MOLECULAR OF (YADA )VIRULANCE GENE IN YERSINIA ENTEROCOLOTICA ISOLATED FROM CHILDREN IN ABO-RISH HOSPITAL, EGYPT

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MOLECULAR OF (YADA )VIRULANCE GENE IN YERSINIA ENTEROCOLOTICA ISOLATED FROM CHILDREN IN ABO-RISH HOSPITAL, EGYPT

Tarek F.H.Alsaadawi¹;Omneia F. Hassan²; Mahmoud M.M. Zaky³ and Doaa M.A. Ghaith⁴

¹Faculty of Science, Al-Azhar University, Egypt

²Animal Health Research Institute, Giza, Egypt

³ Faculty of Science, Port-Said University, Egypt

⁴ Faculty of Medicine, Cairo University, Egypt

Key Words:-Yersinia enterocolotica , Biochemical methods , PCR ,Virulence gene.

ABSTRACT

The genus Yersinia belongs to the family Enterobacteriaceae and it constitutes facultative anaerobic, oxidase negative and catalase positive bacteria. Yersinia cells are Gram-negative straight rods or coccobacilli, carrying DNA with a guanine/cytosine content (the G+C content) of 46-50 mol %. Gastrointestinal disorders refer either to acute enterocolitis, particularly in children, or to pseudo appendicitis, in young adults In this study, pathogenic strains of Y. enterocolitica were identified by microbiological and PCR methods. The samples were collected from stool samples of children suffering from acute diarrhea. The techniques were used to isolate Y. enterocolitica from the samples PSB, and direct on the CIN, biochemical and PCR methods.PCR methods based on the identification of (YadA) virulence gene for detection, were used for the specific detection of the pathogenic strains of Y. enterocolitica. Traditional microbiological methods have been found to be insufficient for the specific identification of the Y. enterocoliticapathogen. Future research focusing on the identification using molecular methods or combining the two methods is necessary. The use of PCR has shown to be a sufficient standard of specific identification and is not time consuming.

INTRODUCTION

The genus Yersinia belongs to the family Enterobacteriaceae and it constitutes facultative anaerobic, oxidase negative and catalase positive bacteria. Yersinia cells are Gram-negative straight rods or coccobacilli, carrying DNA with a guanine/cytosine content (the G+C content) of 46-50 mol %.(Kämpfer 2000 andBottoneet al.,2015).  

Yersiniosis is a rare disease in Muslim countries due to the scarcity of pork consumption. The incidence of yersiniosis is reported to be 10%–30% in European countries and 0.06%–2% in Muslim ones (Sonnenwirthet al.,1990 and Grant et al., 1998).

Y. enterocolitica, a classical enteric pathogen, causes human and animals infections whose symptoms include diarrhea, terminal ileitis, intestinal intussusception,mesenteric lymphadenitis, arthritis, and septicemia (Imotoet al., 2012).

Pathogenic strains of Y. enterocoliticaare transmitted to man mainly from contaminated water or food (meat, milk or vegetables), more specifically raw or undercooked pork, and they may cause various infectious diseases (enteritis, enterocolitis, mesenteric lymphadenitis). (Schmidt andRodrick2003).

Gastrointestinal disorders refer either to acute enterocolitis, particularly in children, or to pseudoappendicitis, in young adults (Bottone, 1997).

Isolation of Y. enterocoliticais include enrichment followed by streaking on selective agar such as cefsulodin, irgasan and novobiocin medium (CIN) (Mehlmannet al., 1978andSchiemann, 1979).

Single colonies of bacterial strains were picked and further grown and sub-cultured several times to obtain a pure culture. Biochemical characterizations of the strains was performed using API 20E system  (Simmons et al., 2014).

PCR and DNA colony hybridization assays have been used to verify the pathogenicity of Y. enterocoliticaisolates rapidly and with high specificity (Bhaduri et al.,1997; Kapperud et al.,1990 and Wren et al.,1990). These methods are based on specific segments, such as yadAand virFgenes, of the virulence plasmid.

