Nov 27, 2008 - (more than 200) within the family Lamiaceae. ...... 34.9% of the population does not use antibiotics for common cold. ..... 2012 Apr. ..... 125- Nikoli c M, Glamo c lija J, Ferreira IC, Calhelha RC, Fernandes Â, Markovi c T,.
ACKNOWLEDGEMENTS First of all we praise and thank Allah the most Gracious and the most Merciful for giving us the capability, strength and will to accomplish this work. We would like to thank our dear Dean Prof Dr. Maged Elghazoly, for his interest, help, support, and facilities in our project. We would express our special thanks, our forever interested, encouraging and always enthusiastic Dr. Ingy El-Soudany. She was always keen to know what we were doing and how we were proceeding, although it is likely that she has never grasped what it was all about. We will miss your screams of joy whenever a significant momentous was reached. We are also grateful to our university PUA staff: T.A. Samar Bassam, for sharing in the aim of the work and supervising the Pharmacognosy part in our project. We also thank T.A.Mona Salah for good helping and support. With a special mention to Microbiology Lab technician for their work majority of our practical part in our project. A very special gratitude to Medical Research Institute, Microbiology Department, Alexandria University and Mustafa Kamel Hospital For Armed Forces for helping and providing the samples for the work. Finally, last but by no means least, thanks to everyone in the impact hub… our parents, family and friends for their endless support.
Thanks for all your encouragement …
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LIST OF CONTENTS LIST OF CONTENTS ………………………………………………….................i LIST OF TABLES …………………………...........................................................ii LIST OF FIGURES…………...……………….…………………………………..iv LIST OF ABBREVIATIONS……………………………………………………..vi
I.
INTRODUCTION ....................................................................................... 1
II.
REVIEW OF LITERATURE ..................................................................... 8
III.
AIM OF THE WORK ................................................................................. 43
IV.
MATERIALS AND THE METHOD ........................................................ 44
V.
RESULTS ..................................................................................................... 51
VI.
DISCUSSION ............................................................................................... 69
VII.
SUMMARY AND CONCLUSION ............................................................ 73
VIII. RECOMMENDATIONS ............................................................................ 75 IX.
REFRENCES .............................................................................................. 76
2
LIST OF TABLES Table 1
Characteristics of intestinal infections caused by Eschericia coli
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2 3 4
Interpretation of the diameters of the inhibition zones of P.aeruginosa. Interpretation of the diameters of the inhibition zones of S.aureus. Interpretation of the diameters of the inhibition zones of Enterobacteriace ( K.pneumonia and E.Coli. ).
46 46 46
5 6 7 8
Distribution of the clinical isolates according to the source of the sample. Distribution of causative bacteria isolate of different samples. Distribution of Causative organism according to source of infection. Collective table for Antibiotic susceptibility test among the isolated bacteria. Antibiotic susceptibility test among isolated bacteria. Distribution of resistant isolate for each causative bacteria. Distribution of different MDR isolates. Antibiotic Resistance Pattern for the 13 MDR isolates. Determination of MIC of LE for E.coli ATCC 25922 and S.aureus ATCC25923. The MIC of LE for the different isolates included in the study. Determination of MIC of 4 thyme samples for E-coli ATCC25922 and S.aureus ATCC25923.
51 52 52 53
MIC of thyme samples (1) and thyme sample (4) for different isolates. Determination of effect of thyme aqueous extact on Levofloxacin the combination.
60 61
9 10 11 12 13 14 15 16 17
3
55 56 56 57 58 58 58
LIST OF FIGURES Figure
Page
1
SEM micrograph of cluster of Escherichia coli bacteria.
8
2
Shows the action of Escherichia coli LT (heat-labile toxin).
10
3
Electron micrograph of E. coli showing virulence factor.
12
4
K.pneumonia culture UniProt. Taxonomy: Species K pneumonia.
15
5
This scanning electron micrograph (SEM) reveals some of the ultrastructural morphologic features of K pneumonia. Courtesy of CDC/JaniceCarr.
15
6
Gm stain of Pseudomonas aeruginosa cells.
18
7
Schematic representation of the arrangement of components in the cell wall of P.aeruginosa. CM=Cytoplasm.
20
8
General structure of prorin protein illustrating the membrane.
21
9
Tripartiate Efflux pump of P. aeruginosa.
22
10
Gm-stain of Staphylococcus aureus.
27
11
Catalase-positive culture of Staphylococcus aureus .
27
12
Yellow colonies of S. aureus in Mannitol Salt Agar.
28
13
Superficial impetigo.
30
14
Staphylococcal Scalded skin syndrome.
30
15
Furuncle
30
16
The effect of selective antibiotic pressure in bacteria.
35
17
Chemical structure of levofloxacin
38
18
Chemical structure of thymol and carvacrol
40
19
Key elements of thyme powder
42
20
Distribution of the clinical isolates according to the source of the sample.
51
21
Distribution of causative bacteria isolate of different samples
52
22
Distribution of Causative organism according to source of infection
53
23
Antibiogram of isolate number 29 (K. pneumonia).
54
4
24
Antibiogram of isolate number 30(E.coli).
54
25
Distribution of different MDR isolates.
57
26
59
28
Detection of MIC of LE and thyme aqueous sample for E-coli and S-aureus standard strains. Detection of MIC of LE, thyme aqueous extract sample 4 and combination for P.aeruginosa isolates. Labiacious glandular hair in thyme.
29
Non-glandular bent hair in thyme.
63
30
Pollen grain with 6 germ pore
63
31
Peltate non-glandular hair in Cascarilla.
63
32
Contaminated sample of thyme (nearby peltate hair prisms of Calcium oxalate).
64
33
Labiacious glandular hair in thyme (sample 4).
64
27
5
61 63
LIST OF ABBREVIATIONS cAMP: cyclic adenosine monophosphate. CF: cystic fibrosis. cGMP: cyclic guanosine monophosphate. Cipro: Ciprofloxacin. E.coli: Escherichia coli. EAEC: enteroaggregative. EHEC: enterohemorrhagic. EIEC: enteroinvasive. EPEC: enteropathogenic. ESBLs: extended-spectrum beta-lactamases. ESBLs: Extended-spectrum plasmid-mediated enzymes. ETEC: Enterotoxigenic. FnBP: Fibrinectin-binding protein. FQ: Fluoroquinolone. Gm: Gram. HUS: hemolytic uremic syndrome. K pneumonia: Klebsiella pneumonia. LE: levofloxacin. LPL: lipopolysaccharides. LT: heat-labile toxin. MDR: Multi Drug Resistance. MIC: minimum inhibitory concentration. MRSA: Methicillin-resistant S. aureus. P. aeruginosa: Pseudomonas aeruginosa. PABA: paraaminobenzoic acid. PBPs: penicillin-binding proteins. PDR: Pan Drug Resistance. PMNs: polymorphonuclear neutrophils. S. aureus: Staphylococcus aureus.
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SHV-1: Sulfhydryl variable. ST: stable toxin. UTI: urinary tract infection. VISA: vancomycin-intermediate S aureus. VRSA: vancomycin-resistant S aureus. XDR: Extensively drug-resistance.
7
REVIEW OF LITERATURE Infectious diseases have been an important cause of morbidity and mortality throughout history. During the last three decades the resurgence of infectious diseases has occurred as the result of the expansion of the antimicrobial era and the challenges to antimicrobial resistance as well as the impact of an increasing immunocompromised population. (1) The most encountered Gram positive drug-resistant pathogens include methicillin-resistant Staphylococcus aureus, multidrug-resistant Streptococcus pneumoniae, and vancomycin- resistant Enterococcus spp. whileAcinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa (P. aeruginosa) represent the multidrug-resistant Gram negative bacteria. Infections caused by P. aeruginosa are associated with significant morbidity and mortality. (1, 2)
Escherichia coli Escherichia coli (E.coli) is Gm negative, nonspre forming, facultatively anaerobic, rod-shaped bacteria from the family Enterobacteriaceae. (1)
Figure (1): SEM micrograph of cluster of E.coli bacteria (2) E. coli is part of the normal flora of the colon in humans and other animals but can be pathogenic both within and outside of the GI tract. [Note: The differences in the degree of virulence of various E. coli strains is correlated with the acquisition of plasmids, integrated prophages, and pathogenicity islands.] E. coli has fimbriae or pili that are important for adherence to host mucosal surfaces, and different strains of the organism may be motile or nonmotile. Most strains can ferment lactose. (3) E. coli produces both acid and gas during fermentation of carbohydrates Over 700 antigenic types (serotypes) of E. coli are recognized based on O, H, and K antigens. At one time serotyping was important in distinguishing the small number of strains that actually cause disease. Thus, the serotype O157:H7 (O refers to somatic antigen; H refers to flagellar antigen) is uniquely responsible for causing HUS .
8
Nowadays, particularly for diarrheagenic strains (those that cause diarrhea) pathogenic E. coli are classified based on their unique virulence factors and can only be identified by these traits. (4) Pathogenic strains of E. coli are responsible for three types of infections in humans: urinary tract infections (UTI), neonatal meningitis, and intestinal diseases (gastroenteritis). The diseases caused (or not caused) by a particular strain of E. coli depend on distribution and expression of an array of virulence determinants, including adhesins, invasins, toxins, and abilities to withstand host defenses. (5)
I)
Infections:
A-Intestinal diseases Transmission of intestinal disease is commonly by the fecal–oral route, with contaminated food and water serving as vehicles for transmission. At least five types of intestinal infections that differ in pathogenic mechanisms have been identified (table: 1): Enterotoxigenic (ETEC), enteropathogenic (EPEC), enterohemorrhagic (EHEC), enteroinvasive (EIEC), and enteroaggregative (EAEC). E.coli all are basically the same organism, differing only by the acquisition of specific pathogenic traits. EHECE. coli infection should be suspected in all patients with acute bloody diarrhea, particularly if associated with abdominal tenderness and absence of fever. (6) Table (1) Characteristics of intestinal infections caused by Eschericia coli (6)
1. ETEC: ETEC are a common cause of traveler’s diarrhea. Transmission occurs through food and water contaminated with human waste or by person-to-person contact. ETEC colonize the small intestine (pili facilitate the binding of the organism to the intestinal mucosa). (7) In a process mediated by enterotoxins ETEC cause prolonged hypersecretion of chloride ions and water by the intestinal mucosal cells, while inhibiting the reabsorption of sodium. The gut becomes full of fluid, resulting in significant watery 9
diarrhea that continues over a period of several days. Enterotoxins include a heatstable toxin (ST) that works by causing an elevation in cellular cyclic guanosine monophosphate (cGMP) levels, whereas a heat-labile toxin (LT) causes elevated cyclic adenosine monophosphate (cAMP) .(Figure:2)(6)
Figure (2): shows the action of E.coli LT (6)
2. EPEC: EPEC are an important cause of diarrhea in infants, especially in locations with poor sanitation. The newborn becomes infected perinatally. The EPEC attach to mucosal cells in the small intestine by use of bundle-forming pili BfpA). Characteristic lesions in the small intestine called attaching and effacing lesions (A/E), in addition to destruction of the microvilli, are caused by injection of effector proteins into the host cell by way of a type III secretion system (T3SS).(8) EPEC cells are presented at the apex of pedestals elicited by dramatic cytoskeletal rearrangements, induced by the T3SS effectors. EPEC are not invasive and, thus, do not cause bloody diarrhea. Toxins are not elaborated by EPEC strains. Watery diarrhea results, which, on rare occasions, may become chronic. (6)
3. EHEC: EHEC bind to cells in the large intestine via BfpA and, similar to EPEC, produce A/E lesions. However, in addition, EHEC produce one of two exotoxins (Shiga-like toxins 1 or 2), resulting in a severe form of copious, bloody diarrhea (hemorrhagic colitis) in the absence of mucosal invasion or inflammation. Serotype O157:H7 is the most common strain of E.coli that produces Shiga-like toxins. This strain is also associated with outbreaks of a potentially life-threatening, acute renal failure (HUS) characterized by fever, acute renal failure, microangiopathic hemolytic anemia and thrombocytopenia in children younger than ages 5 to 10 years. The primary reservoir of EHEC is cattle. Therefore, the possibility of infection can be greatly decreased by thoroughly cooking ground beef and pasteurizing milk. (9)
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4. EIEC: EIEC cause a dysentery-like syndrome with fever and bloody stools. Plasmidencoded virulence factors are nearly identical to those of Shigella species. These virulence factors allow the invasion of epithelial cells (Ipa) and intercellular spread by use of actin-based motility. In addition, EIEC strains produce a hemolysin (HlyA). (6)
5. EAEC:
EAEC also cause traveler’s diarrhea and persistent diarrhea in young children. Adherence to the small intestine is mediated by aggregative adherence fimbriae. The adherent rods resemble stacked bricks and result in shortening of microvilli. EAEC strains produce a heat-ST that is plasmid encoded.(10)
B-Extraintestinal disease: The source of infection for extraintestinal disease is frequently the patient's own flora, in which the individual’s own E. coli is nonpathogenic in the intestine. However, it causes disease in that individual when the organism is found, for example, in the bladder or bloodstream (normally sterile sites). (11) 1. Urinary tract infection: E. coli is the most common cause of urinary tract infection (UTI), including cystitis and pyelonephritis. Women are particularly at risk for infection. Uncomplicated cystitis (the most commonly encountered UTI) is caused by uropathogenic strains of E.coli, characterized by P fimbriae (an adherence factor) and, commonly, hemolysin, colicin V, and resistance to the bactericidal activity of serum complement.Complicated UTI (pyelonephritis) may occur in settings of obstructed urinary flow, which may be caused by non-uro-pathogenic strains.(12) 2-Neonatal Meningitis: Neonatal meningitis affects 1/2,000-4,000 infants. Eighty percent of E.coli strains involved synthesize K-1 capsular antigens (K-1 is only present 2040% of the time in intestinal isolates). E. coli strains invade the blood stream of infants from the nasopharynx or GI tract and are carried to the meninges (5) The K-1 antigen is considered the major determinant of virulence among strains of E. coli that cause neonatal meningitis. K-1 is a homopolymer of sialic acid. It inhibits phagocytosis, complement, and responses from the host's immunological mechanisms. K-1 may not be the only determinant of virulence, however, as siderophore production and endotoxin are also likely to be involved. (5, 6) Epidemiologic studies have shown that pregnancy is associated with increased rates of colonization by K-1 strains and that these strains become involved in the subsequent cases of meningitis in the newborn. Probably, the infant GI tract is the portal of entry into the bloodstream. Fortunately, although colonization is fairly common, invasion and the catastrophic sequelae are rare. Neonatal meningitis requires antibiotic therapy that usually includes ampicillin and a third-generation cephalosporin. 3. Nosocomial (hospital-acquired) infections: These include sepsis/bacteremia, endotoxic shock, and pneumonia. (6)
11
II) Virulence: (5, 13) 1-Virulence Determinants of Pathogenic E. coli
Figure (3): Electron micrograph of E. coli showing virulence factor (6)
Adhesins
CFAI/CFAII Type 1 fimbriae P fimbriae as in UTI S fimbriae Intimin (non-fimbrial adhesin) EPEC adherence factor
Invasins hemolysin Shigella-like "invasins" for intracellular invasion and spread Motility/chemotaxis flagella Toxins LT toxin ST toxin Shiga toxin Cytotoxins Endotoxin (LPS) Antiphagocytic surface properties Capsules K antigens LPS Defense against serum bactericidal reactions LPS K antigens
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Defense against immune responses Capsules K antigens LPS Antigenic variation Genetic attributes Genetic exchange by transduction and conjugation Transmissible plasmids R factors and drug resistance plasmids Toxin And other virulence plasmids Siderophores and siderophore uptake systems Pathogenicity island.
