BENHA VETERINARY MEDICAL JOURNAL, VOL. 34, NO.1:94 -105 , MARCH, 2018

BENHA UNIVERSITY FACULTY OF VETERINARY MEDICINE Phenotypic and genotypic characterization of species isolated from chicken and milk products ¹Ashraf,A.Abd El Tawab, ²Wafaa, M.M Hassan,³Fatma, I.El- Hofy, 4Haidy,T.Zaki

¹Bacteriology,Immunology and Mycology Dep., Fac.Vet.Med.Benha Univ.²Animal Health Research

ABSTRACT

Listeria monocytogenes has became increasingly important as a food-associated pathogen. It can cause a rare but serious disease called listeriosis with high fatality rates (20–30%) compared with

other foodborne microbial pathogens. To estimate the incidence and levels of Listeria spp. in different food sources, A total of 200 random samples which collected from different sources 65 samples isolated from poultry , 45 from poultry byproducts (Frozen chicken meat balls (kofta), Frozen chicken burger and frozen chicken sausages), 20 from pasteurized milk ,30 from milk

products (Feta cheese ,cream cheese )and 40 from poultry eggs .Only 7(14%) of samples that collected from suspected isolates and detected on Oxford agar, PALCAM agar and ALOA agar were suspected to be Listeria spp. In poultry, poultry byproducts, pasteurized milk, milk byproducts and poultry eggs, listeria percentage were 0/65(0%), 4/45(1.8), 0/20(0%), 0/30(0%) ,3/40(1.2%) isolates, respectively. The in-vitro antimicrobial sensitivity test showed that the isolated L.monocytogenes were sensitive to Sulphamethoxazole /trimethoprim, Gentamycin ,Ceftazidime and Cephazoline followed by Sulphamethoxazole, ceftriaxone , weak sensitivity to Penicillin . While they were resistant to Erythromycin, Clindamycin, Ampicillin. The results of virulence tests for isolated listeria strains appeared that all of L.monocytogenes were virulent strains as all of them were positive to CAMP test; showed narrow zone of β–hemolysis on sheep blood agar .The PCR results for Listeria isolates showed that all strains are L.monoctogenes and have Inl A and hly A genes positive as virulence genes while negative Inl B, iap and prf A genes. Keywords: L.monocytogenes, poultry, milk. (http://www.bvmj.bu.edu.eg) (bvmj, 34(1): 94 -105 , MARCH, 2018

1. INTRODUCTION Veterinarians, medical doctors and people is a Gram- involved in food science know listeriosis by positive rod-shaped non-capsulated bacterium various names (circling disease, that form single short chains (Theivagt et al., meningioencephalitis disease ,cheese 2006),facultative anaerobic, non-spore- sickness) but few know who Gustav Hülphers forming, rod-shaped 0.5 mm in width was because he did not preserve his bacterial and 1–1.5 mm in length (Vazquez-Boland et strains, which he named bacillus hepatis, later al., 2001 and Vera et al., 2013). recognized as Listeria monocytogenes Taxonomically, it is divided into six (McLauchlin, 2004& Hülphers, 2004). species (i.e. Listeria monocytogenes, Listeria ivanovii, Listeria seeligeri, Listeria innocua, 94

