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Milk isn’t only a perfect food, but also an ideal media for microbes, thus it is a major pathogen vehicle. This study was conducted to characterize molecularly some of Gram-negative pathogens in random150 raw milk samples (100 from healthy dairy farm animals and 50 from milk vendors) collected from ElmahallaElkobra and Mansoura cities, Egypt, then 20 farm samples with subclinical mastitis were excluded. Gram-negative bacteria were isolated and identified biochemically from the other 130 samples; the predominant species percentages were Escherichia coli 25.4, Serratiamarcescens 13.8, Kluyveraascorbata 8.5, Citrobacterdiversus 7.7, Klebsiellaoxytoca 5.4, and Klebsiellapneumoniae3.8. 18 shigatoxin producingE. coli (STEC) strains were classified serologically to 8 non-O157 serotypes, and the predominant was O26:H11 (87.72%). By PCR;16S-23S rDNA was identified in 3 K. pneumoniae, and shiga toxins (stx1 and 2), αhaemolysin (hlyA) and intimin (eaeA) genes were detected in 15, 13, 7 and 9 STEC respectively. Conclusively, raw milk still contributes a public hazard,so, better effective control measures are required. Molecular Characterization Of Some Gram-Negative Bacteria ... Huda H. A. Borham et., al. Kafr El-Sheikh Vet.Med.J. Vol. 1 No.1 (2003) 56 INTRODUCTION Milk has a marvelous nutritional value, but is also a perfect medium for growth of bacteria,and it can be contaminated from various sources; within the udder, exterior to the udder and from the surface of milk handling and storage equipment (Panigrahiet al., 2018), due to poor handling and storage (Ogotet al., 2015). It is a staple food in epidemiology linked to zoonotic pathogens, as it acts as a dangerous source of pathogenic bacteria (mainly GNB) such as Salmonella spp., E. coli, and other coliforms that have a public human health hazard (Panigrahiet al., 2018), and also a reservoir for a diverse group of microorganisms that can cause spoilage(O’Sullivan and Cotter 2017). Despite all these dangers,there is an increasing trend toward raw milk consumption as its health benefits are believed to be destroyed upon heating (Claeys et al., 2013), and the demand of safe and high quality milk urges for more studies to ensure these demands, therefore this study aimed to characterize molecularly by PCR some of the pathogenic GNB isolated from raw milk samples. MATERIAL AND METHODS Sampling: A total of 150 random milk samples; 100 quarter milk samples from apparently healthy dairy animals (buffalo and cow), and 50 from milk vendors, were collected aseptically in sterile container from Mansoura and ElmahallaElkobra cities in Egypt, from November 2016 to June 2017, and farm samples were collected after cleaning, drying udders and scrubbing the tip of each teat with 70% ethanol, then discarding the first few streams. All samples were transferred to the laboratory of Microbiology department, faculty of Vet. Med., Kafrelsheikh University, in icebox under aseptic conditions, and examined as soon as possible. California mastitis test (CMT): CMT was carried out as described in A.P.H.A.(2004) on farm samples using Schalm reagent, and 20 positive samples were excluded. Isolation and biochemical identification of GNB: After mixing, 1 ml of each sample was nourished in 9 ml nutrient Molecular Characterization Of Some Gram-Negative Bacteria ... Huda H. A. Borham et., al. Kafr El-Sheikh Vet.Med.J. Vol. 1 No.1 (2003) 57 broth, at 37oC for 24 hours, then a loopful was streaked on MacConkey's agar, and incubated at 37oC for 24-48hrs.Purified single colonies were identified based on Gram stain reaction, KOH test, cultural characters and biochemical tests (IMViC, TSI, LIA and urease test) (Quinn et al., 2002). Suspected E. coli and K. pneumoniaeisolates were streaked on EMB agar, and subjected to additional confirmatory tests (gelatin hydrolysis, oxidation–fermentation, nitrate reduction, ODC, ADH, ONPG and sugars fermentation tests) (Kreig and Holt, 1984). Serotyping of E. coli isolates: E. coli isolates were transferred on semisolid agar to Food Analysis Center, Faculty of Veterinary Medicine, Benha University for serotyping of somatic (O) and flagellar (H) antigens by using rapid diagnostic E. coli antisera sets (DENKA SEIKEN Co., Japan) (Kok et al., 1996). Identification of STEC and K. pneumoniae isolates by PCR: Bacterial DNA was extracted from the isolates according to Shah et al. (2009) by QIA amp kit. E. coli isolates were tested by multiplex PCR for stx1, stx2, eaeA andhlyA genes, using Pharmacia Biotech primers (Table 1), and the amplification reaction was performed on a thermal cycler using 25 μlas described by Fagan et al.(1999); initial denaturation at 95°C/3 min, followed by 35 cycles of amplification (denaturation at 95°C/20 sec, annealing at 58°C /40 sec, and extension at 72°C/90 sec) and final extension at 72°C/5 min. The control positive was E. coli O157:H7 Sakai strain. Amplified products were analyzed by 2% agarose gel electrophoresis (Applichem, Germany, GmbH) in 1x TAE buffer stained with ethidium bromide to be visualized under UV transillumator (Sambrook et al., 1989).K. pneumoniaeisolates were tested for 16S-23S rDNA internal transcribed spacer (specific for identification of K. pneumoniae) (Table 1) (Liu et al., 2008). Amplification of PCR; initial denaturation at 94°C/10 min, followed by 35 cycles of denaturation at 94°C/30 sec, annealing at 57°C/20 sec and extension at 72°C/20 sec, followed by a final extension at 72°C/10 min. The control positive wasK. pneumoniaeATCC 700721 (MGH 78578)strain.Amplified products were analyzed by 1% of agarose gel electrophoresis. Molecular Characterization Of Some Gram-Negative Bacteria ... Huda H. A. Borham et., al. Kafr El-Sheikh Vet.Med.J. Vol. 1 No.1 (2003) 58 RESULTS Isolated Gram-negative: Out of 130 non-repeated Gram-negative bacterial isolates; 113 belonged to 20 species were identified according to Farmer et al. (2007), in which E. coli predominated (Table 2).Serotyping of STEC: 18 STECisolates classified with in 8 different non-O157 serotypes (Table 3), and O26:H11 (5) was the predominant. PCR results: stx1, stx2, eaeA and hlyA were detected in 15, 13, 7, and 9 STEC respectively (Fig. 1, and Table 3), and 3 K. pneumoniaeisolates tested positive for 16S-23S rDNA(Fig. 2). Fig. (1): Agarose gel electrophoresis of multiplex PCR of stx1 (614 bp), stx2 (779 bp), eaeA (890 bp) and hlyA(165 bp) genes: M: 100 bp ladder as molecular size DNA marker, C+: Control positive E. coli forstx1, stx2,eaeAand hlyA genes, C-: Control negative: 1 (O26): Positive for stx1, stx2 andeaeA; 2, 3, 5 (O26), 9&11 (O111): Positive for stx1, stx2,eaeAand hlyA; 4 (O26): Positive for stx1and eaeA; 6&7 (O55): Positive for stx1and hlyA; 8 (O86): Positive for stx1;10 (O111): Positive for stx1, stx2and hlyA; 12 (O119), 17&18 (O146): Positive for stx1 and stx2; 13 (O121): Positive for stx2and hlyA; 14, 15&16 (O128): Positive for stx1. 890 779 614

MOLECULAR CHARACTERIZATION OF SOME GRAM-NEGATIVE BACTERIA ISOLATED FROM MILK