Treatment of Ecoli infections: I) Doxycycline Doxycycline inhibits protein synthesis and thus, bacterial growth, by binding to the 30S and possibly 50S ribosomal subunits of susceptible bacteria. It is used to treat traveler's diarrhea. (Main bacterial resistance mechanism to tetracycline is the active efflux effect mediated by tetA and tetB). II) Trimethoprim/sulfamethoxazole: Trimethoprim/sulfamethoxazole inhibits bacterial growth by inhibiting the synthesis of dihydrofolic acid. It is used to treat traveler's diarrhea. Main mechanisms of bacterial resistance: (1) The permeability barrier and/or efflux pumps. (2) Naturally insensitive target enzymes (3) Regulational changes in the target enzymes (4) Mutational or recombinational changes in the target enzymes (5) Acquired resistance by drug-resistant target enzymes.(14)
III)
Ciprofloxacin (Cipro): Cipro is a fluoroquinolone (FQ) that inhibits bacterial DNA synthesis and, consequently, growth. It is used to treat mild-to-moderate UTI .Main mechanism of resistance is mutations in chromosomal gyrA, gyrB, parC genes and presence of plasmid-borne qnrA, qnrB, qnrS, and aac (6'-Ib-cr genes).(15)
IV)
Fluroqinolone (FQ)
FQ is used for infections due to multidrug-resistant Gm-negative organisms. It is used to treat community-acquired pneumonia, acute pyelonephritis and complicated UTI, and traveler's diarrhea.Microbial resistance mutations in the genes encoding the targets gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE), by increased levels of the multidrug efflux pump AcrAB ,and by the presence of plasmid-borne mechanisms QnrA, QnrB, QnrS .(16)
13
V) Beta-lactams: -
Amoxicillin Amoxicillin interferes with the synthesis of cell wall mucopeptides during active multiplication, resulting in bactericidal activity against susceptible bacteria. It is used to treat uncomplicated UTI and complicated UTI or pyelonephritis. (17) Resistance to amoxicillin is due to one of four general mechanisms: (1) Inactivation of antibiotic by Beta-lactamase, (2) Efflux (3) Impaired penetration of drug to target PBPs. (4) Modification of target PBPs. Beta-Lactamase production is the most common mechanism of resistance. Complete cross-resistance has been reported between amoxicillin and ampicillin. (17) -
Aztreonam Aztreonam is a monobactam that inhibits cell wall synthesis during bacterial growth. It is active against aerobic Gm.-negative bacilli. It is used to treat complicated UTIs/pyelonephritis and intra-abdominal infections, and pneumonia. Resistance to aztreonam is primarily through hydrolysis by beta-lactamase, alteration of (PBPs), and decreased permeability. (18)
Klebsiella pneumonia Family: Enterobacteriaceae; Genus: Klebsiella; Species: Klebsiella pneumoniae (K. pneumonia) is a Gm-negative, nonmotile, encapsulated, lactosefermenting, facultative anaerobic, rod-shaped bacterium. A member of the family Enterobacteriaceae. Members of the Klebsiella genus typically express 2 types of antigens on their cell surface. (19) K. pneumoniae is able to grow either with or without free oxygen, deeming it a facultative anaerobe. K. pneumoniae is found in the normal flora of the mouth, skin, and intestinal tract of humans where it initially does not cause disease. The first is a lipopolysaccharide (O antigen); the other is a capsular polysaccharide (K antigen). Both of these antigens contribute to pathogenicity. About 77 K antigens and 9 O antigens exist. The structural variability of these antigens forms the basis for classification into various serotypes. (20) The virulence of all serotypes appears to be similar. Three species in the genus Klebsiella are associated with illness in humans: K.pneumoniae, Klebsiella oxytoca, and Klebsiella granulomatis. Organisms previously known as Klebsiella ozaenae and Klebsiella rhinoscleromatis are considered non fermenting subspecies of K pneumoniae that have characteristic clinical manifestations. With those exceptions, strains within this genus ferment lactose, most produce highly mucoid colonies on plates because of the production of a luxuriant polysaccharide capsule, and all are nonmotile. (21)
14
Figure (4): K.pneumonia culture UniProt. Taxonomy: Species K pneumonia.(22)
Figure (5): This scanning electron micrograph (SEM) reveals some of the ultrastructural morphologic features of K pneumonia e. Courtesy of CDC/JaniceCarr. (22)
Infections K pneumonia is a very common pathogen that is encountered by many health care providers K pneumonia infections is spread through exposure to the bacteria via respiratory tract, which causes pneumonia, or the blood to cause an infection in the bloostream. K pneumonia infections are most well-known in hospitals spread through person-to-person contact by contaminated hands of surrounded people in the hospitals. Healthcare settings are most vulnerable to K pneumonia infections due to the nature of procedures that allow easy access of bacteria into the body. Patients who are on ventilators, catheters, or surgery wounds are highly prone to catching this deadly infection. (23) 1. K.pneumoniae has been considered a respiratory pathogen that causes Pneumoniae, the symptoms include: toxic presentation with sudden onset, high fever, and hemoptysis in the lungs, where they cause destructive changes.
15
Necrosis, inflammation, and hemorrhage occur within lung tissue, sometimes producing a thick, bloody, mucoid sputum described as currant jelly sputum. The illness typically affects middle-aged and older men with debilitating diseases such as alcoholism, diabetes, or chronic bronco pulmonary disease. (Droplet direct contact) (24) 2. A hospital-acquired pathogen that causes several infections: urinary tract,nosocomial pneumonia and intra -abdominal infections.(contamination of respiratory support equipment, use of urinary catheters) 3. Community Acquired K.pneumoniae has been responsible for increased number of bacteremic liver abscess cases (Presence of invasive devices). (25) 4. Patients with K pneumonia liver abscess also showed higher rates of occurrence for the following complications: (Sepsis and septic shock may follow entry of organisms into the blood from a focal source). a. pulmonary emboli or abscess b. brain abscess c. pyogenic meningitis d. Endophthalmitis. (26,27)
I) Virulence factors: 1- Members of the Klebsiella genus typically express two types of antigens on their cell surfaces.
The first, O antigen, is a component of the lipopolysaccharide (LPS), of which 9 varieties exist. (28) The second is K antigen, a capsular polyaccharide with more than 80 varieties. They possess a polysaccharide capsule. (29)
2- This organism is also surrounded by a capsule, which increases its virulence by acting as a physical barrier to evade the host’s immune response the main determinant of their pathogenicity. The capsule is composed of complex acidic polysaccharides.
Its massive layer protects the bacterium from phagocytosis by polymorphonuclear granulocytes. The capsule prevents bacterial death caused by bactericidal serum factors.
3- This is accomplished mainly by inhibiting the activation or uptake of complement components, especially C3b. (30) the bacteria also produce multiple adhesins. These may be fimbrial or nonfimbrial, each with distinct receptor specificity. These help the microorganism to adhere to host cells, which is critical to the infectious process.(31) 4- Lipopolysaccharides (LPS) are another bacterial pathogenicity factor. They are able to activate complement, which causes selective deposition of C3b onto LPS molecules at sites distant from the bacterial cell membrane. This inhibits the formation of the membrane attack complex (C5b-C9), which prevents membrane damage and bacterial cell death. (32) 5- Availability of iron increases host susceptibility to K pneumoniae infection. Bacteria are able to compete effectively for iron bound to host proteins because of the secretion of high-affinity, low molecular weight iron chelators 16
known as siderophores. This is necessary because most host iron is bound to intracellular and extracellular proteins. In order to deprive bacteria of iron, the host also secretes iron-binding proteins. (33)
II) Treatment A.monotherapy K. pneumoniae is resistant to a number of antibiotics, deeming treatment options very limited. Choosing an antibiotic treatment for K. pneumoniae depends on the organ system that has been targeted. The choice is especially modified for people with confirmed bacteremia. Antibiotics with high intrinsic activity against K. pneumoniae include: (34) 1. Cephalosporin (disrupt synthesis of the peptidoglycan layer of bacterial cell walls. Peptidoglycan is a strong structural molecule specific to the cells walls of bacteria. With the cell wall structure compromised, the bactericidal result is lysis and death of the cell.) 2. Carbapenems (kill bacteria by binding to PBPs, thus inhibiting bacterial cell wall synthesis). 3. Aminoglycosides (mode of action as protein synthesis inhibitors). (35) 4. Quinolone (Quinolones and FQ inhibit bacterial replication by blocking their DNA replication pathway). These treatments are initially used as monotherapy or even as a combination. For patients who are severely ill, an initial course, usually between 48-72 hours of combination aminoglycoside therapy, is suggested. This should then be followed by an extendedspectrum cephalosporin. (36)
B. Multiple therapy: Klebsiella organisms are resistant to multiple antibiotics. This is thought to be a plasmid-mediated property. Length of hospital stay and performance of invasive procedures are risk factors for acquisition of these strains. Treatment depends on the organ system involved. In general, initial therapy of patients with possible bacteremia is empirical. The choice of a specific antimicrobial agent depends on local susceptibility patterns. (37) Bacteremia is confirmed, treatment may be modified.Agents with high intrinsic activity against K pneumoniae should be selected for severely ill patients. Examples of such agents include third-generation cephalosporins (eg, cefotaxime, ceftriaxone), carbapenems (eg, imipenem/cilastatin), aminoglycosides (eg, gentamicin, amikacin), and quinolones. These agents may be used as monotherapy or combination therapy. Some experts recommend using a combination of an aminoglycoside and a third-generation cephalosporin as treatment for non–ESBLproducing isolates. Others disagree and recommend monotherapy. (38)
Ceftazidime/avibactam is indicated to treat adults with complicated intraabdominal infections (in combination with metronidazole) and complicated UTIs, including kidney infections (pyelonephritis), who have limited or no alternative treatment options. The bactericidal activity of ceftazidime results from the inhibition of cell wall synthesis via affinity for (PBPs) (39)
17
Aztreonam may be used in patients who are allergic to beta-lactam antibiotics. Quinolones are also effective treatment options for susceptible isolates in patients with either carbapenem allergy or major beta-lactam allergy. (The bactericidal action of aztreonam results from the inhibition of bacterial cell wall synthesis due to a high affinity of aztreonam for penicillin binding protein 3 (PBP3). By binding to PBP3, aztreonam inhibits the third and last stage of bacterial cell wall synthesis. Cell lysis is then mediated by bacterial cell wall autolytic enzymes such as autolysins. It is possible that aztreonam interferes with an autolysin inhibitor.) (39)
III)
Mechanism of resistance antibiotics:
1) K. pneumoniae strains are naturally resistant to ampicillin, carbenicillin and ticarcillin because of production of a chromosomal penicillinase, sulfhydryl variable (SHV-1). 2) K. pneumoniae became the index species for plasmids-encoding extendedspectrum-[beta]-lactamases (ESBLs) conferring resistance to expandedspectrum cephalosporins. Initially, those were Temoniera (TEM). (40) 3) SHV-type ESBLs and coexisted on the plasmids with elements encoding resistance to aminoglycosides, tetracyclines and trimethoprimsulfamethoxazole. 4) The emergence of a new ESBL family, the cefotaximase-M (CTX-M) group, which are currently the dominant ESBLs in K. pneumoniae. These enzymes confer resistance to penicillins and expanded-spectrum cephalosporins but are ineffective against carbapenems. Notably, ESBL-producing organisms have (41) disseminated in the pediatric population as well.
Pseudomonas aeruginosa Pseudomonas aeruginosa (P.aeruginosa) is member of the Gamma Proteobacteria class of Bacteria. It is a Gm negative, aerobic rod belonging to the bacterial family Pseudomonadaceae. P. aeruginosa is a common inhabitant of soil, water, and vegetation.)5, 13)
Figure (6): Gm stain of P. aeruginosa cells. (5) It is found on the skin of some healthy persons and has been isolated from the throat (5 percent) and stool (3 percent) of nonhospitalized patients. In some studies, gastrointestinal carriage rates increased in hospitalized patients to 20 percent within 18
72 hours of admission. Within the hospital, P. aeruginosa finds numerous reservoirs: disinfectants, respiratory equipment, food, sinks, taps, toilets, showers and mops. Furthermore, it is constantly reintroduced into the hospital environment on fruits, plants, vegetables, as well by visitors and patients transferred from other facilities. Spread occurs from patient to patient on the hands of hospital personnel, by direct patient contact with contaminated reservoirs, and by the ingestion of contaminated foods and water.