All pathogenic Y. enterocoliticastrains carry a 70-kb plasmid, the Yersinia virulence plasmid pYV (Gemskiet al.,1980).

Some virulence genes (i.e., yadA,virF, ysa, inv, ail, and yst) located in chromosome or plasmid of pathogenic Y. enterocoliticahas been widely used to identify pathogenic strains in epidemiological studies (Fredrikssonet al., 2006).

The yadAgene encodes the production of the YadA membrane protein, which functions as a protection of Y. enterocoliticacells against the activity of polymorphonuclear leukocytes (Ruckdeschelet al.,1996).

Until recently, pathogenic strains of Y. enterocoliticaused to be identified on the basis of the detection of the virulence plasmid pYV (70 kb). Localised on the plasmid are virulent genes yadAand virF (Cornelis 1998; Thoerneret al., 2003 and Platt- Samorajet al.,2006).

MATERIAL & METHODS

1-Patient:-

The study was conducted during the period from January / 2018 to January / 2019 on 300 patients, from Abu-Resh Children’s Hospital(Cairo- University), Egypt. The patients were suffering from recurrent Gastroenteritis included (diarrhea, vomiting, fever).

  A stool samples (300 samples) following infection control procedures were collected after taking the patient's consent. All patients are with the same age range (from 6 Month -14 years old).

2-3. Characterization of bacterial isolates:-

Y. enterocoliticacolonies are characterized by their unique bull’s-eye morphology on the CIN agar (1.5 mm diameter, deep red/purple center with a sharp edge surrounded by a translucent border). Therefore, only colonies with suspected morphologies were selected and tested by gram staining (Erkmen, et al.,1996 and Falcao et al.,2004).

The colonies were then grown on CIN agar, and the cells with a typical dark red center known as the “red bull’s eye”(Sinhaet al.,2000) were ransferred to blood agar and used for biochemical testing and PCR.

Identification of genus Y. enterocolitica:

2.4-BiochemicalIdentification :-

For confirmation of genus Yersinia Gram’s staining, motility test and various biochemical reactions like oxidase test, urease test, citrate test, acid and alkali reaction on KliglerIron agar (KIA) slants were performed (Cowan, 1975 andBercovier and Mollaret, 1984).

One bacterial colony was resuspended in 10 ml PBS (phosphate- buffered saline) and adjusted to McFarland standard 5.0. The API 20E strips were inoculated following the manufacturer’s instructions. Because of the temperaturedependentVoges–Proskauer test, the API 20E system was incubated for 18–20 h at 25_C (Bercovieret al., 1980). All isolates were Identified & Susceptibility using the database provided by the manufacturer the Vitike 2 compact system.(bioMerieux).

** Methods of genotypic identification:

Primers for identification of Yersinia enterocolitica.:

The PCR primers used in this study (Table 1) of Yersinia and plasmid of yersinia were prepared by sigma company.

Table ( 1 ):- Primer used for identification of plasmid (yadA).

 The first set of the primer was used for detection and identification of the isolated yersiniaenterocolitica and the other for plasmid.

Method for Extraction Plasmid:

1. Pick a single colony from a freshly streaked bacterial plate and use it to inoculate LB (+antibiotics). And then grow at 37 ̊C for 12 ~ 16 hrs with vigorous shaking (OD600 = 1.0 ~ 1.5).

2. Harvest 3 – 5 ml of bacteria culture by centrifugation at 13,000 rpm for 30 sec at RT and discard supernatant. Note: Drain tubes on a paper towel to remove excess media.

