الفهرس 
Literature reviewOnly 14 pages are availabe for public view
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Abstract
Diabetic foot infections (DFIs) have become one of the most important and growing public health concerns that are more difficult to treat in diabetic patients. DFIs not only extend the average length of the hospital stay, resulting in a huge economic burden but also increase the risk of amputation. Pseudomonas aeruginosa is one of the most important opportunistic pathogenic bacterium in the diabetic foot that should never be ignored. It can be responsible for a spectrum of severe complications from superficial colonization of diabetic foot ulcers to extensive tissue damage, including osteomyelitis, septic arthritis, bacteremia and amputation. One of the challenges in managing P. aeruginosa infections is its multiple resistant to the most commonly used antibiotics and possession of virulence factors. Antimicrobial resistance in bacterial pathogens is a significant challenge that considered the main cause of morbidity and mortality in infected patients.
Due to challenges associated with the antimicrobial resistance in bacterial infections and associated diseases in addition to lack of new antibiotics, development of novel effective treatment options and alternative antimicrobial therapies are now highly required.
Probiotics are living microbial species, when administered in adequate amounts confer a health benefit to the host. Probiotics have been proven to be useful and safe in the treatment of several infections (e.g.: diabetic foot ulcers infections), gastrointestinal diseases and metabolic disorders associated with type 2 Diabetes mellitus (T2DM). Moreover, phage therapy is designed to treat bacterial infections using bacteriophages that infect bacteria, and has become a promising alternative to conventional antibiotics. Bacteriophages in many ways are advantageous over antibiotics, as they are self-replicating in the presence of host cells, have low inherent toxicity, highly specific with minimal disruption to normal flora, and disappear with the absence of a host.
The aim of the work presented in this thesis is twofold; the first aim was to study and evaluate the antibacterial activity of probiotics and lytic bacteriophages and their potential against diabetic foot pathogenic bacteria (P. aeruginosa), and the second aim is to evaluate the effect of probiotic bacteria Lactobacillus plantarum on some metabolic disorders associated with T2DM (hyperglycaemia, hyperinsulinemia, dyslipidaemia) and some cardiac markers associated with cardiovascular diseases risk factors.
The obtained results can be summarized as following:
1- A total number of 185 clinical swaps were aseptically collected from 185 patients with diabetic foot ulcers (DFUs). The ages of patients ranged between 41 to 85 years old. Out of total 185 collected specimens from different diabetic foot ulcer patients, 50 predominant isolates (27%) produced positive growth on selective medium (cetrimide agar) were identified as Pseudomonas aeruginosa, which were further confirmed through a series of different biochemical tests. The majority of isolates (n=17, 34%) were derived from older patients aged 61-70 years, of which 22% were from males and 12 % were from females. More strains were isolated from males (n=34, 68%) than from females (n=16, 32%) and males predominated in all age groups.
2- The isolated P. aeruginosa isolates showed maximum susceptibility towards colistin (80%), imipenem (72%),
amikacin (66%) and piperacillin/tazobactam (62%), while, these isolates showed moderate susceptibility towards ceftazidime (58%), cefepime (52%) and gentamicin (46%). On the other hand, the tested isolates showed complete resistance (100%) to ampicillin, cefaclor, and sulphamethoxazole/trimethoprim and high resistance to clindamycin (90%) and amoxicillin/clavulanic acid (84%).
Finally, it was noted that, P. aeruginosa isolates showed increased resistance to quinolones (ciprofloxacin (60%) and levofloxacin (54%)).
3- The identification of the most resistant isolates was confirmed using 16S rRNA gene sequencing as: Pseudomonas aeruginosa DF8, Pseudomonas aeruginosa DF15, Pseudomonas aeruginosa DF24, and Pseudomonas aeruginosa DF39. The sequences were submitted to GenBank at NCBI web site (www.ncbi.nlm.nih.gov) in accession numbers: MT158652, MT158653, MT158654, and MT158655, respectively.
4- The probiotic bacteria Lactobacillus plantarum produced the highest antibacterial activity (highest inhibition zones) than L. delbrueckii subsp. bulgaricus against all isolates of pathogenic P. aeruginosa isolated from DFU patients. The inhibition zone diameters produced by L. plantarum ranged from 13mm to 24mm. On the contrary, the inhibition zone diameters of L. delbrueckii subsp. bulgaricus ranged from 9mm to 16mm. Also, L. plantarum showed antagonistic activity against 92% (46/50) of tested P. aeruginosa isolates, while, L. delbrueckii subsp. bulgaricus showed antagonistic activity against 36% (18/50) of tested P. aeruginosa isolates. The minimum inhibitory concentration MIC and minimum bactericidal concentration MBC of L.
planatrum against the two 100% multi-drug resistant pseudomonas strains were detected as 62.5 g/ml for strain DF15 and 62.5, 125 g/ml respectively for strain DF39.