I) Virulence factor: Antigenic Structure and Toxins Pili (mbriae) extend from the cell surface and promote attachment to host epithelial cells.
Exopolysaccharide is responsible for the mucoid colonies seen in cultures from patients with CF. Lipopolysaccharide, which exists in multiple immunotypes, is responsible for many of the endotoxic properties of the organism.
P.aeruginosa can be typed by lipopolysaccharide immunotype and by pyocin (bacteriocin) susceptibility. Most P aeruginosa isolates from clinical infections produce extracellular enzymes including elastases, proteases, and two hemolysins (a heat-labile phospholipase C and a heat-stable glycolipid). (13) Many strains of P. aeruginosa produce exotoxin A, which causes tissue necrosis and is lethal for animals when injected in puried form. The e toxin blocks protein synthesis by a mechanism of action identical to that of diphtheria toxin, although the structures of the two toxins are not identical. Antitoxins to exotoxin A are found in some human sera, including those of patients who have recovered from serious P aeruginosa infections. P. aeruginosa is pathogenic only when introduced into areas devoid of normal defenses, such as when mucous membranes and skin are disrupted by direct tissue damage as in the case of burn wounds; when intravenous or urinary catheters are used; or when neutropenia is present as in cancer chemotherapy. The bacterium attaches to and colonizes the mucous membranes or skin, invades locally, and produces systemic disease. These processes are promoted by the pili, enzymes, and toxins described earlier. Lipopolysaccharide plays a direct role in causing fever, shock, oliguria, leukocytosis and leukopenia, disseminated intravascular coagulation, and adult respiratory distress syndrome. (5)
II) P. aeruginosa and Resistance to antimicrobial agents: P. aeruginosa is a notoriously difficult organism to control with antibiotics or disinfectants.Recent reports on the antibiotic sensitivity patterns of P. aeruginosa in the UK have highlighted the problem of antibiotic resistance in cystic fibrosis (CF) strains in comparison with other hospital isolates. Extensive use of antibiotics to treat P. aeruginosa in CF has generated the selective pressure to encourage resistance development. (42)
19
Its general resistance is due to a combination of factors: It is intrinsically resistant to antimicrobial agents due to low permeability of its cell wall. It has the genetic capacity to express a wide repertoire of resistance mechanisms. It can become resistant through mutation in chromosomal genes which regulate resistance genes It can acquire additional resistance genes from other organisms via plasmids, transposons and bacteriophages (43)
Mechanisms of resistance: There are three basic mechanisms by which organisms resist the action of antimicrobial agents: restricted uptake and efflux; drug inactivation and changes in targets.
1-Restricted uptake of antibacterial agent: All of the major classes of antibiotics used to treat P. aeruginosa infections have to cross the cell wall to reach their targets (Figure 7). Failure of antibiotics to accumulate within the organism is due to a combination of restricted permeability of the outer membrane and the efficient removal of antibiotic molecules that do penetrate by the action of efflux pumps. (44)
Figure (7): Schematic representation of the arrangement of components in the cell wall of P.aeruginosa. CM=Cytoplasm (44)
20
A-Alginate as a barrier: A characteristic feature of many P. aeruginosa strains in CF is the production of a loosely associated layer of the anionic polysaccharide, alginate, which surrounds the cells and binds them together in aggregates. Although it has been shown that alginate can bind cationic antibiotics such as the aminoglycosides and restrict their diffusion.The effect on the overall sensitivity of mucoid P. aeruginosa is probably minimal. Indeed some mucoid isolates are fully sensitive to aminoglycosides. (45)
B-The outer membrane as a barrier: The outer membrane of P. aeruginosa presents a significant barrier to the penetration of antibiotics, restricting the rate of penetration of small hydrophilic molecules and excluding larger molecules (Figure 7). Small hydrophilic antibiotics such as the B-lactams and quinolones can only cross the outer membrane by passing through the aqueous channels provided by porin proteins. These are barrel-shaped molecules which span the outer membrane, usually associated as trimmers. (Figure 8). (46)
Figure (8): General structure of prorin protein illustrating the membrane. Spanning
B-barrel structure in profile (a) and viewed from above (b) showing the aqueous channel through which hydrophilic antibiotic cross the outer membrane. Most porins from trimmers in the outer membrane (c). (46) P. aeruginosa produces several different porins, oprF being the major species present in all strains6. Although mutants lacking oprF have been reported, loss of oprF has not been found to be a major cause of antibiotic resistance, presumably because such strains have restricted ability to take up hydrophilic nutrients. OprD is a specialized porin which has a specific role in the uptake of positively charged amino acids such as lysine. Loss of oprD is frequently associated with resistance to imipenem, which requires this porin to cross the outer membrane. 21
Interestingly, meropenem is not affected by loss of oprD, indicating that the carbapenems have crossed the outer membrane by different channels. The aminoglycosides and colistin do not cross the outer membrane via porin channels. Instead they promote their own uptake by binding to the lipopolysaccharide (LPS) on the outer face of the membrane. This destroys the permeability barrier of the outer membrane and allows the antibiotics to penetrate through the wall to the cytoplasmic membrane. The aminoglycosides are then actively transported into the cells where they interfere with protein synthesis at the ribosomes. Colistin exerts its bactericidal action through disruption of the cytoplasmic membrane. Resistance to aminoglycosides and colistin has been observed in laboratory strains of P. aeruginosa due to overexpression of an outer membrane protein, oprH, which protects the LPS from binding the antibiotics However, this form of resistance has not been encountered widely in clinical isolate.
2-The role of efflux systems in resistance: The multidrug efflux systems are composed of three protein components, an energy-dependent pump located in the cytoplasmic membrane, an outer membrane porin and a linker protein which couples the two membrane components together.This tripartite arrangement forms an efficient extrusion system for toxic molecules present in the cytoplasm, the cytoplasmic membrane or the periplasm, i.e. the region between the outer and cytoplasmic membranes. (47) (Figure: 8).
Figure (9): Tripartiate Efflux pump of P. aeruginosa (48) Four different antibiotic efflux systems have been described in P. aeruginosa: mexAB-oprM, mexXY-oprM, mexCD-oprJ and mexEF-oprN. The efflux pump is tripartiate shown in (Figure 9). All classes of antibiotics except the polymyxins are susceptible to extrusion by one or more of the efflux systems. MexAB-oprM is responsible for extrusion of blactams, quinolones and a range of disinfectants. MexXY-oprM extrudes aminoglycosides and mexEF-oprN extrudes carbapenems and quinolones. The genes for the systems are present in all strains but they are not expressed at high levels. However, increased expression can result from mutation in regulatory genes such as mexR, which controls expression of the mexAB-oprM genes. 22
3-Inactivation and modification of antibiotics: All P.aeruginosa strains possess the ampC gene for the inducible chromosomal b-lactamase. However, induction alone probably does not account for resistance. Instead, over-expression of the enzyme results from spontaneous mutation in the regulatory gene, ampR. This has occurred particularly where heavy reliance has been placed on ceftazidime therapy. (5, 45) Over-production of the ampC b-lactamase poses a particular threat to cephalosporins. Other b-lactamases produced by P. aeruginosa include extendedspectrum plasmid-mediated enzymes (ESBLs) active against penicillins and cephalosporins. Use of b-lactamase inhibitors (clavulanic acid with ticarillin and tazobactam with piperacillin) provides protection of these antibiotics against some of the plasmid-mediated enzymes, but not the ampC enzyme. Inhibitor-resistant enzymes have also been reported. Specific carbapenemases in P. aeruginosa are of two types, serinebased enzymes and metallo-enzymes (class D). Inactivation of aminoglycosides occurs through production of enzymes which transfer acetyl, phosphate or adenylyl groups to amino and hydroxyl substituents on the antibiotics. (5, 45) Prior to the recognition that aminoglycosides are susceptible to efflux, inactivation was regarded as the major mechanism of resistance for this group of antibiotics. The modifying enzymes use cytoplasmic cofactors (acetyl co-enzyme A or ATP) to supply the substituents added to 24 the aminoglycosides so the modification process occurs within the cytoplasm. (46)
4-Changes in targets: This mechanism of resistance results from mutational changes in target enzymes which result in maintenance of their vital role in cell metabolism but resistance to the action of selective inhibition by antibiotics. In P. aeruginosa it is most commonly encountered with the quinolones through mutation in the gyrA gene encoding the A subunit of the target enzyme, DNA gyrase. Together with active efflux this accounts for the current level of resistance seen in CF strains. Changes in the structure of the ribosome 30S subunit (the aminoglycoside target) influence streptomycin sensitivity but not that of the antipseudomonal aminoglycosides. Alteration in the PBPs of P. aeruginosa resulting in resistance to b-lactams has been reported .24
5-Biofilms and resistance: In CF lung infections P. aeruginosa grows as aggregates of cells (microcolonies) encased in a protective alginate polysaccharide. This mode of growth also occurs on surfaces. The characteristic property of all biofilms is their remarkable resistance to eradication by physical and biochemical treatments, including antibiotics. Although this recalcitrance has been recognized for many years its biological basis has still not been thoroughly explained. Factors which might partly explain the resistance phenotype include the high bacterial cell density and physical exclusion of the antibiotic.
23
Physiological changes might occur in cells within the biofilm involving a general stress response, in which key metabolic processes are shut down and protective mechanisms induced. It is clear that cells in the biofilm, like free-living ‘planktonic’ cells, can sense the presence of other cells (quorum sensing) and alter their properties accordingly. Finally, the population of cells within a biofilm is heterogeneous, containing fast- and slow-growing cells, some resistant through expression of inactivating enzymes and efflux pumps, others conspicuously not expressing such systems. (23)
III)
Infection caused by P. aeruginosa
Ear infections including external otitis: P. aeruginosa is the predominant bacterial pathogen in some cases of external otitis, including "swimmer's ear". The bacterium is infrequently found in the normal ear, but often inhabits the external auditory canal in association with injury, maceration, inflammation, or simply wet and humid conditions. (3)
Eye infections: P. aeruginosa can cause devastating infections in the human eye. It is one of the most common causes of bacterial keratitis, and has been isolated as the etiologic agent of neonatal ophthalmia. Pseudomonas can colonize the ocular epithelium by means of a fimbrial attachment to sialic acid receptors. If the defenses of the environment are compromised in any way, the bacterium can proliferate rapidly through the production of enzymes such as elastase, alkaline protease and exotoxin A, and cause a rapidly destructive infection that can lead to loss of the entire eye.(3)
Respiratory infections: Respiratory infections caused by P.aeruginosa occur almost exclusively in individuals with a compromised lower respiratory tract or a compromised systemic defense mechanism. Primary pneumonia occurs in patients with chronic lung disease and congestive heart failure. Bacteremic pneumonia commonly occurs in neutropenic cancer patients undergoing chemotherapy. Lower respiratory tract colonization of CF patients by mucoid strains of P. aeruginosa is common and difficult, if not impossible, to eradicate. (1, 3)
Bacteremia and septicemia: P.aeruginosa causes bacteremia primarily in immune compromised patients. Predisposing conditions include hematologic malignancies, immunodeficiency relating to AIDS, neutropenia, diabetes mellitus, and severe burns. Most P.seudomonas bacteremia is acquired in hospitals and nursing homes. P.seudomonas accounts for about 25 percent of all hospital acquired Gm-negative bacteremias. (3)
Central nervous system infections: P.seudomonas aeruginosa causes meningitis and brain abscesses. The organism invades the CNS from a contiguous structure such as the inner ear or paranasal sinus, or is inoculated directly by means of head trauma, surgery or invasive diagnostic procedures, or spreads from a distant site of infection such as the urinary tract.
24
Bone and joint infections: Pseudomonas infections of bones and joints result from direct inoculation of the bacteria or the hematogenous spread of the bacteria from other primary sites of infection. Blood-borne infections are most often seen in IV drug users and in conjunction with urinary tract or pelvic infections. P. aeruginosa has a particular tropism for fibrocartilagenous joints of the axial skeleton. P.aeruginosa causes chronic contiguous osteomyelitis, usually resulting from direct inoculation of bone and is the most common pathogen implicated in osteochondritis after puncture wounds of the foot. (1, 3)
Urinary tract infections: Urinary tract infections (UTI) caused by P. aeruginosa are usually hospitalacquired and related to urinary tract catheterization, instrumentation or surgery. P. aeruginosa is the third leading cause of hospital-acquired UTIs, accounting for about 12 percent of all infections of this type. The bacterium appears to be among the most adherent of common urinary pathogens to the bladder uroepithelium. As in the case of E. coli, UTI can occur via an ascending or descending route. In addition, Pseudomonas can invade the bloodstream from the urinary tract, and this is the source of nearly 40 percent of Pseudomonas bacteremias. (13)
Gastrointestinal infections: P. aeruginosa can produce disease in any part of the gastrointestinal tract from the oropharynx to the rectum. As in other forms of Pseudomonas disease, those involving the GI tract occur primarily in immunocompromised individuals. The organism has been implicated in perirectal infections, pediatric diarrhea, typical gastroenteritis, and necrotizing enterocolitis. The GI tract is also an important portal of entry in Pseudomonas septicemia and bacteremia. (5)
Skin and soft tissue infections, including wound infections, pyoderma and dermatitis: P. aeruginosa can cause a variety of skin infections, both localized and diffuse. The common predisposing factors are breakdown of the integument which may result from burns, trauma or dermatitis; high moisture conditions such as those found in the ear of swimmers and the toe webs of athletes, hikers and combat troops, in the perineal region and under diapers of infants, and on the skin of whirlpool and hot tub users. Individuals with AIDS are easily infected. Pseudomonas has also been implicated in folliculitis and unmanageable forms of acne vulgaris. (13)
Endocarditis: P. aeruginosa infects heart valves of IV drug users and prosthetic heart valves. The organism establishes itself on the endocardium by direct invasion from the blood stream. (3)
IV)
Treatment of Pseudomonas infections :
I) Aztreonam (Azactam): Monobactam inhibits cell wall synthesis during bacterial growth. It is active against Gm-negative bacilli but very limited Gm-positive activity and not useful for anaerobes. It lacks cross-sensitivity with beta-lactam antibiotics. It may be used in patients allergic to penicillins or cephalosporins.