3. Resuspended pelleted bacterial cell thoroughly in 250 μl of resuspension buffer by vortexing until no clumps remain. Note : Ensure that RNase A solution has been added to resuspension buffer. It is essential to completely resuspended the cell pellet. It may affect the lysis efficiency

4. Add 250 μl of Lysis Buffer to resuspended cells and mix by inverting the tube 10 times. DO NOT VORTEX and incubate for 3 min at RT. Note : The optimal lysis time allows maximum release of plasmid DNA without release of chromosomal DNA, while minimizing the exposure of the plasmid to denaturing conditions. Long exposure to alkaline condition may cause the plasmid to become irreversibly denatured. It is important to proceed to next step immediately after the lysate becomes clear without any cloudy clumps. Do not vortex, it may cause shearing of genomic DNA. Note: If the Lysis buffer becomes too cold, SDS precipitation may occur, leading to poor cell lysis. If a precipitate has formed, warm the Lysis buffer to 37ºC with gentle shaking.

5. Add 350 μl of Neutralization Buffer and gently mix by inverting the tube 10 times then incubate the tube in ice for 5 min. Note : After addition of Neutralization Buffer, the solution should become cloudy and a fluffy white form. Incubation on ice may help precipitating the denatured cell components more efficiently. The precipitated material contains genomic DNA, protein, cell debris, and SDS. Note : If LysisViewer reagent has been used, the suspension should be mixed until all trace of pink has gone and the suspension is colorless. A homogeneous colorless suspension indicates that the SDS has been effectively precipitated.

6. Centrifuge at 13,000 rpm for 10 min at 4 ̊̊C . While waiting for the centrifugation, insert a column into collection tube.

7. After centrifugation, transfer supernatant promptly into the column. Note : Cell debris, protein and genomic DNA will form a compact white pellet in the tube. Do not transfer with white pellet.

8. Centrifuge at 13,000 rpm for 1 min. Remove the column from collection tube, discard filtrate in collection tube. And then place the spin column back in the same collection tube.

9. (Optional) Add 500 μl of Washing Buffer A and centrifuge at 13,000rpm for 1 min. Remove the column from collection tube, discard filtrate in collection tube. And then place the spin column back in the same collection tube. Note : This step is necessary to remove trace nuclease activity. endA+ strains, such as BL21, HB101, JM series, or any wild-type strains, have high level of nuclease activity that can degrade plasmids. But endA-strains, such as DH5α, XL1-blue and etc, do not require this additional washing step.

10. Add 700 μl of Washing Buffer B and centrifuge at 13,000rpm for 1 min., discard filtrate in collection tube and then place the spin column back in the same collection tube.

11. Centrifuge at., to dry the filter membrane.

12. Transfer the spin column to a new 1.5 ml or 2 ml microcentrifuge tube, and add 50 µl elution buffer or distill water to the upper reservoir of the column and let to stand for 1 min at room temperature. Centrifuge for 13,000rpm for 1 min.

DNA plasmid samples were tested in 50 µl reaction volumes in a 0.2 ml. eppendorf tube, containing 25 µl Fermentas Dream Tag Green PCR Master Mix, 1 µl of each primers, 3 µl target DNA or DNA plasmid, complete to a final volume of 50 µl with sterile deionizer PCR water.

Table ( 2 ): PCR protocol for amplification conditions of plasmid

10 μl of the amplified products were visualized by electrophoresis in a 2.0% gel of high strength analytical grade agarose (Bio-Rad, California USA) in 1×TBE.

Identification of the PCR products by agarose gel electrophoresis:

PCR products were analyzed for the presence of specific fragments of the expected length in a 1.5% agarose gel electrophoresis stained with Ethidium bromide.

1. Agarose gel preparation:

Powdered electrophoresis agarose was prepared of 1× electrophoresis buffer in a flask with a loose fitting cap to reach the required concentration (1.5%).The buffer should not occupy more than 50 % of the flask.

The mixture was heated in a microwave with periodical agitation until the agarose dissolves.

Solution was cooled to 60ºC, and then ethidium bromide was added from a stock solution of 1 ul /gel and mixed thoroughly.

