospholipase 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.

Materials and methods

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 Gram-positive 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 10⁸ CFU/ml . To each isolate, three Balb/c mice weighing 18-20g were injected intraperitoneally (I/p) with approximately 5×10⁷ 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.

PCR targeting virulence genes

A listed of specific primers (Table 1) were used for the detection of virulence genes and to distinguish L. monocytogenes from L. innocua using PCR L. innocua associated genes (Lin0372, Lin0454-lin0455) and L. monocytogenes associated genes (prfA, actA, hlyA) were detected. The PCR reaction mixture containing a total volume of 20 µl, included: PCR (premix10 µl, 1 µl of each forward primer and reverse primers, nuclease free water 6 µl and DNA 2 µl).The amplification was transported to thermo-cycler (BioRad, USA) to start the Program: initial denaturation 2 min at 95 ºC, 1 cycles; denaturation for 30sec at 95 ºC, 35 cycles; annealing for 45sec at 57 ºC for Lin0372, Lin0454-lin0455 and prfA, and annealing of actA and hlyA 60 ºC for 45sec, 35 cycles; extension 1min at 72 ºC, 1cycle; final extension 7min at 72 ºC, 1cycle;hold at 10°C for 10 min,1cycle.After PCR amplifycation,1%agarosegel electrophoresis was adopted to detect the presence of amplification.

Table 1: Primers used in this study