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II) Ciprofloxacin (Cipro, Cipro XR): It exerts bactericidal effect against both actively dividing and dormant bacteria. It is effective against pseudomonads. (18)
III)
Cefepime (Maxipime)
Cefepime is a zwitterion that rapidly penetrates Gm-negative cells. Best betalactam for IM administration. Poor capacity to cross blood-brain barrier precludes use for treatment of meningitis.
IV)
Ceftazidime (Fortaz, Tazicef)
Ceftazidime (Fortaz, Tazicef) is a third-generation cephalosporin with high activity against P.seudomonas. It arrests bacterial growth by binding to 1 or more PBPs.
V) Meropenem (Merrem) It is an ultra–broad-spectrum beta-lactam semisynthetic carbapenem antibiotic that inhibits bacterial cell wall synthesis. (50)
VI)
Doripenem (Doribax)
It binds to several of PBPs, which in turn inhibit bacterial cell wall synthesis. Bacteria eventually lyse due to ongoing cell wall autolytic enzymes.
VII)
Piperacillin and tazobactam (Zosyn)
It is an antipseudomonal penicillin plus beta-lactamase inhibitor. Inhibits biosynthesis of cell wall and is effective during stage of active multiplication. (51)
Staphylococcus aureus: Bacillales Family: Staphylococcaceae Genus: Staphylococcus Species: Staphylococcus aureus Staphylococcal. The main groups of medically important Gm-positive infections. They can be very difficult to treat. The most virulent of the genus, Staphylococcus aureus (S. aureus), they are routinely cultured on enriched media containing nutrient broth or blood Staphylococci are facultative anaerobic organisms. (52) Gm-positive bacteria are frequently seen in grapelike clusters. S. aureus is distinguished from the coagulase-negative staphylococci primarily by coagulase positivity. In addition, S. aureus colonies tend to be yellow (hence “aureus,” meaning golden) and hemolytic rather than gray and non-hemolytic like the coagulase-negative staphylococc. (Staphylococcus aureus) is able to ferment mannitol. sugar is fermented acid is produced and changes the pH of medium to acidic the medium is a yellow color). S.aureus is also distinguished from most coagulase-negative staphylococci by being mannitol-positive. They produce catalase so they cause disease and lack coagulase are often referred to as coagulase negative staphylococci. Staphylococci are hardy, being resistant to heat and drying is one of the most common causes of bacterial infections, and is also an important cause of food poisoning and toxic shock syndrome. (53)
26
Figure (10): Gm-stain of Staphylococcus aureus. (54) Staphylococci generally stain darkly Gm positive. They are round rather than oval and tend to occur in bunches like grapes.
Figure (11): Catalase-positive culture of S.aureus (54)
27
Figure (12): Yellow colonies of S. aureus in Mannitol Salt Agar. (55)
I) Mode of transmission:
Many bacteria are transmitted from one person to another on hands. A person with S aureus carriage in the anterior nares may rub his nose, pick up the staphylococci on the hands, and spread the bacteria to other parts of the body or to another person, where infection results direct contact (nosocomial infection). (56) Droplet and cough (respiratory disease as Pneumonia) Infected instrument contaminated needle (septicemia/nosocomial infection/septic joint /acute endocarditis). (57) Contaminated food feco-oral (toxic shock syndrome) (58) Use private tools for infected patient (Localized skin infections). (59)
Infection caused by S.aureus: Host compromise is required for S. aureus infection, such as a break in the skin or insertion of a foreign body (for example, wounds, surgical infections, or central venous catheters), an obstructed hair follicle (folliculitis), or a compromised immune system. S. aureus disease may be: Largely or wholly the result of actual invasive infection, overcoming host defense mechanisms, and the production of extracellular. 1) Substances which facilitate invasion. 2) A result of toxins in the absence of invasive infection.
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A combination of invasive infection and intoxication lead to infection/ disease like as: I) skin infection 1) S. aureus causes disease by infecting tissues, typically creating abscesses by producing toxins. 2) Localized skin infections: The most common S. aureus infections are small, superficial abscesses involving hair follicles or sweat or sebaceous glands. (60) 3) A carbuncle is a skin infection that often involves a group of hair follicles. The infected material forms a lump, which occurs deep in the skin and often contains pus. When a person has many carbuncles, the condition is called carbunculosis. Carbuncles can develop anywhere. But they are most common on the back and the nape of the neck. Men get carbuncles more often than women 4) A carbuncle is a cluster of several skin boils (furuncles). The infected mass is filled with fluid, pus, and dead tissue. Fluid may drain out of the carbuncle, but sometimes the mass is so deep that it cannot drain on its own. (61) 5) Multi located skin infections that can lead to bacteremia and require antibiotic therapy and debridement. Impetigo is usually a localized, superficial, spreading crusty skin lesion generally seen in children.
II) Systemic infection Deep localized infections: S.aureus is the most common cause of acute and chronic infection of bone marrow infection of joint space in children (septic joint). 6) Acute endocarditis: the bacteria can be introduced into soft tissues and the bloodstream, even when a sterilized needle is used. An abscess in any organ or tissue is cause to suspect S. aureus, although many other bacteria can cause abscesses. 7) Septicemia is a generalized infection with sepsis or bacteremia that may be associated with a known focus (for example, a septic joint) or not. (62) 8) Pneumonia: S. aureus is a cause of severe, necrotizing pneumonia. III) Toxinoses: a) Toxic shock syndrome: results in high fever, rash, vomiting, diarrhea, hypotension, and multiorgan involvement (especially GI, renal, and/or hepatic damage). b) Staphylococcal gastroenteritis: This is caused by ingestion offood contaminated with enterotoxin-producing S. aureus. Often contaminated by a food handler, these foods tend to be protein rich (for example, egg salad or cream pastry) or salty, like ham (S. aureus is salt tolerant), and improperly refrigerated. c) Scalded skin syndrome. (63) 9) Nosocomial infections: S. aureus is one of the most common causes of hospital-associated infections. (62) 29
Figure (13): Superficial impetigo. (64)
Figure (14): Staphylococcal Scalded skin syndrome . (65)
Figure (15): Furuncle. (66)
30
IV)
Virulence factors
Virulence factors are the genetic, biochemical, or structural features that enable an organism to produce disease. Depends on the virulence of the pathogen and the opposing effectiveness of the host defense mechanisms. S. aureus expresses many potential virulence. (66) 1) Coagulase activity: a) Results in localized clotting, which restricts access by polymorphonuclear neutrophils (PMNs) and otherimmune defenses. b) Coagulase a virulence factor mutants lacking the ability to make this factor remain virulent in animal models. For the majority of diseases caused by S. aureus, pathogenesis depends on the combined actions of several virulence factors. 2) Cell wall virulence factors: a) Capsule: express a polysaccharide “microcapsule” of Types 5 or 8. The capsule layer is very thin but has been associated with increased resistance to phagocytosis. b) Protein A: Protein A is a major component of the S. aureus cell wall. It binds to the Fc region of IgG, exerting an anti-opsonin strongly antiphagocytic effect. c) Fibronectin-binding protein: Fibrinectin-binding protein (FnBP) d) staphylococcal surface proteins promote binding to mucosal cells and tissue matrices. Clumping factor: This FnBP enhances clumping of the organisms in the presence of plasma. (67) 3) Cytolytic exotoxins: α, β, γ, and δ Toxins attack mammalian cell (including red blood cell) membranes, and are often referred to as hemolysins. α Toxin is the best studied, and is chromosomally encoded. It polymerizes into tubes that pierce membranes, resulting in the loss of important molecules and, eventually, in osmotic lysis. (68) 4) Panton-Valentine leukocidin: This pore-forming toxin lyses PMNs. Production of this toxin makes strains more virulent. This toxin is produced predominantly by community-acquired methicillin-resistant S. aureus (MRSA) strains. (69) 5) Superantigen exotoxins: These toxins have an affinity for the Tcell receptor–major histocompatibility complex Class II antigen complex. They stimulate enhanced T-lymphocyte response (as many as 20 percent of T cells respond, compared with 0.01 percent responding to the usual processed antigens). This difference is a result of their ability to recognize a relatively conserved region of the T-cell receptor. This major T-cell activation can cause toxic shock syndrome, primarily by release into the circulation of inordinately
31
large amounts of T-cell cytokines, such as interleukin-2 (IL-interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α). (70) a) Enterotoxins (six major antigenic types: A, B, C, D, E, and G) are produced by approximately half of all S.aureus isolates. When bacteria contaminate food and are allowed to grow, they secrete enterotoxin, ingestion of which can cause food poisoning. b) Enterotoxins are superantigens that are even more heat-stable than S. aureus. Organisms are not always recovered from incriminated food but the toxin may be recovered. (71) c) Toxic shock syndrome toxin (TSST –1): This is the classic cause of toxic shock syndrome (TSS). Because of similarities in molecular structure, it is sometimes referred to as staphylococcal enterotoxin F, although it does not cause food poisoning when ingested. Exfoliatin (exfoliative toxin, ET) is also a superantigen. It causes scalded skin syndrome in children. The toxin cleaves desmoglein 1, which is a component of desmosomes (cellstructures specialized for cell-to-cell adhesion). Cleavage results in loss of the superficial skin layer. (72)
V)
Treatment:
Serious S. aureus infections require aggressive treatment systemic antibiotics.Virtually all community and hospital-acquired S. aureus infections are now resistant to penicillin G due to penicillinase-encoding plasmids or transposons. This has required the replacement. (73) The initial agent of choice: 1) Penicillin G, by β-lactamase-resistant. 2) penicillins, such as methicillin or oxacillin (Exerts bactericidal activity via inhibition of bacterial cell wall synthesis by binding one or more of the penicillin binding proteins (PBPs)). (73) Hospital-acquired MRSA: Inrecent decades, a high percentage (often in the range of 50 percent) of hospital S. aureus isolates has been found to be also resistant to methicillin or oxacillin. Antibiotic resistance is caused by chromosomal acquisition of the gene for a distinct penicillinbinding protein (PBP), PBP-2a. This protein codes for a new peptidoglycan transpeptidase with a low affinity for all currently available β-lactam antibiotics, and thus renders infections with MRSA unresponsive to β-lactam therapy. (74)
3) Vancomycin acts by inhibiting cell wall synthesis of bacteria. Peptidoglycan layer of the cell wall is rigid due to its highly cross-linked structure. During the synthesis of the peptidoglycan layer of bacteria, new building blocks of peptidoglycan get inserted (i.e. monomers of N-acetylmuramic acid and Nacetylglucosamine) into the membrane.(75)
32
Vancomycin-Intermediate S aureus (VISA): S aureus is considered to be susceptible to vancomycin if the minimum inhibitory concentration (MIC) is 2 μg/mL or less; of intermediate susceptibility if the MIC is 4–8 μg/mL; and resistant if the MIC is 16 μg/mL or greater. Strains of S aureus with intermediate susceptibility to vancomycin have been isolated in Japan, the United States.These are often known as (VISA). They generally have been isolated from patients with complex infections who have received prolonged vancomycin therapy. Often there has been vancomycin treatment failure. (76) The mechanism of resistance is associated with: a) Increased cell wall synthesis b) Alterations in the cell wall and is not caused by the van genes found in enterococci. S. aureus strains of intermediate susceptibility to vancomycin usually are nafcillin resistant but generally are susceptible to oxazolidinones and to quinupristin–dalfopristin.