5- Our results revealed a significant (P = 0.0001) reduction in body weight of streptozotocin (STZ)-induced diabetic rats (DC) compared to normal control (NC). However, the administration of L. plantarum probiotic (TD) protects diabetic rats from body weight decreasing induced by (STZ) and significantly (P < 0.001) recover the body weight after the first week of treatment.
6- Results from the present study showed a significant increase (P = 0.0001) in blood glucose levels in rats of (DC) group compared with (NC) group after T2DM- induction by STZ and remained over 300 mg/dl up to the end of study. Oral administration of 107 CFU/ ml L. plantarum in (TD) group caused a significant (P = 0.0001) decrease of serum glucose after the first week of treatment by 22.34% from the initial concentration value, and at the end of experiment by 40.44% from the initial concentration value in diabetic rats.
7- Furthermore, at the end of experiment, we observed a significant (P < 0.05) decrease in HbA1c (19.1%) and increase (P = 0.0001) of serum insulin level in
(TD) group by (50.90%) compared to (DC). A significant reduction (P = 0.0001) in CK (39.32%) and LDH (28.28%), TC (24.27%), TG (44.5%),
VLDL-C (44.5%) and LDL-C (36.9%) associated with elevation of HDL-C level (33.52%) was also observed in the (TD) group compared to (DC) group. The probiotic L. plantarum significantly improves the onset of hyperglycaemia, hyperinsulinemia, dyslipidaemia, and may contribute to the improvement of cardiovascular disease risk factors in STZ-induced diabetic rats, indicating a lower risk of diabetes and its complications.
8- In the present study, two sewage samples out of four (Zagazig university hospital and 10th of Ramadan wastewater treatment plant) gave lytic activity against pathogenic P. aeruginosa clinical isolates from DFU patients. Presence of bacteriophages specific for P. aeruginosa isolates were confirmed by plaque assay test. Two plaques with different diameters (2 mm and 4 mm) and clear appearance were selected, purified and characterized.
9- In this study, ϕPAE1 and ϕPAE2 phages showed a broad host range against P. aeruginosa clinical strains. ϕPAE2 infected 74% (37/50) and ϕPAE1 58% (29/50) of P.
aeruginosa clinical strains of which multi-drug resistant isolates. The data obtained in this study showed that, phages ϕPAE1 and ϕPAE2 differed in lytic activity against
P. aeruginosa strains but complemented each other in the lysis of at least 86% (43/50) of the used P. aeruginosa strains. This can be considered an important positive result of the design of therapeutic bacteriophage based preparations.
10- Furthermore, our results reveal that both phages (ϕPAE1 and ϕPAE2) were found to be host specific (family: Myoviridae under TEM), infecting only P. aeruginosa strains and could not infect other bacterial species in the cross infectivity studies.
11- Effect of some physical and chemical factors on the infectivity and stability of phages were investigated. Both phages (ϕPAE1 and ϕPAE2) were found to be relatively heat stable as over a period of 1 h, after exposure to temperature range of 37–50°C, not a significant loss in phage activity was observed. However, at 60°C after 1h, a 36.58% and 21.29% reduction in phage activity for ϕPAE1 and ϕPAE2 respectively, was observed. On the other hand, the lowest activity was observed at 70°C (39.15%) for ϕPAE1 where as it was totally inactivated at 75°C while,
the lowest activity was observed at 75°C (38.01%) for ϕPAE2 where as it was totally inactivated at 80°C.
12- Isolated phages were able to survive and lyse host bacteria over a wide pH range. The optimum pH range for infection was from 6 to 8, with maximum activity observed at pH 7. Furthermore, ϕPAE1 lost its ability to lyse at pH 2, 3, 11 and 12 whereas; ϕPAE2 lost its infectivity at pH 2, 3 and
12. Furthermore, we observed that, bacteriophages tolerate the alkaline medium with rate more than the acidity medium.
13- Regarding the effect of organic solvents on the infectivity of isolated phages, Chloroform was the most effective solvent that reduced the infectivity of ϕPAE1 and ϕPAE2 to 63.27% and 77.88%, respectively. On the other hand, petroleum ether showed the lowest inhibiting effect on the infectivity of ϕPAE1 and ϕPAE2, it was reduced to 96.4% and 97.48%, respectively followed by acetone and ethyl alcohol.
