الفهرس 
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Abstract
Histamine is one of the biogenic amines that formed in foods largely from the growth of microorganisms that possess histidine decarboxylase enzyme. Histamine has been implicated in several outbreaks of food poisoning as chemicals intoxication resulting from the ingestion of foods containing excessive amount of histamine.
This study aimed to bacteriological and molecular identification of different species of histamine producing bacteria, quantification of histamine produced by different species of histamine producing bacteria, antimicrobial susceptibility of the most common isolated histamine producing bacteria and lastly histopathological studies about the effect of histamine on the gastrointestinal tract of albino rats.
Using Modified Niven’s medium in primary identification is more useful, accurate and practical with regard to commercial applications for identification of histamine producing compared to Niven’s medium, yet it is more sensitive but less specific.
Bacterial histamine production more common to be by Gram negative producing bacteria than Gram positive producing bacteria
Incidence of histamine producing bacteria in the frozen fish exceeds the incidence of these bacteria in the frozen meat and sausage.
Bacteriological examination of frozen fish, meat and sausage for detection of histamine producing bacteria revealed that 72.5% of the examined frozen fish, 27 % of the examined frozen meat and 41.6 % of the examined frozen sausage were positive for isolation of histamine producing bacteria using Niven’s agar medium and confirmed by bioMérieux VITEK instrument.
Most frozen fish species contaminated by histamine producing bacteria was tuna followed by herring and mackerel.
Incidence of histamine producing bacteria in the frozen fish was as 57.1% in frozen sardine, 100% in frozen tuna, 68.5% in frozen mackerel and 80% in frozen herring.
The most isolated species from the examined fish samples were Morganella morganii as (39.8%), Klebsiella pneumoniae as (14.5%), Serratia marcescens as (11.6%), Enterobacter cloacae as (7.6%), Escherichia coli as (5.5 %), Citrobacter brakii as (4.6%) , Citrobacter freundii as (3.6%) ,Serratia liquificiens as (5.5%), Aeromonas hydrophila as (1.9 %), Shwanella algae as (1.9 %) , Hafnia alvei as (1.9 %) , Serratia plymuthica as (0.9 %), Bacillus subtillus as (0.9%) and Rothia dentocariosa as (1.9%) .While, isolated species from frozen meat were Klebsiella pneumoniae as (65.6%), Morganella morganii as (25%), Pseudomonas aeroguinosa as (3.1%), Proteus vulgaris as (3.1 %) and unknown isolate as (3.1%).Lastly, isolated species from frozen sausage were Escherichia coli as (41.9%), Klebsiella pneumoniae as (22.5%), Morganella morganii as (16.1%), Proteus pennerii as (12.9 %) and Serratia rubidae as (6.4%).
Molecular based method by using convential PCR have been developed for detection of Gram negative and Gram positive histamine producing bacteria is necessary for confirmation of bacterial cultures which screen positive on Niven’s media.
Molecular examination for confirmation of histamine producing bacteria species using convential PCR technique revealed that the Positive PCR results for isolates from the fish samples were Morganella morganii was (100%), Klebsiella pneumoniae was (80%), Serratia marcescens was (100%), Enterobacter cloacae was ( 62.5%), Escherichia coli was (83 %), Citrobacter brakii was (zero %) , Citrobacter freundii was (50 %) ,Serratia liquificiens was (50%), Aeromonas hydrophila was (zero %), Shwanella algae was (100 %) , Hafnia alvei was (50 %) , Serratia plymuthica was (zero %), Bacillus subtillus was (100%) and Rothia dentocariosa was (zero %) .While, in species isolated from the examined meat samples were Klebsiella pneumoniae was (81%), Morganella morganii was (100%), Pseudomonas aeroguinosa was (zero%), Proteus vulgaris was (zero %) and unknown isolate was (zero %).Lastly, in species isolated from the examined sausage samples were Escherichia coli was (85%), Klebsiella pneumoniae was (57%), Morganella morganii was (100%), Proteus pennerii was (50 %) and Serratia rubidae was (zero%).
Convential PCR technique used for targeting hdc gene in the extracted plasmid DNA from the following isolates ( Citrrobacter braakii, Citrobacter freundii, Pseudomonas aeroguinosa and Aeromonas hydrophila ) revealed negative results which was the same results when targeting this gene using chromosomal DNA .
By using ELISA technique, two isolates (Morganella morganii and Klebsiella pneumoniae) examined for their abilities to produce histamine. The results showed that ELISA technique failed to measure the level of histamine produced by the two mentioned isolates at different dilutions giving over flow on ELISA reader.
By using HPLC to measure histamine level produced by random histamine producing bacteria species were, Morganella morganii (5931 ppm), Klebsiella pneumoniae (3446 ppm), Serratia marcescens (1238 ppm), Enterobacter cloacae (1028 ppm), Escherichia coli (438 ppm), Citrobacter brakii (95 ppm) , Citrobacter freundii (279 ppm) , Serratia liquificiens (histamine not detected), Aeromonas hydrophila (histamine not detected), Shwanella algae (720 ppm) , Hafnia alvei (963 ppm) .While, histamine level in three fish samples, were ( 119 ppm) in mackerel fish, ( not detected) in horse mackerel fish and ( 226 ppm ) in mackerel fish mixed with 1 ml of broth contained concentration10⁷of Morganella morganii .
HPLC is more effective and accurate technique for detection of histamine level than ELISA technique.
Neither the potentiometric nor the PCR method was acceptable for the identification of low histamine-producing bacteria which may give false negative results.
Sensitivity of random isolates of histamine producing bacteria to the different types of antibiotics showed that most isolates were sensitive to Enrofloxacin, Ceftriaxone, Chloramphenicol and Ciprofloxacin, while most isolates were resistant to Erythromycin and Ampicillin.
Histopathological study on the effect of histamine on the gastrointestinal tract of albino rats to illustrate the changes occurred due to ingestion of food with high level of histamine showed that the three control groups used (rats feed on bread then killed after 3 weeks, rats feed on horse mackerel fish species then killed after 30 minutes and rats feed on horse mackerel fish species then killed after 3 weeks) showed normal intestinal villi and crypts lined by mucous secreting columnar epithelium with goblet cells and no inflammatory cells in lamina propria while stomach showed normal gastric mucosa formed of pits and foveolar cells, the surface epithelium is lined by mucous secreting columnar epithelium and the glands are tubular and round lined by mucous secreting columnar epithelium. While, Histopathological examination of gastrointestinal tract for group no.4 (rats feed on fish mixed with 1 ml from broth contained concentration 10⁷ from Morganella morganii bacterial species then killed after 30 minutes) and group no.6 (rats feed on mackerel fish then killed after 30 minutes ) revealed that the intestinal villi show mild surface ulceration and erosion, the lamina propria contains mild acute inflammatory cells while stomach showed mild acute inflammatory cells formed of lymphocytes, histiocytes , polymorphs and eosinophils with mild surface ulceration and erosion. Lastly, histopathological examination of gastrointestinal tract for group no.5 (rats feed on mackerel fish mixed with 1 ml from broth contained concentration 10⁷ from Morganella morganii bacterial species then killed after 3 weeks) and group no.7 (rats feed on mackerel fish then killed after 3 weeks) revealed that Intestine contain mild to moderate chronic inflammatory cells formed of lymphocytes, histiocytes and eosinophils, there is mild surface ulceration while stomach showed that mild to moderate chronic inflammatory cells formed of lymphocytes, histiocytes and eosinophils with mild surface ulceration and erosion.