Vancomycin-Resistant S. aureus (VRSA): Several isolates of (VRSA) strains were isolated from patients.The isolates contained the vancomycin resistance gene vanA from enterococci and the nafcillin resistance gene mecA. Both of the initial VRSA strains were susceptible to other antibiotics. Vancomycin resistance in S aureus is of major concern worldwide. (77)
Antibacterial resistance (ABR) It is the ability of bacteria to protect themselves against the effects of an antibiotic. It involves bacteria that cause many common and life-threatening infections acquired in hospitals and in the community, for which treatment is becoming difficult, or in some cases impossible. Antibiotic resistance leads to treatment failures, threatens our ability to perform modern medical procedures, It occurs when bacteria change in response to the use of these medicines. (78) Resistant bacteria to the antibiotic lead to rapid growth of microorganisms and spread them in to other organs than one class of antibacterial agent to more leaving few treatment option. Antibiotic resistance leads to higher medical costs, prolonged hospital stays, and increased mortality. (79) Among all of the bacterial resistance problems, Gm-negative pathogens are particularly worrisome, because they are becoming resistant to nearly all drugs that would be considered for treatment. The most serious Gm-negative infections are healthcare-associated, and the most common pathogens are Enterobacteriaceae, P. aeruginosa, and Acinetobacter. (80) MDR Gm-positive organisms are major human pathogens, causing both healthcareand community associated infections. Among them, MRSA, vancomycin-resistant Enterococcus faecium (VRE), and drug-resistant Streptococcus pneumonia. (81)
33
Causes of antibiotic resistance: The antibiotic resistance crisis has been attributed to the overuse and misuse of these medications, as well as a lack of new drug development by the pharmaceutical industry due to reduced economic incentives and challenging regulatory requirements. (82) The unnecessary prescription of antibiotics for viral infections, against which they have no effect. The too frequent prescription of “broadspectrum antibiotics”, in place of a better targeted antibiotic, through more precise diagnosis. The inadequate use by the patient, not respecting either dosage or duration of the treatment, which means that some of the bacteria may survive and become resistant. (83)
Mechanism of bacterial resistance to antimicrobial agents: Bacteria have evolved sophisticated mechanisms of drug resistance to avoid killing by antimicrobial molecules. (84) ● Modifications of the antimicrobial molecule. ● Prevention to reach the antibiotic target (by decreasing penetration or actively extruding the antimicrobial compound). ● Changes and/or bypass of target sites. ● Resistance due to global cell adaptive processes. ● Alteration of metabolic pathway
Examples for these methods of resistance that bacteria can make against antibiotic agents: 1.FQ resistance can occur due to three different biochemical routes, all of which may coexist in the same bacteria at a given time, one of the ways that bacteria can resist FQ is some mutations in genes encoding the target site of FQs (DNA gyrase and topoisomerase IV), over-expression of efflux pumps that extrude the drug from the cell. (85) 2. The predominant mechanism of resistance to β-lactams in Gm-negative bacteria is the production of β-lactamases, whereas resistance to these compounds in Gmpositive organisms is mostly achieved by modifications of their target site as
alteration of PBP-the binding target site of penicillin's-in MRSA and other penicillin-resistant bacteria, or modification in structure of ribosomal protection proteins.(86) 3. Absence of paraaminobenzoic acid (PABA), this is precursor for the synthesis of folic acid and nucleic acids. (84, 85) 4. By decreasing drug permeability or increasing active pumping out of drugs through cell membrane bacteria can reduce drug accumulation. (84, 86)
34
Selection pressure: Suppression of the susceptible organisms and favoring the growth of resistant mutants in the presence of the antimicrobial agent. The influence exerted by antibiotic on natural selection to promote one group of organisms over another. (87) In the case of antibiotic resistance, antibiotics cause a selective pressure by killing susceptible bacteria, allowing antibiotic-resistant bacteria to survive and multiply. It can be regarded as a force that causes a particular organism to evolve in a certain direction. (88)
Figure (16): The effect of selective antibiotic pressure in bacteria. (89)
Cross Resistance Microorganisms resistant to certain drugs may also be resistant to other drugs that share similar mechanisms of action or binding as macrolides and lincosamides or that are chemically related as different aminoglycosides. (90) Cross-resistance is a single resistance mechanism confers resistance to an entire class of antibiotics. An example is the aminoglycoside-modifying enzymes which may confer resistance to several members of the aminoglycoside family. It can also occur across different classes of agents - a result of either overlapping drug targets as is the case with macrolides and lincosamides or if there is a drug efflux pump with a broad range of activity (i.e. capable of exporting different classes of drugs). (91)
Multi Drug Resistance (MDR) It is acquired resistance to at least one agent in three or more antimicrobial categories. MDR is a problematic because Infections with MDR bacteria are hard to treat since few or even no treatment options remain. In some cases health care providers have to use antibiotics that are more toxic for the patient. (92)
35
MDR facilitates spread of antibiotic resistance. When MDR plasmids are transferred to other bacteria, these become resistant to many antibiotics at once. In environments where bacteria are continuously exposed to antibiotics, like in hospitals or some large production animal farms, MDR may be favorable and therefore selected and spread further. (93) MDR complicates efforts to reduce resistance. When many different antibiotics select for the same resistant bacteria or plasmids, reducing use of one type of antibiotic is not enough to reduce resistance to that antibiotic. (94)
Extensively drug-resistance (XDR) In the medical literature XDR has been used as an acronym for several different terms such as ‘extreme drug resistance’, ‘extensive drug resistance’, ‘extremely drug resistant’ and ‘extensively drug resistant’. (95) Initially, the term XDR was created to describe extensively drug‐ resistant Mycobacterium tuberculosis and was defined as ‘resistance to the first‐line agents isoniazid and rifampicin, to a FQ and to at least one of the three‐second‐line parenteral drugs (i.e. amikacin, kanamycin or capreomycin). (96, 97) Subsequent to this, definitions for strains of non‐mycobacterial bacteria that were XDR were constructed according to the principle underlying this definition for XDR Mycobacterium tuberculosis (i.e. describing a resistance profile that compromised most standard antimicrobial regimens). (98)
Pan Drug Resistance (PDR) It is acquired resistance to at least one agent in all antimicrobial categories. From the Greek prefix ‘pan’, meaning ‘all’, pandrug resistant (PDR) means ‘resistant to all antimicrobial agents’. Definitions in the literature for PDR vary even though this term is etymologically exact and means that, in order for a particular species and a bacterial isolate of this species to be characterized as PDR, it must be tested and found to be resistant to all approved and useful agents. (98)
Control of antibacterial resistance The WHO, the ECDC (European Centre for Disease Prevention and Control) considers that three strategic areas of intervention should be prioritized and that each one can play an important role. (99) 1. Prudent use of available antibiotics and, when possible, infection prevention through appropriate vaccination and complete take course of antibiotic even became healthy. 2. Hygienic precautions for the control of cross transmission of resistant strains between persons, including screening for resistant strains and isolation of carrier patients. 3. Research and development of antibiotics with a novel mechanism of action.
36
The most effective interventions to limit the clonal spread of resistant organisms are effective infection control measures. Hospital antibiotic formulary restriction is the only control measure with proven effectiveness to control resistance related to antibiotic use. (100) Hospital formularies should eliminate or restrict antibiotics with a highresistance potential (eg, ceftazidime, ciprofloxacin, and imipenem), and should be replaced with equivalent antibiotics with a low-resistance potential (eg, cefe-pime, levofloxacin (LE), and meropenem). Such low-resistance-potential antibiotics can either prevent or eliminate resistance problems associated with K .pneumoniae, Enterobacter species, or P. aeruginosa. (101, 102) High-resistance-potential antibiotics, particularly Cipro and ceftazidime, also may indirectly increase the prevalence of highly resistant organisms (eg, MRSA), vancomycinresistant enterococci [VRE]). Vancomycin use should be restricted, not because it increases enterococcal resistance per se, but because it selects out naturally resistant enterococcal strains (eg, Enterococcus faecium that are vancomycin resistant). (102) For antibiotic resistance control interventions to be effective, they must be applied simultaneously to all antibiotics with activity against the specific resistance pathogen at the hospital formulary level. Multiple antibiotic substitutions are usually necessary to eradicate resistance problems caused by a particular pathogen. Multiple drugs of the same spectrum and low-resistance potential are necessary to eliminate resistance problems; single antibiotic substitutions are not effective. (103)
Fluoroquinolones The FQ class of antibiotics promises to become as diverse and as important as beta-lactam agents. The FQS inhibit bacterial DNA gyrase and are bactericidal. All FQS have potent activity against most Gm-negative bacteria; Cipro is the most active against P. aeruginosa. Activity against Gm-positive organisms is variable; MRSA has acquired resistance to the FQS at an alarming rate. (104) The first FQS, nalidixic acid, was used transiently until ocular toxicity was reported. Shortly afterwards, second-generation agents were developed, epitomized by Cipro. This agent has a wider spectrum of in vitro antibacterial activity, in particular against Gm-negative bacteria, and is effective in the treatment of many types of infection. (105) Despite excellent results in many respiratory infections, reports of failure in pneumococcal infection have limited its use in this area. Subsequently, newer agents were developed that had increased antimicrobial activity against Gm-positive pathogens; these included sparfloxacin and grepafloxacin. However, sparfloxacin has now largely been abandoned because of significant phototoxicity and potential for serious cardiac dysrhythmias, secondary to its effects on the QT interval. The 8-chloro compounds, such as Bay y 3118, clinafloxacin and sitafloxacin, were even more active, but also photoreactive. Only sitafloxacin remains in limited development. Moxifloxacin and gatifloxacin, both 8-methoxyquinolones, are the most recent and most potent FQS. (106, 107) Naphthyridone derivatives emerged in parallel with the moxifloxacin lineage. Enoxacin and tosufloxacin (available in Japan) were the first naphthyridones; they were followed by trovafloxacin and, more recently, gemifloxacin . (108)
37
Levofloxacin LE is a third generation FQS of antibacterial agent with a broad spectrum activity against Gm-positive and Gm-negative bacteria and atypical pathogens. It provides clinical and bacteriological efficacy in a range of infections, including those caused by both penicillin-susceptible and -resistant strains of S.pneumoniae. (109)
Figure (17): Chemical structure of LE (110)
Mechanism of action: LE interferes with the bacterial DNA synthesis via inhibition of the DNA gyrase in Gm-ve bacteria or topoisomerase II in Gm+ve bacteria. Inhibition of topoisomerase (DNA gyrase) enzymes, which inhibits relaxation of supercoiled DNA and promotes breakage of double stranded DNA. (111)
Mechanism of bacterial Resistance: Resistance is becoming a prevalent clinical issue that is threatening the use of that drug. Resistance mechanisms are grouped into three distinct categories. The cellular alterations associated with each mechanism are not mutually exclusive and can accumulate to create strains that exhibit very high levels of LE resistance. (112)
A) Target Mutation-Mediated LE Resistance: LE resistance is most often associated with specific mutations in gyrase and/or topoisomerase IV. Generally, mutation of one type II enzyme confers ≤10-fold drug resistance. Selection for higher levels of resistance (∼10–100-fold) usually yields strains with mutations in both enzymes. (113)
B) Plasmid-Mediated Quinolone Resistance: Plasmids that carry quinolone resistance genes have been identified as an emerging clinical problem that generally cause low-level (≤10-fold) resistance However, resistance as high as ∼250-fold has been reported. Unlike targetmediated resistance, which is transmitted vertically from generation to generation, plasmid-mediated quinolone resistance can be transmitted horizontally (through bacterial conjugation) as well as vertically. Plasmids that confer quinolone resistance typically carry additional genes that cause resistance to other drug classes. (114)
38
C) Restricted Uptake-Mediated Quinolone Resistance: The cellular concentration of quinolones is regulated by the opposing actions of diffusion-mediated drug uptake and pump-mediated efflux. In contrast to Gm-positive species, the outer membrane of Gm-negative bacteria poses an additional barrier that drugs must cross to enter the cell. Therefore, drug influx in Gm-negative species is facilitated by protein channels called porins. If the expression of porins is down regulated, it can lead to low-level resistance to quinolones. (115)
D) Efflux Pump Mediated Quinolone Resistance: In addition to the introduction of plasmid-encoded efflux pumps, enhanced expression of chromosome-encoded efflux pumps also can lead to quinolone resistance. Most commonly, the up regulation of these pumps is caused by mutations in regulatory proteins. In general, changes in quinolone uptake and retention cause low-level resistance and, in the absence of additional resistance mechanisms, do not appear to be a major clinical issue. However, lowering the cellular concentration of quinolones creates a favorable background for other forms of resistance to develop and propagate. (116, 117)
Thyme Thymus is one of the most important genera as regards the number of species (more than 200) within the family Lamiaceae. This genus belongs to the tribe Mentheae, subfamily Nepetoideae. (118) The genus Thymus It is divided into eight sections: Micantes, Mastichina, Piperella, Teucrioides, Pseudothymbra, Thymus, Hyphodromi, and Serpyllum. (118) Thymus species Thymus vulgaris, Thymus serpyllum, Thymus pulegioides, and Thymus glabrescens. (119) Active constituent Oils are very complex natural mixtures which can contain about 30 –60 components at quite different concentrations. Generally, these major components determine the biological properties of the essential oils.
Chemical composition of thyme essential oil: Tujene ,α-Pinene , Camphene, Sabinene, 3-Otenol, 3-Otanone , β-Myrcene 3-Otanol, α-Pellandrene , δ-3-Carene , α-Terpinen. (120) Thyme essential oils have been reported to possess antimicrobial activities, most of which are mediated by thymol and carvacrol, as the phenolic components of the oil. (118)
39
Carvacol
thymol
Figure (18): Chemical structure of thymol and carvacrol (121)
Uses: i. ii. iii.
iv.
v. vi.
Thyme has been thought to be antiseptic, antimicrobial, astringent, anithelmintic, carminative, disinfectant.(122) Thyme and thyme oil have been used as fumigants, antiseptics, disinfectants, and mouth washes. (124) The pleasant-tasting infusion can be taken for minor throat and chest infections, and the fresh leaves may be chewed to relieve sore throats. Thyme is prescribed with other herbs for asthma, hay fever, and is often used to treat worms in children. (124) Thyme is a rich source of flavonoid phenolic antioxidants, Thyme is also a good source of vitamins. It is particularly rich in Vitamin A and Vitamin C. Vitamin A is an antioxidant, vital for maintaining healthy mucus membranes (125) and skin as well as good vision. Thyme may also improve liver-function and act as an appetite stimulant. Thyme is also a good source of vitamins. (125) The antiseptic and tonic properties of thyme make it a useful tonic for the immune system in chronic, especially fungal, infections as well as an effective remedy for chest infections such as bronchitis, whooping cough, and pleurisy.(126)
Thyme and its antibacterial effect: The importance of the plant extract when associated with antibiotics, to control resistant bacteria, which are becoming a threat to human health. Furthermore, in a few cases, this plant extract was active against antibiotic resistant bacteria under very low concentration, thus minimizing the possible toxic effects. (127) i. ii.