14- The ability of P. aeruginosa phages to form plaques after different storage temperatures (4°C, 30°C, 37°C and 44°C) for a month was slightly affected. The storage of ϕPAE1 and ϕPAE2 at 4ºC showed the least effect on its infectivity after extended incubation for one to four weeks while, the
storage of ϕPAE1 and ϕPAE2 at 44ºC showed the highest reduction on its infectivity. Moreover, Phage counts were slightly decreased by increasing storage period and temperature.
VΙI- Conclusion
• With increased burden of nosocomial infections and antimicrobial resistance, it has become difficult for healthcare administrations and infection control committees to reach the goal for elimination of intervals. However, by practicing sound and healthy ways for care delivery designed by infection control committees, controlling transmission of these infections using appropriate methods for antimicrobial use, the resistance in emerging pathogens can be reduced.
• Increased antibiotic use leads to antibiotic resistance, so unnecessary use must be avoided. Intrinsic and acquired resistance mechanisms should be considered when prescribing antibiotics. Furthermore, infection control measures are important in reducing the spread of resistant infections.
• Diabetic foot infections are one of the major causes of social and economic problem. P. aeruginosa were found to be one of the most predominant bacterial pathogens from diabetic foot infections in this study. Knowledge on the antibiotic susceptibility pattern of these isolates is very crucial in planning DFI management and also to create a national antimicrobial treatment guideline. This would not only limit
the emergence of antibiotic resistance but also reduce the burden of health care cost.
• It is recommended that antimicrobial sensitivity testing is necessary for initiating appropriate antimicrobial regimen which will help to reduce the drug resistance and minimize the healthcare costs.
• Isolation of the difficult-to-treat organisms in the present study constringe the concept of treatment with costly antibiotics like colistin or carbapenems because of multi-drug resistant nature of the pathogens, which puts substantial financial burden of the disease on the patient.
• The results of this study indicated that Lactobacillus strains showed strong antagonistic activities against a wide range of P. aeruginosa strains, they could be considered as good potential probiotic choice for treatment and prevention of infections. They should be studied further as bio-therapeutic agents for treatments of specific disease conditions. The strains should also be investigated further for other probiotic bioactivities that have human health benefits or combination.
• The study suggests that the indigenous lactobacilli might play an important role in the protection of host against MDR P. aeruginosa infection, and such probiotic strains may beneficially be employed as bio-therapeutic agents in partial replacement or adjunct to antibiotic therapy in the treatment of
MDR P. aeruginosa infection. Accordingly, the test lactobacilli might be useful in the field of food as well as pharmaceutical industries because of their (lactobacilli) therapeutic potentiality; however, further studies are warranted to extend and authenticate the current indications.
• Based on a wide range of investigations, L. plantarum should be considered an important LAB species with excellent antimicrobial activity. Because of that some L. plantarum strains can be used as effective bio-preservatives in food industry or supporting therapeutic agents in treatment of infections caused by susceptible microorganisms. Antimicrobial compounds produced by L. plantarum such as bacteriocins and organic acids, could also be applied as alternatives of preservatives and therapeutics.
• A regular and adequate consumption of probiotics may produce a number of health benefits including reducing cardiovascular risk factors. Probiotics have been shown to lower the cardiac markers levels, lower LDL-cholesterol, and improve the LDL/HDL ratio as well as lower blood glucose levels. All of these markers have been shown to individually or in combination participate in pathogenesis of CVD. Additional studies are needed to understand the mechanisms by which probiotics may beneficially impact various aspects of
cardiovascular function. There is also an immense scope for research on how probiotics could be incorporated into food through newer techniques such as microencapsulation and cell immobilization as a daily supplement generally and specially for diabetic patients
• Bacteriophage therapy is a rapidly growing field with a proven potential for utility in treating antibiotic resistant bacterial infections in the era of burgeoning multidrug-resistance and slowing production of new chemical antibiotics. In order to fully ascertain the potential of becteriophage drugs, the collective findings of the scientific community and the long experience of phage therapy all over the world must be leveraged to devise and test new phage therapeutics that can best address the medical needs of our era as a safe, efficient, cost-effective therapy.
• Bacteriophage therapy becomes the main way to deal with MDR P. aeruginosa infections. Thus, it is currently important for phage therapy to become a routine medical procedure in opposition to what it is now: demonstrations of success in some applications. It is evident that such a transition will require an accumulation of large collections of phages specific for P. aeruginosa and deep studies of these phages. It was concluded from this study that waste water is good source for isolation of phages against MDR bacteria. The isolated phages
ϕPAE1 and ϕPAE2 were lytic phages showing a good heat and pH stability which is a very promising parameter for using it as a potential candidate for phage therapy.