2. Placement of combs and gel pouring:

The combs were placed 0.5-1.0 mm above the plate so that a complete well was formed when the agarose was added.

The gel mixture was then poured directly into movable casting apparatus. The gel thickness was 3-5 mm with no air bubbles under or between the teeth of the comb. After the gel was completely set (30-45 minutes at room temperature), the comb was carefully removed. Electrophoresis buffer was added to cover the gel to a depth of about one mm.

3. Loading:

The PCR product (5µl) and molecular weight maker were loaded. The PCR products were loaded into the slots of the submerged gel using micropipette.

4. Electrophoresis time and voltage:

The lid of the gel tank was closed and attached to the power supply. The running parameters were 1-5 volt/cm (measured as distances between electrodes). The gel was run until the bromothymol blue and xylene cyanol had migrated the appropriate distance through the gel (2/3 of the gel length).

5. Gel examination:

After the electric current was turned off, the lid of the gel was removed and the gel was transferred to trans-illuminator to observe the amplified DNA on the gel in comparison to molecular weight markers. The gel was photographed.

Finally we made sequencing to the PCR product on GATC Company by use ABI 3730xl DNA sequencer by using forward and reverse primers.

RESULTS

1-      Isolation of yersinaia:

The cold enrichment culture was done for all 300 patients studied. The total number of Yersinia isolates from the stool culture of all patients studied was 5 (1.66%); The 5 isolates of Y. enterocolitica were recovered from children 3 male 190 (63.33 % ) aged (1, 2, 5 ) years old , while the 2 female age 110 (36.66 % ) (1, 5 ) years old. ( Table3).

Table (3) Incidence of Yersinia spp. isolated from the stool culture of 300 patients :-

Table (4) Age & sex Distribution of 300 Patient of children with acute diarrhea.

2-      Cultural / colony characteristics:-

Typical dark red colonies surrounded by transparent border; usually flat with smooth border and entire edge giving a characteristic “Bulls-Eye” appearance, were observed after 72 hrs of incubation on CIN agar (Fig. A) in case of positive samples, whereas in MacConkey agar, the colonies were pale pink (Fig. B). Gram staining revealed gram negative coccobacillary morphology.

FigureA                           Figure B

Figure1 :-A   (colonies of Yersinia enterocolotica on CIN)

                             B    ( Yersiniaentercolotica on MacConkey Agar )

3-      Biochemical Test :-

The isolates recovered under the present study were positive Indole, Voges-Proskauer, Urea, M.R and were negative Citrate, Oxidase the isolates were confirmed as Y. enterocolitica(Table 3).And confirm the with API20E BY Vitek 2 Compact (bioMérieux).

Table 5. Biochemical properties of Y. enterocolitica

4-      API 20 E & Antibiotic Susceptibility 

Five bull’s-eye colonies were identified on CIN media and therefore were suspected to be Y. enterocolitica. Gram stain­ing showed that all suspected colonies are negative rods. Finally, API test had confirmed isolates as Y. enterocolitica by Vitek2 Compact system.(BioMérieux).

Molecular result:-

In our  we made sequencing to the PCR product on GATC Company by use ABI 3730×l DNA sequencer by using forward and reverse primers.

• To detect the gene (yadA,) using the PCR methods, specific sequences of genes were amplified and individual amplified fragments were detected by agarose gel electrophoresis  (Figure 1).

Fig( 2): electrophoretic pattern of PCR product of plasmid yersinia Lane 1: 100 bp plus DNA, Gene Ruler™, Fermentas, Lane 2: control positive , Lane3: control Negative , Lane 4: isolate 1, Lane 5: isolate 2.

DISCUSSION

Yersinia enterocoliticais a food-borne pathogen which can cause acute gastroenteritis and mesenteric lymphadenitis, which resembles appendicitis. Yersiniosis is thought to be the third most common intestinal zoonosis in the European Union, after campylobacteriosis and salmonellosis (Grahek-Ogden et al., 2007 and EFSA and ECDC, 2016).