The main component of the essential oil of Thyme, thymol, is active against Salmonella, Enterococcus, E.coli and Staphylococcus bacteria. (128) Its antibacterial effect proved stronger than that of standard concentrations of benzoyl peroxide, the active ingredient used in most creams and washes that are recommended for acne. (129)
40
The effect of thyme in combination with antibiotics: Thyme can have a synergistic effect in combination with antibiotics. Several modes of action have been put forward by which antibiotics and the essential oil components may act synergistically, such as by affecting multiple targets; by physicochemical interactions and inhibiting antibacterial-resistance mechanisms. (129) Thyme extracts use in combination with antibiotics against drug-resistant bacteria, such combinations have proven their effectiveness by reducing the minimum effective dose of conventional antimicrobial drugs used in the treatment of human infectious diseases and in food preservation. (130) Synergistic effect of Thymus essential oil with the other components demonstrated the potential of Thymus to enhance the antimicrobial activity of FQS antibiotics against staphylococcus aureus. As these antibiotics are effluxed by NorA pump, this increased sensitivity to antibiotics is possibly due to NorA efflux pump inhibitors. (130)
Microscopical examination of thyme (x330): (131) (Figure 19) 1. Upper epidermis of the leaf in surface view a. Diacytic stomata, a capitate glandu b. A multicellular glandular trichome c. Conical covering trichomes. The calyx. 2. Part of the lamina in sectional view showing 10 Outer epidermis of the calyx in surface view conical trichomes in the upper epidermis with capitate glandular trichomes and part of a (u.ep.). Covering trichome. 3. Covering trichomes on the lower epidermis 11 A multicellular glandular trichome in surface (ep.) of the leaf. View with surrounding epidermal cells (ep.). 4. Lower epidermis of the leaf in surface view. 12 A multicellular glandular trichome in sectional 5. Part of a group of fibres from the stem. View. 6. Outer epidermis of the corolla in surface view 13 Epidermis of the stem in surface view showing a multicellular glandular trichome and cicatrices (cic).part of a covering trichome 7. Pollen grains showing 8. Inner epidermis of the corolla in oblique surlar trichome and part of the underlying palisade face view. (pal) 9. Covering trichomes from the inner epidermis.
41
Figure (19): Key elements of thyme powder (131)
42
AIM OF THE WORK The aim of this work is to estimate the effect of aqueous extract of thyme as antibacterial agent on MDR isolates and also to test its effect on the LE antibacterial activity against MDR isolates.
43
Materials and methods 1- Collection of samples:
-
Samples were collected from Mostafa Kamel military hospital and Medical research institute hospital in the time range starting from February 2018 to April 2018. A total of 36 isolates were collected. Isolates were from different clinical sources: blood, urine, wound, abscess, ear, respiratory isolates (sputum and endotracheal tube).
2- Identification of bacterial isolates (136): Conventional methods for identification and characterization of isolate were employed including colonial morphology, Gm stained preparation and biochemical examinations such as oxidase, catalase and coagulase tests. The identification of Gm negative bacilli were also confirmed by set of biochemical reactions. E.coli isolates were identified as (132): 1-Gm negative bacilli. 2-Lactose fermenting colonies on MacConkey agar. 3-IMVC a) Indole Test positive b) Methyl Red Test positive c) Voges Proskaur Test negative d) Citrate test negative. 4-Urease test negative. 5-Oxidase test negative. 6-Triple sugar iron test (Acidic slant/acidic butt (A/A) gas positive and (H2S negative) K pneumonia isolates were identified as: 1-GM negative bacilli 2-Lactose fermenting mucoid colonies on MacConkey agar 3-IMVC a) Indole test negative b) Methyl red test negative c) Voges Proskaur Test positive d) Citrate Test positive 4-Urease Test positive. 5- Oxidase test negative. 6-Triple sugar iron test (Acidic (yellow) slant/acidic (yellow) butt (A/A) gas positive and H2S positive (133). P. aeruginosa isolates were identified as (134): 1-Growth on MacConcky agar identified as large smooth, mucoid and non_lactose fermenting colonies with flat edges and an elevated appearance. 2-Oxidase test (oxidase positive). 3-Growth at 42⁰C. 4-Triple sugar iron agar, giving a red slant and unchanged butt.
44
S. aureus isolates were identified as (64, 135): 1- Gm positive cocci. 2- Catalase test (catalase positive). 3- Coagulase test (coagulase positive ) 4- Beta Hemolyrtic colonies on blood agar. 5- Mannitol salt agar (Yellow colonies with yellow zones). Bacterial storage and revival: One ml of fresh saturated bacterial culture grown on Luria Bertani (LB) broth was added to 1 ml of 10% sterile glycerol solution in screw capped tubes. The tubes were stored at -20° C. For bacterial revival, one loopful was streaked over blood agar and incubated at 37°C.
3- Antibiotic sensitivity testing using Disk Diffusion Susceptibility Testing (Kirby-Bauer Method) : (136) Materials Needed:
Sterile saline in 2-ml tubes 0.5 McFarland standard Mueller-Hinton(MH) agar (Oxoid, UK) Caliper or ruler Antibiotic disks Forceps 18- to 24-hour old pure culture of isolates Vortex Sterile swabs Inoculating loop or needle 35°C to 37°C non-CO2 incubator
First the inoculums were prepared where four or five isolated colonies were touched using a sterile inoculating loop or needle. The organism was suspended in 2 ml of sterile saline and the saline tube was vortexed to create a smooth suspension. The turbidity of this suspension was adjusted to a 0.5 McFarland standard by adding more organism if the suspension is too light or diluting with sterile saline if the suspension is too heavy. This suspension was used within 15 minutes of preparation. The dried surface of a MH agar plate was inoculated by streaking the swab three times over the entire agar surface; the plate was rotated approximately 60 degrees each time to ensure an even distribution of the inoculum. Then, placement of the antibiotic disks; nine commercially available antibiotic disks were used. Amoxicillin (AMX), Ceftriaxone (CTR), Vancomycin (VA), Meropenem (MRP), Aztreonam (AT), Co-trimoxazole (COT), LE (LE), Amikacin (AK) in addition to Erythromycin (E). All discs were provided by Oxoid, UK. Following incubation, the zone sizes were measured to the nearest millimeter using a ruler or caliper; including the diameter of the disk in the measurement. All measurements were made with the unaided eye while viewing the back of the petri dish. The plate was held a few inches above a black, non-reflecting surface illuminated with reflected light. Growth up to the edge of the disk was reported as a zone of 0 mm. 45
Table (2): Interpretation of the diameters of the inhibition zones of P.aeruginosa (136)
Antimicrobial Agent
Disk Content
Zone Diameter Interpretive Criteria (nearest whole mm) Sensitive
Intermediate
Resistant
Meropenem
10 µg
≥19
16-18
≤15
Aztreonam
30 µ g
≥22
16-21
≤15
LE
5µg
≥17
14-16
≤13
Amikacin
30µg
≥17
15-16
≤14
Table (3): Interpretation of the diameters of the inhibition zones of S.aureus (136) Antimicrobial agent
Disk content
Amoxicillin Co-trimoxazole LE Amikacin Erythromycin
30µg 1.25/23.75µg 5µg 30µg 15µg
Zone Diameter Interpretive Criteria (nearest whole mm) Sensitive Immediate Resistant ≥22 ≤21 ≥16µg 11-15 ≤10 ≥19 16-18 ≤15 ≥17 15-16 ≤14 ≥23 14-22 ≤13
Table (4): Interpretation of the diameters of the inhibition zones of Enterobacteriace (K.pneumonia and E.Coli.): (136) Antimicrobial agent Amoxicillin Ceftriaxone Meropenem Aztreonam Co-trimoxazole LE Amikacin
Disk content
10µg 30µg 10µg 30µg 1.25/23.75µg 5µg 30µg
Zone Diameter whole mm) Sensitive ≥17 ≥23 ≥23 ≥21 ≥16 ≥17 ≥17
46
Interpretive Criteria (nearest Immediate 14-16 20-22 20-22 18-20 11-15 14-16 15-16
Resistant ≤13 ≤19 ≤19 ≤17 ≤10 ≤13 ≤14
4- Determination of minimum inhibitory concentration (MIC) of LE
using microdilution test: (136) Materials Needed: Polystyrene 96-well microtitre plates sterile with lid and u shaped wells. (Cellstar®, Greiner Bio-One, Germany) Sterile wasserman tubes Nutrient broth (Oxoid, UK) LE antibiotic powder (Pharoneya Pharmaceuticals, Egypt) 0.5 McFarland standard Blood agar as media for subculture.(5-10% de-fibrinated human blood in Nutrient agar) Standard strain E.coli ATCC 25922 and S.aureus ATCC 25923 Checkerboard titration assay was performed to assess MIC of LE for the 22 MDR clinical isolates in order to detect the MIC. Broth microdilution tests were performed according to the Clinical and Laboratory Standard Institute CLSI recommendations to determine the isolates susceptibility. Each isolate was freshly thawed and subcultured prior to testing the pure colonies were suspended in nutrient broth and adjusted to 0.5 McFarland standard. The isolates were tested in a microtitre plate against the LE (in twofold dilutions in nutrient broth) at concentrations ranging from 2µg/ml to 250µg/mL. A twofold serial dilution of LE in nutrient broth was prepared in sterile wasserman tubes (concentrations ranging from 500µg/ml to 4µg/ml).Each well of the microtitre plate will contain 100µl nutrient broth and 100µl of the antibiotic concentration in question so that the actual concentrations are ranging from 250µg/mL to 2µg/ml. Positive control wells contained broth only. Each isolate (or standard strain) was inoculated where the inoculum size was 10µgl. Results were read visually after overnight incubation at 37°C. 5- Determination of minimum inhibitory concentration (MIC) of
thyme aqueous extract using microdilution test: (136) The 4 thyme samples were collected from Al-doha company (sample 1) Fathallah hypermarket (sample 2), Carrefour hypermarket (sample 3), A herbalist in smouha (sample 4).
Thyme samples were prepared by decoction to produce stock of 150mg/mL. Checkboard titration assay was performed to assess MIC of thyme aqueous extract of the 4 samples to the standard strains (S.aureus and E.coli). Checkboard titration assay was also performed to assess MIC of thyme aqueous extract of 2 samples (sample 1 and 4) for the 13 MDR clinical isolate. Broth microdilution tests were performed according to the Clinical and Laboratory Standard Institute CLSI recommendations to determine the isolates susceptibility. Each isolate was freshly thawed and subcultured prior to testing the pure colonies were suspended in nutrient broth and adjusted to 0.5
47
McFarland standard. The isolates were tested in a microtitre plate against the thyme (in twofold dilutions in nutrient broth) at concentrations ranging from 1.17mg/mL to 150mg/mL. Each well of the microtitre plate will contain 200µl thyme aqueous extract concentration in question. Positive control wells contained broth only. Each isolate (or standards strain) was inoculated where the inoculum size was 10µgl. Results were read visually after overnight incubation at 37°C. 6- Determination of minimum inhibitory concentration (MIC) of
combining effect of thyme aqueous extract and LE using microdilution test: (136) Checkerboard titration assay was performed to assess MIC of the combination of LE aqueous extract of sample 4 and thyme for the 13 MDR clinical isolates in order to detect the MIC. Broth microdilution tests were performed according to the Clinical and Laboratory Standard Institute CLSI recommendations to determine the isolates susceptibility. Each isolate was freshly thawed and subcultured prior to testing the pure colonies were suspended in nutrient broth and adjusted to 0.5 McFarland standard. The isolates were tested in a microtitre plate against the LE (in twofold dilutions in nutrient broth) at concentrations ranging from 2µg/ml to 250 µg/ml and thyme at concentrating ranging from 1.17mg/ml to 150mg/ml. Each well of the microtitre plate will contain 100µl LE and 100µl of the thyme aqueous solution concentration in question. Positive control wells contained broth only. Each isolate was inoculated where the inoculum size was 10µgl. Results were read visually after overnight incubation at 37°C.
Detecting MIC of LE, thyme aqueous extract and combination: By definition: The MIC was the lowest concentration of the antimicrobial agent at which no growth was detected visually. (136)
7- Microscopical examination of thyme samples: The 4 samples were inspected microscopically to confirm the presence of key elements of thyme and to determine the presence of any contamination.
8- The Questionnaires: Two Questionnaires were included one for the antimicrobial use and the other for the herbal use. Age
40
Educational level No education Student low education level high education level Gender
Male
Area
%
Female
48
Question
Yes No
Do you use antibiotics? Do you use it only when prescribed by doctor? Do you store the syrup antibiotic in refrigerator after reconstitution Do you give the antibiotic prescribed by doctor to your son to his brothers? Do you know the side effects of antibiotic Do you know What precautions should be taken when using antibiotic? Do you use antibiotic for common cold? Have you ever made culture sensitivity test? Do you have resistance to any antibiotic? Does the pharmacist tell you how to use the antibiotic? Do you buy one tablet only? Do you complete the course or stop it when you get better? Does the pharmacist reconstitute the syrup antibiotic? Do you shift from one antibiotic to another? Do you take more than antibiotic together? If the doctor didn't prescribe it for you do you take the drug? Which dosage form do you prefer?
What is the most antibiotic you use it
%
Augmentin Hi-biotic Azithromycin not specific antibiotic When do you take non prescribed antibiotic?
%
Common cold Pharyngitis inflammation or sore throat Fever Tooth pain Not specific condition
Duration of using antibiotics
Less than 1 week
1 week
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2 weeks or more
as prescription
Question
Yes
Do you use medicinal herbal in general? What are the medicinal herbals you used? Do you use medicinal herbs when you have bacterial infection as pharyngitis? Do you use thyme when you have bacterial infection? When do you use thyme? How Many Times you have in day or week? Where do you buy thyme? Do you notice any difference when you buy from different sources? Did you know that thyme has antibacterial effect? Do you think to substitute antibiotic with thyme up to now? Do you think to have a cup of thyme dailyup to now ? Which place you prefer? herbalist
50
No
TV Ads
packets
RESULTS Distribution of the clinical isolates according to the source of the sample Table (5) figure (20) shows the source of different clinical isolates. Urine samples were 14 ( 38.8% ) , wound samples were 7 (19.4%) , abscess were 7 (19.4%) , 1 ear swab ( 2.7 % ) and blood samples were 5 (13.8%). Respiratory isolates were 2 (5.4 %). 1 sputum sample and 1 from endotracheal tube. Table (5): Distribution of the clinical isolates according to the source of the sample. Type of sample
Urine
Wound
Abscess
Blood
Respiratory sample
Left ear
Total
No. of isolates
14 (38.8%)
7 (19.4%)
7 (19.4%)
5 (13.8%)
2 (5.4%)
1 (2.7%)
36 (100%)
*Respiratory isolates are endotracheal tube and sputum.