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Listeria welshimeri and Listeria grayi), of polymerizing actin to form long tails ,the which only L. monocytogenes and L. ivanovii actin-assembly-inducing protein (ActA) are pathogenic. While L. monocytogenes which propels bacteria through the cell and infects both man and animals, L. ivanovii is into neighbouring cells, (Todar ,2008) principally an animal pathogen that rarely Each step requires expression of occurs in man. (Robinson et al., 2000). specific virulence factors. The major The organism can survive at varying virulence genes are clustered together on the temperatures ranging from 4 to 37C chromosomes and regulated by the positive (Janakiraman, 2008). The bacterium can regulatory factor A protein.PrfA tolerate a wide range of pH and temperatures. (Positive Regulatory Factor A (PrfA). (Scortti Optimum growth occurs at 30-37C but the et al.,2007 and Freitag et al.,2009). organism can multiply at4-45C. A typical There is interplay between temperature tumbling motility is observed around25C. It and L.monocytogenes motility. L. can grow at pH 4.5-9.6, although the growth monocytogenes can employ either multi- is minimal at low pH and low temperatures flagellar based motility or actin based (OIE, 2014). propulsion depending on its environment. Listeriosis is often noticed along with When L. Monocytogenes lives outside a host other poultry diseases such as coccidiosis (<30 °C), it is flagellated and utilizes these ,infectious coryza, verotoxic E.Coli flagella for movement. When the bacteria ,salmonellosis, campylobacteriosis and encounters higher temperatures, like those in a parasitic infections ,signifying the host, the flagellar expression is halted and the opportunistic nature of the organism (Adzitey bacteria are non-flagellated (Williams et al., et al.,2012) 2005). Egg and egg products have never been The present study was conducted to estimate caused listeriosis but is most frequently the prevalence of listeria species in poultry isolated from egg shells and in the and milk products with special interest to environment of laying hens.(Chemaly et al., L.monocytogenes. In addition to clarify the 2008) L. monocytogenes can Survive 90 days virulence of isolated strains and to carry out on stored egg at 5C and for 15 days at 10C the antibiotics sensitivity testing of them . In (Gandhi and chikindas, 2007 ). addition ,detection of some virulence factors Briefly, Invasiveness by virulence of L.monocytogenes by PCR technique. factors, L.monocytogenes expresses cell- surface and secreted proteins that enable 2. MATERIAL AND METHODS attachment to host cells, escape from the 2.1. Samples collection: phagocytic vacuole by Internalin A (inIA) and Two hundred random samples which Internalin B (inIB) mediate the attachment of collected from different sources 65samples L. monocytogenes to the surface of host cells. isolated from poultry , 45 from poultry Once ingested the bacterium produces byproducts (Frozen chicken meat balls listeriolysin (LLO) to escape from the (kofta),Frozen chicken burger and frozen phagosome and lyses the phagosomal chicken sausages),20 from pasteurized milk membrane,The bacterium then multiply ,30 from milk products (Feta cheese ,cream rapidly in the cytoplasm and moves through cheese) and 40 from poultry eggs. The the cytoplasm to invade adjacent cells by samples were collected in sterile plastic bags, 95

Phenotypic and genotypic characterization of listeria species isolated from chicken and milk products kept in ice box and transferred with a 24-48hours at 35C ,enhanced zone of β- minimum delay to the laboratory for studying hemolysis considered as a positive reaction. the presence of listeria species. 2.5. Genotypic detection of isolated 2.2.Bacteriological examination: L.monocytogenes and some virulence in them A-Primary stage: One ml of sample using polymerase chain reaction (PCR) was inocluted into 9 ml fraser broth 1,half PCR using six sets of primers was used for fraser broth (without supplement) and genotypic detection of L.monocytogenes incubated aerobically at 30±1c for 24±3 hours strains and five virulence genes of .B-Secondary stage : one ml of incubated L.monocytogenes. The virulence genes were broth was inoculated into 9ml fraser broth 2 , 16SRNA;internalin A (inlA); internalin B full strength fraser broth (with supplement) (inlB); heamolysin(hlyA) ;invasion associated and incubated at 37 C for 48±3 hours .C- protein (iap) and positive regulatory factor A Third stage :0.1ml of incubated fraser broth (prfA). was streaked onto the following media : It was applied on seven isolated ALOA agar; PALCAM agar and Oxford agar L.monocytogenes following QIAamp® DNA plates then the plates were incubated at Mini Kit instructions (Catalogue no. 37±1Cfor 48 hours and examined after 24±3 51304),Emerald Amp GT PCR master mix hours. The listeria like colonies were picked (Takara) Code No. RR310A and Agarose 1% and streaked onto Tryptic Soy agar ( Bio-life (Sambrook et al., 1989).The PCR condition ) with 0.6% yeast extract (TSA,YA) then, have specific sequence and amplify specific incubated at 35C for 48 hours. The isolates products as shown in Table (2). Temperature were morphologically identified by Gram and time condition of the primers during PCR stain and biochemical tests according to are shown in table (3) according to specific (Markey et al.,2013) authors and Emerald Amp GT PCR master 2.3. In –Vitro anti-microbial sensitivity tests : mix (Takara) kit. The isolated L.monocytogenes strains 2. RESULTS were subjected to the sensitivity test against Of the total 200 different samples different antibiotics, using the disc and agar analyzed,L. monocytogenes was detected in 7 diffusion method: antibiotic discs are samples with prevalence of 14% ; represented Erythromycin(E), Penicillin (P), as in poultry, poultry byproducts, pasteurized Sulphamethoxazole (SM), ceftriaxone (CRO), milk, milk byproducts and poultry eggs, Gentamycin (CN), Clindamycin (DA), listeria percentage were 0/65(0%), 4/45(1.8), Ampicillin (AM), Cefazoline (KZ), 0/20(0%), 0/30(0%) ,3/40(1.2%) isolates, Sulphamethoxazole/ trimethoprime (SXT), respectively. as Table (1) The isolated ceftazidime (CAZ). (Finegold and Martin, colonies grow well on ALOA agar producing 1982 and NCClS, 1999) blue-green colonies surrounded by opaque 2.4. Virulence tests : halo and on PALCAM agar gives grey-green Hemolytic activity: all isolates were with black center and black halo against cultured on 5% sheep blood agar to determine cherry-red background and on Oxford agar their hemolytic activity . Also, they were gives grayish colonies surrounded by black subjected to CAMP test (Mckellar,1994) by halos. They were Gram – positive or streaking staphylococcus aureus strains in coccobacilli ;motile showing Umbrella – single straight lines in parallel on sheep blood shaped motility. On biochemical reactions agar plates , the isolated listeria strains streaks ,they produce acid from L-rhamnose, dextrose perpendicularly,(1-2mm).then incubated for and but not with D-xylose and mannitol. The 96 Abdel Tawab et al., 2018,bvmj, 34(1): 94 -105 results of virulence tests showed that , all enhanced hemolysis at junction of tested isolated L.monocytogenes strains showed strains and S. aureus strain. The result of in - narrow zone of β-hemolysis on 5% sheep vitro sensitivity test showed that the isolated blood agar, positive in CAMP test and L.monocytogenes showed as arrow-shaped zone of weak were sensitive to Sulphamethoxazole / ceftriaxone (42.8%), weak sensitivity to trimethoprim (100%), followed by Penicillin (14.2%). While the isolated strains Gentamycin, Ceftazidime and Cephazoline were resistant to Erythromycin, Ampicillin (71.4%), followed by Sulphamethoxazole, and Clindamycin as Fig (1)