This predominant bioserotype, however, has also been recovered from domestic animals, such as dogs and cats, suggesting that pets may also represent a source of human infection, especially for young children (Fredriksson-Ahomaaet al.,2006).

In this study yersiniaenterocolotica was isolate from 5 of our patient in who the other enteric bacterial pathogens mentioned were excluded  our findings clearly show that y.ent is present as a pathogen of diarrhea in this country and can be isolated from the stool samples of children suffering from acute diarrhea we found a frequency of (1.66%) for this organism which near the frequency of about (1 %) , (2.7 % )reported by other studies (Soltan-Dallal and Moezardalan,2004 andKanan and Abdulla,2009).

Y. enterocoliticais divided into 70 serotypes among which only few are pathogenic to humans (19). Harmful serotypes are divided into two groups: the American group (O:8, O:13a, O:13b, O:20, and O:21) and the European group (O:3, O:5,27, and O:9) (18). However, serotypes O:3, O:8, O:9, and O:5, 27 are the most common causes for yersiniosis worldwide (McNally et al., 2004 and Fabrega and Vila 2011).

This study was conducted throughout the year where the microbial activity was observed in the winter more than any other season and is absent in the summer based on the fact that this organism increases significantly compared to other species in the cold seasons and this is consistent with other studies (Cover and Aber 1993 and Naqvi,1993)

The clinical manifestations of infection in our children were mild as 5 cases of fever, vomiting and diarrhea were severe but we did not find bloody stools according to the Naqvi study. (Naqvi, 1993) .

The diagnosis of enteritis caused by Y. enterocolitica is mainly carried out by culture on laboratory plates. Cefsulodin–irgasan–novobiocin (CIN) agar is the media most frequently used and is based on Y. enterocolitica’s resistance to the abovementioned antibiotics (Schiemann, 1979).

Plates are incubated at 22–28°C for 48h, preferentially, or at 30°C for 24h. Y. enterocolitica colonies appear as a dark red “bull’s eye” with a translucent edge allowing easy identification. Corroboration is carried out by the specific biochemical properties of Y. enterocolitica such as motility at 25°C but not at 37°C, production of urease, lack of oxidase activity and lactose fermentation, as well as absence of either gas or hydrogen sulfide on Kligler’s iron agar. Alternatively, the API 20E system can also be used to identify the Y. enterocolitica (Bottone, 1997).

Additional methods to identify pathogenic Y. enterocolitica include both detection of the presence of virulence genes and tests to assess phenotypic characteristics associated with the production of plasmid-encoded virulence factors (Fredriksson and Korkeala, 2003; Lambertzet al.,2008).

Lambertz et al. (2008), showed that current methods for detection of the pathogenic Y. enterocolitica bacterium in food are time consuming and inefficient. Thus, they developed and evaluated an in-house TaqMan probe-based real-time PCR method for the detection of this pathogen. The complete method consists of overnight enrichment, DNA extraction, and real-time PCR amplification. In addition, the method was tested on naturally contaminated food. (Lambertzet al.,2008).

Virulence genes can be applied as distinguishing markers and indicators of the potential virulence of Y. enterocoliticastrains. Plasmid-encoded targets, like virF and yadA genes, and chromosomally encoded targets, like ail , inv, ystB, and HPIINT genes, have also been used in PCR to identify Y. enterocoliticastrains in clinical, food, and environmental samples (Bhaduriet al., 2005 andThisted and Danielsson, 2005).

YadA is responsible for other activities including mediating autoagglutination. The head domain possesses self-affinity and interacts in an antiparallel zipper-like manner (the more distal part of the protein interacts) causing bacterial flocculation (Hoiczyket al., 2000).