Distribution of the clinical isolates according to the source of the sample
urine wound abcess blood respiratory samples ear
Figure (20): Distribution of the clinical isolates according to the source of the sample.
Distribution of causative bacterial isolates of different sample. Table (6) shows the identification of causative bacteria from different clinical samples. 14 (38.8%) of the isolates were identified as S.aureus 2 of them are MRSA. 12(33.33%) of the isolates were identified as E.Coli. 3(8.3%) of the isolates were identified as P.aeruginosa 7(19.4 % ) of the isolates were identified as K.pneumonia.
51
Table (6): Distribution of causative bacteria isolate of different samples. Causative E.Coli K.pnemoniae P.aeruginosa S.aureus MRSA Total organism 12(33.33%) 7(19.4%) 3(8.3%) 12(33.33%) 2(5.5) 36(100%) %
Distribution of causative bacteria isolate of different samples E.coli K.pneumonia P.aeruginosa S.aureus MRSA
Figure (21): Distribution of causative bacteria isolate of different samples.
Distribution of causative organism according to source of infection Table (7) shows the distribution of causative organism according to source of infection. The 14 urine samples had 11 E.coli isolates. 5 out of the 7 wound samples were S.aureus , 5 out of the 7 abscess isolates were S.aureus. The blood samples, respiratory samples and the ear swab were 2 S.aureus, 2 K.pnemonea and 1 P.aeruginosa respectively. Table (7): Distribution of Causative organism according to source of infection.
Type of causative organism E.Coli S.aureus K.pnemoneasi ella P.aruginosea monas MRSA Total
Urine
Wound
Abscess
Blood
Respiratory sample
Ear
Total
11 1 2
5 1
5 -
1 2 1
2
-
12(33.33%) 12(33.33%) 7(19.4%)
-
1
1
-
-
1
3(8.3%)
14(38.8%)
7(19.4 %)
2(5.4%)
1(2.7%)
2(5.5%) 36(100%)
1 7(19.4%) 5(13.8%)
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12 10 8 6 4 2 0
E.coli S.aureus Klebsiella Pseudomonas MRSA
Figure (22): Distribution of Causative organism according to source of infection.
Antibiotic susceptibility test among the isolated bacteria Table (8): Collective table for Antibiotic susceptibility test among the isolated bacteria.
*VA for Gm. positive bacteria **E not for P.aeruginosa.
53
Figure (23): Antibiogram of isolate number 29 (K. pneumonia)
Figure (24) Antibiogram of isolate number 30 (E-.coli) Table (9) shows that amoxicillin was applied for 34 isolates and 30 (88.23%) of the isolates were resistant, 17/25 (68%) of the isolates were resistant to ceftriaxone. Whereas cefoxitin was applied for 6 isolates and 2(33%) of the isolates were resistant and two were identified as MRSA. All the isolates of S.aureus showed inhibition zones. The two MRSA isolates showed inhibition zone for the vancomycin and according the CLSI (The disk test does not differentiate vancomycin –susceptible isolates of S. aureus from vancomycin-intermediate isolates, all of which give similar size zones of inhibition). MIC tests should be performed to determine the susceptibility of all isolates of staphylococci to vancomycin. (136)
54
Meropenem was applied for 27 isolates and 7 (25.92%) of the isolates were resistant, and 26/34 (76.47%) of the isolates were resistant to aztreonam. Co-trimoxazole was applied for 33 isolates and 14 (42.42%) of the isolates were resistant. LE was applied for 35 isolates and 18(51.42%) of the isolates were resistant. Amikacin and erythromycin showed resistance in 9/32(28.125%) and 11/19 (57.89) respectively. Table (9): Antibiotic susceptibility test among isolated bacteria Antibiotic Amoxicillin (amx) (Total 34) Ceftriaxone (CTR) (Total 25) *Cefoxitin (FOX) (Total 6 ) **Vancomycin(VA) ( total 6) Meropenem (MRP) (Total 27)( Aztreonam (AT) (Total 34) Co-trimoxazole (COT) (Total 33) Levofloxacin (LE) (Total 35) Amikacin (AK) (Total 32) ***Erythromycin (E) (Total 19)
Sensitive 3 (8.8%)
Intermediate 1 (2.94%)
Resistant 30(88.23%)
8 (32%)
0 (0%)
(68%) 17
0
0
2 ( 33% )
100 (6%)
0
0
0
7 (25.92%)
0 6 (17.64%)
2(5.88%)
26 (76.47%)
18(54.54%)
1 (3.03%)
14 (42.42%)
15(42.85%)
2(5.71%)
18 (51.42%)
20(62.5%)
3(9.37%)
9 (28.125%)
7(36.84%)
1(5.26%)
11(57.89%)
*FOX for S.aureus only. **VA for GM positive bacteria. ***E not for P.aeruginose.
Table (10) shows the different resistance patterns for the different bacterial isolates. For K.pneumonae isolates, they showed the highest resistance for Amoxicillin (100%), Co-trimoxazole (100%) and were least resistant to Erythromycin (28.57%), Amikacin (57.14%) and Meropenem (57.14%). E.coli isolates were highly resistant to Amoxicillin(75%) and least resistant to Meropenem(16.67).The study included 3 P.aeruginosa isolates that showed 66.67% resistance to each of Amoxicillin, Ceftriaxone, Co-trimoxazole and LE. The 14 S.aureus isolates included 2 MRSA isolates.
55
Table (10): Distribution of resistant isolate for each causative bacteria. organism
Gram negative
Gram positive
K.pneumonae (n=7)
E-coli (n=12)
P.aeruginosa (n=3)
S.aureus (n14)
7 (100%)
9 (75%)
2 (66.67%)
6 (85.71%)
8 (66.67%)
2 (66.67%)
12 (85.71%) 1 (8.33%)
_
_
_
0 (0%)
Meropenem (MRP)
4 (57.14%)
2 (16.67%)
0 (0%)
1 (8.33%)
Aztreonam (AT)
5 (71.42%)
7 (58.33%)
1 (33.33%)
13 (92.86%)
Co-trimoxazole (COT)
7 (100%)
5 (41.67%)
2 (66.67%)
1 (8.33%)
Levofloxacin (LE)
6 (85.71%)
7 (58.33%)
2 (66.67%)
1 (8.33%)
Amikacin (AK)
4 (57.14%)
3 (25%)
0 (0%)
2 (14.29%)
Erythromycin (E )
2 (28.57%)
8 (66.67%)
_
1 (8.33%)
Antibiotic
Amoxicillin (amx) Ceftriaxone (CTR) Vancomycin(VA)*
Table (11) shows the distribution of MDR isolates. Out of the 36 collected isolates 13 isolates (36.11%) were MDR; resistant to 3 or more different antimicrobial classes. Within the MDR isolates 46.15% were K.pnemonea, 30.78% were E.coli and 7.69% were P.aeruginosa. On the other hand 2 isolates were identified as MRSA. Table (11): Distribution of different MDR isolates MDR
K.pneumonia
E-coli
P.aeruginosa
S.aureus
Total (13)
(6) 46.15 %
(4) 30.78%
(1) 7.69%
(2) 15.38 %
56
Distribution of different MDR isolates
klebsiella E.coli P.aeruginosa S.aureus
Figure (25): Distribution of different MDR isolates Table (12) shows antibiotic resistance patterns for the different MDR bacterial isolates included in the study. All the MDR isolates were 100% resistant to amoxicillin and LE. MDR K.pneumonae and P.aeruginosa were 100% resistant to ceftriaxone and co-trimoxazole. MRSA isolates showed the highest resistance against aztreonam The best susceptibility among the MDR strains was for the meropenem where K.pneumonae, E.coli, P.aeruginosa and MRSA were 66.07%, 25%, 0% and 50% respectively. Table (12): Antibiotic Resistance Pattern for the 13 MDR isolates. organism
Gram negative
Gram positive
Antibiotic
K.pneumonae (n=6)
E-coli (n=4)
P.aeruginosa (n=1)
MRSA (n=2)
Amoxicillin (amx)
6 (100%)
4 (100%)
1 (100%)
2 (100%)
Ceftriaxone (CTR)
6 (100%)
3 (75%)
1 (100%)
0 (0%)
Meropenem (MRP)
4 (66.67%)
1 (25%)
0 (0%)
1 (50%)
Aztreonam (AT)
5 (83.33%)
3 (75%)
0 (0%)
2 (100%)
Co-trimoxazole (COT)
6 (100%)
4 (100%)
1 (100%)
0 (0%)
LE (LE)
6 (100%)
4 (100%)
1 (100%)
2 (100%)
Amikacin (AK)
4 (66.67%)
1 (25%)
0 (0%)
1 (50%)
Erythromycin ( E )
2 (33.33%)
3 (75%)
0 (0%)
1 (50%)
57
Determination of MIC of LE and thyme aqueous extracts. For validation of the methodology, the MIC of LE was determined for standard strains E.coli ATCC25922 and S.aureus ATCC25923. Table (9). The MIC was 1.5µg/ml for each of the E.coli ATCC25922 and S.aureus ATCC25923 which is below the CLSI break points 8µg/ml and 4µg/ml for each organism respectively. (136) Table (13): Determination of MIC of LE for E.coli ATCC 25922 and S.aureus ATCC25923. Strain
E-coli ATCC 25922
S.aureus ATCC25923
MIC
1.5 µg/mL
1.5 µg/mL
Table (14) shows the MIC rage of LE to different bacteria isolates which was from 15.5 to >250µg/mL with median125µg/ml and mean 128.8µg/ml. Table (14): The MIC of LE for the different isolates included in the study.
MIC of LE(µg/ml)
Rang 15.5->250
Mean 128.82
Median 125
For assessment of the antibacterial action of the 4 thyme samples, MIC for each sample was established to the standard strains as shown in the table both sample1 and sample 4 showed the best MIC for S.aureus (Gm. positive) E.coli (Gm. negative) (75mg/ml for each) Table (15): Determination of MIC of 4 thyme samples aqueous extract for E-coli ATCC25922 and S.aureus ATCC25923 Sample number
Sample1
Sample2
Sample3
Sample4
MIC for Strain MIC for E-coli MIC for S.aureus
75mg/ml 75mg/ml
75mg/ml >75mg/ml
>75mg/ml >75mg/ml
75mg/ml 75mg/ml
58
Figure (26): Detection of MIC of LE and thyme aqueous sample for E-coli and S-aureus standard strains. Table (16) shows the MIC for 2 thyme samples for some isolates (MDR and some non-MDR) Sample (1)aqueous extract showed MIC less than 150mg/ml in 5 out of the 13 MDR isolates (38.5%) and 6 out of the 7 of the non-MDR isolates (85.7%). Sample (4) aqueous extract showed MIC less than 150mg/ml in 7 out of the 13 MDR isolates (53.8%) and 5 out of 8 the 7 non- MDR isolates (7.14%).
59
Table (16): MIC of thyme samples (1) and thyme sample (4) for different isolates MDR/non MDR MDR MDR MDR MDR MDR MDR MDR MDR MDR MDR MDR MDR MDR Non MDR Non MDR Non-MDR Non-MDR Non-MDR Non-MDR Non-MDR
Staph26
MIC Thyme(1)mg/m l 150
Staph 31
>150
150
k.pnemonea7 k.pnemonea1 1 k.pnemonea 10 k.pnemonea2 7 k.pnemonea2 9
>150
150
>150
150
>150
150
>150
> 150
>150
> 150
E.coli 30
150
> 150
E.coli 32
150
> 150
E.coli 33 P.aruginosea 36 k.pnemonea 35
125
75
150
150
>150
> 150
E.coli 8
>150
> 150
Staph2
150
150
E.coli 25 P.aruginosea 13
150
75
150
> 150
E.coli 9
>150
150
E.coli1 19
150
> 150
E.coli 20
150
150
staph 23
150
125
Isolate number
MIC Thyme (4)mg/ml 150
Table (17) shows the MIC in LE (with median125mg/ml) and MIC of LE in combination (with median62.5µg/ml). 4out of 13 isolates showed 2 fold decrease,3out of 13 isolates showed 4 fold decrease, one isolate showed 83.3 fold decrease,. These isolates showed clear synergism. Meanwhile 2 isolates showed increased MIC i.e. antagonism.
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Table (17): Determination of the effect of thyme aqueous extract on LE the combination. Isolate number
MIC of LE µg/ml
MIC of LE in Combination µg/ml
Fold decrease in MIC*
Staph26
15.5
15.5
1
MIC of thyme in combination mg/ml 8
Staph 31
31.25
15.5
2.02
8
K.pnemonea 7
125
1.5
83.33
1
k.pnemonea11
250
62.5
4
38
k.pnemonea10
125
62.5
2
38
k.pnemonea27
125
31.25
4
17
k.pnemonea 35
>250
>250
1
150
k.pnemonea29
250
62.5
4
38
E.coli 30
62.5
62.5
1
38
E.coli 32
15.5
>250
150
E.coli 33
62.5
31.25
(increased MIC) 2
E.coli 8
250
>250
150
P.aruginosea 36
62.5
31.25
(increased MIC) 2
*fold decrease in MIC =MIC of LE alone/ MIC of LE in combination.
Figure (27) Detection of MIC of LE, thyme aqueous extract sample 4 and combination for P.aeruginosa isolates.
