الفهرس 
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Abstract
Summary
K. pneumoniae is an opportunistic MDR pathogen, which may result in difficult treatment of human and animal infections due to poor antibiotic response. K. pneumoniae capsules can lead to colonization in the respiratory, gastrointestinal, and urinary tracts, as well as the development of invasive infections. It also plays an important role in the pathogenicity and chronigenicity of K. pneumoniae infections. As a result, it has been initiated for searching for a new, better, and affordable antibiotic derived from medicinal plants as alternatives or complementary treatments to overcome multiple drug resistance.
Therefore, this study was conducted to investigate the prevalence of MDR klebsiella isolates in animal and human sources in Egypt. Further, to validate the efficiency of the herbal extract alone and in combination with the drug of choice on multidrug-resistant klebsiella isolates and their ability to inhibit capsule synthesis.
The bacteriological investigations revealed that K. pneumoniae was recovered from 25 out of 200 samples with an overall prevalence of 25%.
Nine of the isolates were recovered from 50 analyzed samples from animal sources (18%) comprising minced meat, sausage, kofta, burger, and luncheon. The other 16 isolates were recovered from 50 samples from human sources (32 %) comprising sputum, blood cultures, lung, trachea, and urine samples.
This study highlights the most alarming situation of highly diverse antibiotic resistance.
It was found that most klebsiella isolates showed absolute resistance to cefotaxime followed by ceftriaxone and cephalexin (92%). More than half of the isolates represented resistance to the rest tested antibiotics with the overall proportion of MDR isolates reaching 100%. The MDR pattern may be attributed to the unregulated use of antibiotics in veterinary and human medicine in Egypt.
Regarding K. pneumoniae isolates from human sources, absolute resistance to cefotaxime and erythromycin was detected while the erythromycin resistance percentage for those from animal sources was 55.5 %.
In contrast, K. pneumoniae isolates from animal sources showed a low resistance rate to meropenem and chloramphenicol (27 and 55%, respectively), however, their percentages were high in isolates recovered from human sources (80 and 62 %, respectively).
In total, klebsiella isolates from human sources represent a great pattern of resistance and almost all isolates gave high frequencies to more than seven drugs.
The antimicrobial activity of the essential oils by agar well diffusion method among ten herbal oils demonstrated that tea tree oil (TTO) was the strongest one followed by thyme oil. Both oils showed inhibition zones ranging from 14 to 32 mm for tested klebsiella isolates.
Conversely, moringa and linseed oils didn’t exhibit any antibacterial activity against the tested K.pneumonia.
The minimum inhibitory concentration was determined by serial dilution technique using 96-well microtiter plates and the results showed that tea tree oil was the most effective against MDR strains as MICs ranged from 2 to 32 µg/ml for K. pneumoniae strains, and MBC ranged from 4 to 64 µg/ml. While the thyme extract has an effect with MICs ranging from 0.5 to 256 µg/ml. Amikacin exhibited a good value of MICs (2-4 µg/ml) and MBC values (4-8 µg/ml).
The checkerboard broth microdilution method was used for the determination of synergy between the amikacin and the effective plant extracts. Combinations of tea tree-amikacin and thyme-amikacin exerted synergetic consequences against almost isolates. The best synergistic capacity appeared with tea tree oil followed by thyme oil. FIC index values of the tea tree combination ranged from 0.1325 to 0.53 and the values of the thyme combination ranged from 0.15 to 0.53. Of interest, antagonism was not detected against any isolate in our study.
Identification of different constituents of tea tree oil and thyme oil was determined by Gas chromatography–mass spectrometry analysis (GC-MS). This analysis showed the presence of a high number of bioactive constituents in all tested fractions, this could give a clue to the wide medicinal activity they may possess. A preliminary investigation of the extract of both tea tree oil and thyme oil showed the presence of acids, alcohols, aldehydes, aromatic compounds, esters, and ketones. Data revealed the presence of seven bioactive compounds in the tea tree oil. These compounds are Terpinene-4-ol which showed the highest peak area followed by P-cymene, Alpha-Terpineol, Beta-pinene, beta-myrcene, Limonene, and Farnesol.
Regarding thyme oil, the analysis revealed the presence of ten chemical compounds. Thymol was the principal bioactive component followed by p-cymene, Gamma terpinene, Linalool, and Eugenol.
These forced us to validate the efficacy of the combination of amikacin with each effective oil on the most virulent factor, i.e. capsule.
Capsular polysaccharide (CPS) is one of the most important virulence factors of K. pneumoniae in causing infections. CPS is essentially the outer layer of the pathogen containing polymorphonuclear cells, which creates resistance against phagocytosis.
Transmission electron micrographs of K. pneumoniae cells were detected and the results showed that untreated cells of K. pneumoniae had normal cell conditions with the rugose, rigid surface, and rod shape. After treatment with thyme oil and amikacin, the cell surface became crumpled and shrunken. The cells also showed irregular shapes. Some of them opened outwards and left cleavage in the cell surface. After treatment with tea tree oil and amikacin especially, severe damage to cells was observed as the cell envelope was broken and there was leakage of cytoplasm. Lysed cells were devoid of cytoplasmic contents, with emptied and broken cell envelopes and subsequently, the cell collapsed completely.
Additionally, the expression of five virulence genes (wcaG, rmpA, magA,uge, and wabG), that were involved in pathogenicity and capsule formation in Klebsiella was estimated before and after each treatment by quantitative real-time PCR.
The tea tree-amikacin mixture significantly downregulated the mRNA expression levels of the previous five genes with efficacy more than thyme oil-amikacin combination.