Both genes (yadA and virF) are contained on the virulent plasmid pYV, and the fact that the passaging of strains may lead to the loss of the plasmid may explain the less frequent detection of pathogenic strains of Y. enterocolitica. In their comparative study on the prevalence of the ail and yadAgenes in Y. enterocoliticastrains and non-pathogenic Yersinia spp. strains,(Blais&Phillippe, 1995).

In our study the samples were collected from stool samples of children suffering from acute diarrhea. The techniques were used to isolate Y. enterocolitica from the samples PSB, and direct on the CIN, biochemical and PCR methods.PCR methods based on the identification of (Yada) virulence gene for detection, were used for the specific detection of the pathogenic strains of Y. enterocolitica. Detect the gene (yadA,) using the PCR methods, specific sequences of genes were amplified and individual amplified fragments were detected by agarose gel electrophoresis  (Figure 2).

Finally we made sequencing to the PCR product on GATC Company by use ABI 3730xl DNA sequencer by using forward and reverse primers. Only by combining the traditional Sanger technology with the new 454 technology, can genomes now be sequenced and analyzed in half the usual project time, with a considerable reduction in the number of coatings and gaps. In addition, considerable cost advantages now make genome sequencing with the 454 technology accessible to the research community.

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جزیء فیروس (YADA) الجینی فی الیرسینیا المعویة المعزولة من الأطفال فی مستشفى أبو الریش ، مصر

طارق فاروق حنفی السعداوی¹، امنیة فکری حسن²، محمود محمد زکی³،

دعاء محمد عبدالعاطی غیاث⁴

1 کلیة العلوم جامعة الازهر مصر

2 معهد بحوث صحة الحیوان – مرکز البحوث الزراعیة مصر

3 کلیة العلوم جامعة بورسعید مصر

4 کلیة الطب جامعة القاهرة مصر

ینتمی جنس الیرسینیا لعائلة انتیروبکتیریاسی وهی تشکل بکتریا إیجابیة لاهوائیة ,وسالبة مع تفاعل الاوکسیدیز وایضا سالبة مع صبغة جرام. خلایا الیرسینیا عبارة عن قضبان مستقیمة سالبة الجرام أو عصیات عصویة ، تحمل الحمض النووی مع محتوى جوانین / سیتوزین (محتوى G + C) بنسبة 46-50 مول٪. تشیر اضطرابات الجهاز الهضمی إما إلى التهاب الأمعاء والقولون الحاد ، خاصة عند الأطفال ، أو إلى التهاب الامعاء الکاذب عند البالغین. فی هذه الدراسة، تم التعرف على سلالات مسببة للأمراض من الیرسینیا المعویة عن طریق الطرق المکروبیولوجیةوکذلک تقنیة تفاعل البلمرة المتسلسل. تم تجمع العینات من عینات براز من الأطفال الذین یعانون من الإسهال الحاد. تم عزل الیرسینیاالمعویةمن عینات البراز بواسطة الزراعة المباشرة على  CIN  میاسرة. والتعرف علیها بواسطة التفاعلات الکیمیائیة وتقنیة تفاعل البلمرة المتسلسل المبنیة على تحدید جین YadA من جینات عوامل الضراوة الموجودة فی الیرسینیا المعویة. بناءاً على ما سبق فان الطرق المکروبیولوجیة التقلیدیة غیر کافیه للتعرف على البکتیریا المعویة الممرضة. واخیرا قمنا بتصنیع تتابع البادئ بواسطة شرکة جاتا باستخدام تتابع ABI 3730xl DNA باستخدام بادئ أمامی وخلفی. فقط من خلال دمج تقنیة Sanger التقلیدیة مع تقنیة 454 الجدیدة ، یمکن الآن ترتیب تسلسل الجینوم وتحلیله فی نصف الوقت المعتاد، بالإضافة إلى ذلک ، فإن مزایا التکلفة الکبیرة تجعل الآن تسلسل الجینوم مع تقنیة 454 فی متناول جمیع الباحثین.