Table (1): Total number and percentage of positive samples for listeria isolation from examined samples. Positive percentage Number of Number of Samples positive %1 %2 %3 samples samples Poultry 65 zero zero zero zero Frozen wings 35 zero zero zero zero Frozen breast 30 zero zero zero zero Poultry products 45 4 8.9 57.1 2.0 Frozen chicken meat 10 2 4.4 28.5 1.0 balls (kofta) Frozen chicken burge 15 1 2.2 14.2 0.5 frozen chicken sausages 20 1 2.2 14.2 0.5 Pasteurized Milk 20 zero zero zero zero Milk products 30 zero zero zero zero Feta cheese 10 zero zero zero zero Cream cheese 20 zero zero zero zero Poutry eggs 40 3 7.5 42.8 2.5 Total 200 7 3.5 100.0 3.5 1 Percentage in relation to total number of samples in each raw. 2 Percentage in relation to total number of positive samples (7). 3 Percentage in relation to total number of collected samples(200).

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Phenotypic and genotypic characterization of listeria species isolated from chicken and milk products

Fig.(1): In-vitro antimicrobial sensitivity test for isolated L.monocytogenes .

Table (2): Oligonucleotide primers sequences sources Amplifie Primer Sequence Reference d product ggA CCg ggg CTA ATA CCg AAT gAT AA Kumar et 16S rRNA 1200 bp TTC ATg TAg gCg AgT TgC AgC CTA al., 2015 CTG CTT GAG CGT TCA TGT CTC ATC Soni et al., Iap CCC C 131 bp 2014 CAT GGG TTT CAC TCT CCT TCT AC TCT-CCG-AGC-AAC-CTC-GGA-ACC Dickinson prfA 1052 bp TGG-ATT-GAC-AAA-ATG-GAA-CA et al., 1995 ACG AGT AAC GGG ACA AAT GC inlA 800 bp CCC GAC AGT GGT GCT AGA TT Liu et al., Ctggaaagtttgtatttgggaaa 2007 inlB 343 bp tttcataatcgccatcatcact GCA-TCT-GCA-TTC-AAT-AAA-GA Deneer and hlyA TGT-CAC-TGC-ATC-TCC-GTG-GT 174 bp Boychuk, CCYTTTTATGTACCCAYGA 1991

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Table (3): cycling conditions of the different primers during cPCR Primary Secondary No. of Final Gene Annealing Extension denaturation denaturation cycles extension 16S 94˚C 94˚C 60˚C 72˚C 35 72˚C rRNA 5 min. 30 sec. 1 min. 1 min. 12 min. Iap 94˚C 94˚C 60˚C 72˚C 35 72˚C 5 min. 30 sec. 30 sec. 30 sec. 7 min. prfA 94˚C 94˚C 50˚C 72˚C 35 72˚C 5 min. 30 sec. 50 sec. 1 min. 10 min. inlA 94˚C 94˚C 55˚C 72˚C 35 72˚C 5 min. 30 sec. 45 sec. 45 sec. 10 min. inlB 94˚C 94˚C 55˚C 72˚C 35 72˚C 5 min. 30 sec. 40 sec. 40 sec. 10 min. hlyA 94˚C 94˚C 50˚C 72˚C 35 72˚C 5 min. 30 sec. 30 sec. 30 sec. 7 min.

