الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Human Campylobacteriosis is a common food-borne bacterial disease in many developed and developing countries. It is an infectious disease caused by bacteria of the genus Campylobacter. Most people who become ill with campylobacteriosis get diarrhea (frequently with blood in the feces), abdominal pain, headache, nausea, vomiting, cramping, and fever within two to five days after exposure to the organism. The illness typically lasts about one week. Zoonotic Campylobacter spp are a significant public health problem and rapid detection of Campylobacter is an important criterion in quality control of poultry.
This cross sectional study was carried out over a period of 14 months, from October 2016 – December 2017 at the Microbiology Laboratory of the High Institute of Public Health The present study compared the performance of conventional culture on CCDA for isolation and identification of Campylobacter spp, from chicken meat and parts, followed by confirmation of Campylobacter spp by MALDI-TOF technique, versus, the isolation of Campylobacter spp directly from chicken broth using PCR. The antimicrobial resistance of the Campylobacterspp isolates was further identified using disk diffusion method.
The study involved 100 chicken meat samples, which were randomly collected from various markets and supermarkets from 3 districts in Alexandria (Middle, East and West).
Ten grams of each sample were homogenized in 90 mL of 1% bufferedpeptone water.This broth was incubated for 48 hoursto be used later used for culture and PCR. After incubation, serial 10- fold dilutions were done until reaching a dilution of 10-6. An additional volume of broth (10-1 dilution) was frozen at -20°C for further use in PCR.
Regarding culture, a direct plating method was performed, where 100 μL from the previous dilutions were spread on the surface of CCDA agar plates with antimicrobial supplements (SR0155E, Oxoid, United Kingdom) and then incubated under microaerophilic conditions at 42°C for 48 h. The total number of colonies forming units (CFU/g) was determined from countable plates which the bacterial counts averaged (30 to 300 CFU/g) and multiplied by the dilution factor.
Identified isolates as Campylobacter were further cultured on Columbia agar and incubated at 42 °C for 48 h. Microscopic examination and biochemical tests were done to identify isolates according to the conventional microbiological methods in theISO protocol (2006). Isolates that were typical on CCDA and had suggestive morphology by microscopy were then subjected to the oxidase test.
Isolates which were oxidase positive were then sub-cultured on triple sugar iron (TSI) agar. Campylobacter on TSI agar showed either no change or alkaline/ no change. Isolates were also subjected to indole, methyl red, Voges-Proskauer, citrate;(IMViC),urease and catalase tests to confirm the identificationof Campylobacter. All reactions were recorded after incubation at 42°C.
Isolates which were biochemically positive were subjected to MALDI-TOF identification. All confirmed isolates were subjected to antimicrobial susceptibility testing using the following 5 antimicrobial agents,erythromycin (E) (15μg), tetracycline (TE) (30μg), chloramphenicol (C) (30μg), ciprofloxacin(CIP: 5 μg), and nalidixic acid (NA) ( 30 μg). Furthermore, Campylobacter was detected in chicken broth by polymerase chain reaction (PCR). DNA was extracted using proteinase K solvent and Tris EDTA buffer. The amplification reaction was performed using multiplex PCR technique. PCR assay was done using specific primers to the unique regions of Campylobacter genus and to the unique regions of different Campylobacter species. The test aimed to detect the presence of 16rRNA, Askand cj0414 genes of Campylobacter in chicken samples. Discrete intense bands were observed using an ultraviolet transilluminator scanner.
The results of this study could be summarized as follows:
1. Campylobacter spp was detected in 79% of samples by culture on CCDA, but only 21 samples (21%) were biochemically proven to be Campylobacter spp. Furthermore, this number decreased to 15 (15%) when MALDI TOF was used as a confirmatory tool following culture. Even lesser numbers of Campylobacter spp were detected by PCR from chicken broth, where only 9 samples out of 100 broth samples were positive by PCR.
2. C.coli constituted 60% of Campylobactersppisolated using culture,while C.jejuniconstituted 40% of all confirmed isolates.
3. Out of the 40 intestine samples, Campylobacterspp was confirmed in11 samples (27.5% ), and in 2 samples (10%) of each of necks and wingsand 0 % of thigh samples
4. All Campylobacterisolates from wing samples were C.coli, while all isolates from the neck samples were C. jejuni. Intestine samples harboured both species, where 30% of intestineisolates were C. jejuniand 70%of them were C.coli.
5. There was no statistical significance in the distribution of different Campylobacter spp among different parts of chicken samples.
6. Campylobacter colony counts (>105) were only encountered in the broth of intestinal samples (72.7%)
7. Forty percent of isolates (6 isolates) that were positive by culture, had negative results forCampylobacter by PCR.
8. There was an almost perfect agreement (kappa=0.704, p = 0.000) between CCDA culture and PCR from broth, as regards the identification of Campylobacter spp. The positive agreement between both methods was 75%.
9. The sensitivity and specificity of PCR were 60% and 100.00% respectively.
10. PCR detected 2 mixed samples (22.22%) of C. jejuniand C. coli.
11. C. jejuni responded better to erythromycin, ciprofloxacin and chloramphenicol (susceptibility = 100%, 80% and 80% respectively).C. coli had a poorer susceptibility profile when compared toC. jejuni, since 70% of C. coli isolates responded to ciprofloxacin while the rest of antibiotics were less effective, ranging between 10% and 40% sensitivity.
12. Tetracycline had unsatisfactory results where only 40% of C. jejuni isolates and 30 % of C. coli isolates were sensitive to it.Nalidixic acid was the poorest antibiotic since none of C. jejuni and only 10% of C. coli were susceptible to it.