Fig. (2): 16SrRNA genes . Lane L:100-1500bp Fig.(3): inlA gene. Lane L:100-1000bp Ladder. Ladder. Neg:Negative control . Pos. :positive Neg:Negative control . Pos:Positive control at (1200bp) .Lanes 1 to 7 control at (800bp ). Lanes 1 to 7

:L.monocytogenes (16SrRNA) gene positive :L.monocytogenes (inlA )gene positive.

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Phenotypic and genotypic characterization of listeria species isolated from chicken and milk products

Fig.(4): hlyA gene. Lane L:100-600bp Ladder. Neg: Fig. (5): inlB gene. Lane L:100-600bp Ladder. Neg: Negative control . Pos:Positive control at Negative control . Pos:Positive control at (174bp ). Lanes 1 to 7 :L.monocytogenes (343bp ). Lanes 1 to 7 :L.monocytogenes (hlyA )gene positive. (inlB) gene negative

Fig.(6): prfA gene. Lane L:100-1500bp Ladder. Neg: Negative Fig.(7): iap gene. Lane L:100-600bp Ladder. Neg: control . Pos:Positive control at (1052bp ). Lanes 1 Negative control . Pos:Positive control at to 7 :L.monocytogenes (prfA )gene negative (131bp ). Lanes 1 to 7 :L.monocytogenes (iap)gene negative

4. DISCUSSION contamination of fermented dairy products Most human listeriosis cases appear to made from raw milk may be due either to use be linked to consumption of ready-to-eat of contaminated raw milk or to (RTE) food products (Lunde´n et al., 2004). postprocessing contamination from In particular, L. monocytogenes 100 Abdel Tawab et al., 2018,bvmj, 34(1): 94 -105 environmental sources not directly linked to percentage (0%) that totally disagreed with raw milk (Kells and Gilmour, 2004). (Reiter et al., 2005) Listeriosis is of great public health The result of in vitro sensitivity test concern because of its high mortality (20 to showed that ,the isolated L.monocytogenes 30%) and its common source epidemic were highly sensitive to Sulphamethoxazole potential. The most important aspect in food /trimethoprim (100%), followed by hygiene is the ability of the bacteria to survive Gentamycin Ceftazidime and Cephazoline in a wide range of temperatures and to make (71.4%), followed by Sulphamethoxazole, biofilms on various environmental surfaces, ceftriaxone (42.8%), weak sensitivity to which serve as natural habitats or reservoirs Penicillin (14.2%). While the isolated strains (Duggan and Phillips, 1998). were resistant to Erythromycin, Ampicillin Listeriosis is caused by members of and Clindamycin. And these results came in the genus Listeria, which has now 17 species. accordance with those recorded by (Zeinali et However, only two species are considered al., 2017) and disagreed with (Hof, 2004; pathogenic. Altuntas et al.,2012). L. monocytogenes is considered The heamolytic activities of listeria pathogenic to human beings and several species determine its pathogenicity (Maarouf animal species, whereas L. ivanovii is et al., 2007). The result of virulence tests for pathogenic especially to ruminants but isolated listeria showed that ,all occasionally to humans (McLauchlin& L.monocytogenes were positive to CAMP test Martin, 2008). and showed narrow zone of β-heamolysis in Ability of L. monocytogenes to cause sheep agar, the similar results were reported disease depends upon the expression of by (Boland et al., 2001 and Marrouf et al ., virulence factors and immune status of 2007) individuals. Usually individuals having The PCR results for L.monocytogenes weakened cell-mediated immunity are more isolated showed that(16SrRNA ;inl A and susceptible to L. monocytogenes. (Lecuit et hlyA) genes were detected in all seven strains. al., 2004) While inl B; iap and perfA genes are not The result of isolated samples as in detected in all isolated that dissimilar to that Table (1) revealed that L. monocytogenes was reported by (Xiaolong et al., 2017) detected in 7 samples out of 200 samples Regarding to the occurance of (14%)represented as in poultry, poultry 16SrRNA genes in L.monocytogenes isolates , byproducts, pasteurized milk, milk the obtained result revealed that , it was byproducts and poultry eggs, listeria amplified in all seven tested isolates 100% percentage were 0/65(0%), 4/45(1.8), ,that agreed with those recorded by 0/20(0%), 0/30(0%) ,3/40(1.2%) isolates, (Gelbicova and Karpiskova ,2012 and Ciolacu respectively that disagreed with that recorded et al .,2015) by (Alsheikh et al.,2013 and Zeinali et al., The result of PCR for amplification of 2017) internalin A (inlA) gene in L.monocytogenes In this research listeria (Fig.3) showed that , inlA gene was amplified monocytogenes was found in Frozen wings in all seven tested isolates giving product of of poultry, frozen breast of poultry by 800bp,that similar recorded by (Gelbicova

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