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17
17
Microscopical examination of thyme samples: The microscopial examination of the 4 samples revealed the following: Sample 1: Key elements of thyme; labiacious glandular hair (figure28), non-glandular bent hair (figure 29)and other non-glandular trichomes were found. Pollen grains with 6 germ pores were also present (figure30). However, the examination also revealed the presence of peltate non-glandular hair (figure31), lignified cork cells and prisms of Calcium oxalate. These key elements are related to cascarilla bark. Thus, this indicates contamination Cottony unicellular hair was also present . Sample 2: Key elements of thyme; labiacious glandular hair(figure 28 ), non-glandular bent hair (figure29)(more than sample 1) and other non-glandular trichomes were found. Diacytic stomata and pollen grains with 6 germ pores were also present (figure30 ). However, the examination also revealed the presence of peltate non-glandular hair (figure31) which belongs to cascarilla bark. Thus, this indicates contamination . Cottony unicellular hair was also present . Sample 3: Key elements of thyme; labiacious glandular hair (figure28), non-glandular bent hair (figure 29) and other non-glandular trichomes were found. Pollen grains with 6 germ pores were also present (figure 30). However, the examination also revealed the presence of peltate non-glandular hair (figure 31) and lignified cork cells which belong to cascarilla bark. Thus, this indicates contamination . Cottony unicellular hair was also present in addition to very few spiny pollen grains with 3 germ pores characteristic of family Compositae. This indicates further contamination.)figure 32) Sample 4: The sample was composed mainly of dried stems which lead to a coarse powder even after grinding . Key elements of thyme; labiacious glandular hair (figure 28 ), non-glandular bent hair (figure29 )and other non-glandular trichomes were found. Pollen grains with 6 germ pores were also present (figure30 ). The labiacious glandular hair appeared orange in color indicating the high content of volatile oil in the sample (figure33).Peltate hair was very rare almost absent.
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Figure (28): Labiacious glandular hair in thyme .
Figure (29): Non-glandular bent hair in thyme .
Figure (30): Pollen grain with 6 germ pore
Figure (31): Peltate non-glandular hair Cascarilla 63
Figure(32): Contaminated sample of thyme (nearby peltate hair , prisms of Calcium oxalate )
Figure (33): labiacious glandular hair in thyme (sample 4)
Conclusion: All samples contained thyme but only sample 4 was the least contaminated.
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Analysis of the questionnaires : Age
Educational level
40
9%
83.90%
7.10%
No education
Student
0
56%
low education level 3.20%
Male
Female
22.60%
77.40%
Gender
high education level 40.60%
Which dosage form do you prefer ?
%
Syrup
4.50%
Ointment
6.50%
Suppository
0%
Tablets
88.40%
Syringe
9.70%
What is the most antibiotic you use it
%
Augmentin
29%
Hi-biotic
5.10%
Azithromycin
3%
not specific antibiotic
63%
Duration of using antibiotics
Less than 1 week
1 week
2 weeks or more
as prescription
%
27.10%
25.80%
7.80%
38.10%
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Question
Yes
No
some times
Do you use antibiotics ?
97.40%
0.60%
Do you use it only when prescribed by doctor? Do you store the syrup antibiotic in refrigerator after reconstitution ? Do you give the antibiotic prescribed by doctor to your son to his brothers ? Do you know the side effects of antibiotic ?
22.60% 61.90%
5.10% 72.30% 20.60% 17.40%
16.10%
48.40% 35.50%
65.80%
34.20%
Do you know What precautions should be taken when using antibiotics ? Do you use antibiotic for common cold ?
55.50%
44.50%
32.90%
34.90% 32.90%
Have you ever made culture sensitivity test ?
7.10%
92.90%
Do you have resistance to any antibiotic ?
4.80%
94.20%
Does the pharmacist tell you how to use the antibiotic ? Do you buy one tablet only ?
57.40%
42.60%
4.50%
82.60% 12.90%
Do you complete the course or stop it when you get better ? Does the pharmacist reconstitute the syrup antibiotic ? Do you shift from one antibiotic to another ?
46.50%
34.20% 19.40%
11%
76.80% 12.30%
20%
40.60% 39.40%
Do you take more than antibiotic together ?
7.70%
81.30% 11%
If the doctor didn't prescribe it for you do you take the drug ? There is any antibiotic which isn't effective every use ?
9%
78.10% 12.90%
23.80%
76.20%
When do you take non prescribed antibiotic ?
%
Common cold
36.25%
Pharyngitis inflammation or sore throat
22.30%
Fever
6.40%
tooth pain
3.20%
Not specific condition
31.80%
66
Question
Yes
No
Sometimes
Do you use medicinal herbal in general?
23.90%
36.10%
40%
Do you use medicinal herbs when you have bacterial infection as pharyngitis? Do you use thyme when you have bacterial infection? Do you notice any difference when you buy from different sources? Did you know that thyme has antibacterial effect? Do you think to substitute antibiotic with thyme up to now? Do you think to have a cup of thyme daily up to now ? Which place you prefer?
23.90%
53.50%
22.60%
4.20%
90.30%
5.50%
35.40%
42.20%
22.40%
11.60%
88.40%
9%
49.70%
41.30%
6.50%
61.90%
31.60%
herbalist
TV ads
packets
Which is the most used herb ?
Thyme
Others
6.20%
93.02%
What is the difference you notice when using thyme from different sources ? Quality
% 21.50%
Taste
29.11%
odor
15.18%
other
34.21%
When you use thyme as antibacterial ?
%
Common cold
4.82%
Infection
6.02%
not used
59.03%
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How many times do you take thyme as treatment daily?
%
once
3.75%
twice
8.75%
3 times
1.25%
not used
86.25%
This questionnaire was answered by (155) from the analysis of the questionnaire, we can highlight the following:
Only 22.6% of the population use the antibacterial agents only when prescribe by doctor. 34.9% of the population does not use antibiotics for common cold. Only 7.1% use the antibiotics based sensitivity culture test. 34.2%do not complete the antibiotic course. 20% can shift from one antibiotic to another. Based on the questionnaire, the most common used antibiotics are amoxicillin–clavulinic acid combination (29%) and (39%) claimed their use for any nonspecific antibiotics. 36% of the population use medicinal herbal in general. 90.3% don’t use thyme as antibacterial agents &88.4% don’t know that thyme has antibacterial effect.
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DISCUSSION Multi-drug-resistant bacteria represent a major medical threat, contributing to the deaths of increasing numbers of patients worldwide. (137) Alternative approaches to treatment of infectious diseases are being investigated; examples include the use of plant extracts individually and/or in combination with antibiotics. This latter approach (i.e., combination therapy or synergistic therapy) may lead to new ways of treating infectious diseases and could be useful for patients with serious infections caused by drug resistant pathogens. (138) The aim of this study was to estimate the effect of aqueous extract of thyme as antibacterial agent on MDR isolates and also to test its effect on the levofloxacin antibacterial activity against MDR isolates. This study included 36 isolates of different clinical samples. 38.8% were urine samples, 19.4% wound samples, 19.4% abscess, 2.7 % ear swab and 13.8% blood samples and 5.4 % respiratory isolates (the respiratory isolates were sputum sample and sample from endotracheal tube). The most identified causative bacteria from different clinical samples were S.aureus 38.8% followed by E.Coli 33.33%, then K.pneumoniae 19.4 % and finally P.aeruginosa 8.3%. Custovic et al., (2014) reported in an epidemiological surveillance of bacterial nosocomial infections in the surgical intensive care unit that K. pneumoniae was the commonest (51.0%), followed by Proteus mirabilis (21.3%) and P. aeruginosa (10.6%). (MRSA) (16%) was among the commonest Gm-positive bacteria. (139) The most identified bacteria in urine samples was E.coli (11/14) and from the wound samples was S.aureus (5/7). From blood samples, respiratory samples and abscess the most frequent bacteria was S.aureus (2/5), Klebsiella (2/2) and S.aureus (5/7), respectively. Custovic et al., (2014) also reported high prevalence of K. pneumoniae among patients with respiratory tract infections but Klebsiella spp. (60%) as the most common cause of the urinary tract infections. (139)On the contrary, Shaikh et al,(2008) detected E. coli as the most frequent pathogen (26.3%) for urinary tract infections and Klebsiella spp. was responsible for only 5.2 %.(140) The antibiotic susceptibility test revealed that the different isolates showed the highest resistance to amoxicillin and aztreonam, but they showed the highest sensitivity to amikacin and co-trimoxazole. K.pneumonae isolates showed the highest resistance for amoxicillin (100%), co-trimoxazole (100%). E.coli isolates were highly resistant to amoxacillin(75%). The P.aeruginosa isolates showed 66.67% resistence to each of amoxacillin, ceftriaxone, co-trimoxazole and LE. The 14.3% of S.aureus isolates included in the study where identified as MRSA.
69
Alam et al., (2017) found that 48.6% of E. coli isolates from urine were resistant to co-trimoxazole, 49.3% of Klebsiella isolates were resistant to ampicillin while 50.3% S. aureus isolates were MRSA.(141) Kresken et al.,(2014) have observed that 42.9% of E. coli isolates from urinary tract were resistant to amoxicillin while resistance to co-trimoxazole was in 30.9% of the isolates.(141) In another study, the prevalence of MRSA was reported to be 39.5% where infection was commonly associated with wound, skin, and soft tissues.(142) The 36 isolates included in the study had 36.11% MDR isolates .Within these MDR isolates 46.15% were Klebsiella, 30.78% were E.coli and 7.69% were P.aeruginosa, besides the 2 MRSA. In a study for prevalence of MDR in bacterial uropathogens, Baral et al.,(2012) reported 41.1% isolates were MDR; 81.3%, of them were E. coli. (143)In another study for antibiotic resistance pattern of MDR bacteria among blood isolates, Anjum et al.,(2014) reported 14.39% MDR of the blood culture positive isolates.(144) In an Egyptian study on MDR pathogens of bacterial meningitis, Abdelkader et al.,(2017) reported 36.6% MDR isolates which is very similar to our findings. (145) All the MDR, isolates were 100% resistant to amoxicillin and LE. MDR K.pneumonae and P.aeruginosa were 100% resistant to ceftriaxone and cotrimoxazole. MRSA isolates showed the highest resistance against aztreonam. The best susceptibility among the MDR isolates was for the meropenem where K.pneumonae, E.coli, P.aeruginosa and MRSA were 66.67%, 25%, 0% and 50% respectively. Baral et al.,(2012) found that the majority of MDR E. coli isolates showed high resistance to different classes of antibiotics used: co-trimoxazole (86.8%), amoxicillin, (94.1%), ciprofloxacin (92.6%), ceftriaxone (100%) and amikacin (6.2%).(143) Abdelkader et al.,(2017) observed that all MDR isolates (100%) showed resistance to penicillin and ampicillin, however, they showed lower resistance to meropenem (50%), levofloxacin (50%), amikacin (48%).(10) This high incidence of MDR and antimicrobial resistance is contributed to many reasons related to miss-use or overuse. (146) .This was confirmed according to the questionnaire included in the study. The questionnaire included a population of 155 where 22.6% of the population use the antibacterial agents only when prescribe by doctor and 34.2% do not complete the antibiotic course. Only 34.9% of the population does not use antibiotics for common cold and only 7.1% use the antibiotics based sensitivity culture test. Since 100% of MDR isolates were resistant to LE and since it is a broad spectrum antibiotic that is widely used, therefore it was the antibiotic of choice in our study. First, the MIC of LE was determined for standard strains E.coli ATCC25922 and S.aureus ATCC25923. The MIC was below the CLSI break points; 8µg/ml and 4µg/ml for each organism respectively (1.5µg/ml for each). Then, MIC of LE was
70
detected for each MDR isolate. The MIC ranged from 15.5 µg/mL to >250µg/mL, with 125µg/ml median and 128.8µg/ml mean. Many plants have been used because of their antimicrobial traits, which are due to compounds synthesized in the secondary metabolism of the plant. These products are known by their active substances, for example, the phenolic compounds which are part of the essential oils, as well as in tannin (147, 148). Thymus vulgaris (thyme) extracts and oil are known with antimicrobial properties, hence can be of great significance in therapeutic treatments. (149) Meanwhile, according to the questionnaire included in this study most of the population does not know that the thyme has antibacterial activity. Based on the fore mentioned reasons, this study included 4 thyme samples of different sources in the market in Alexandria. The samples were collected from Aldoha company (sample 1) Fathallah hypermarket (sample 2) , Carrefour hypermarket (sample 3) , a herbalist in smouha (sample 4).These samples were examined microscopically , then assessed for their antimicrobial activity against the standard strains E.coli ATCC25922 and S.aureus ATCC25923. The microscopical examination revealed that all the samples contained thyme but only sample 4 was the least contaminated. In sample 4, the labiacious glandular hair appeared orange in color indicating the high content of volatile oil in the sample. On the other hand, the aqueous extracts of sample 1 and sample 4 showed the best MIC for S.aureus ATCC25923and E.coli ATCC25922 (75mg/ml for each). Therefore, the MIC of the aqueous extracts of samples 1 and 4 was determined for the MDR isolates. Sample 1 aqueous extract showed MIC less than 150mg/ml in 38.5% of MDR isolates and 85.7% of the non-MDR isolates. Whereas, sample 4 aqueous extract showed MIC less than 150mg/ml in of the 53.8% MDR isolates and 71.4% of nonMDR isolates. The higher effect of sample 4 against MDR isolates can be contributed to the higher content of the volatile oil, as it was proved by the microscopical examination. El-Astal et al., (2005) showed that the different concentrations of aqueous extract of thyme had inhibitory action on E. coli, K. pneumoniae, and S. aureus. The detected MIC for these organisms was >20 mg/ml.(150) In contrast with our findings Nzeako et al., (2006) found that thyme water extract possessed antimicrobial activity only against S. aureus and concluded that the extract has limited or narrowed antimicrobial activity. (151) The difference in the individual findings may be due to the differences in the methods of extraction. Nzeako et al., (2006) prepared the water extract by boiling which may decrease the volatile oil content of the extract with the antimicrobial activity (151) in our study, the extract was prepared by decoction of thyme in hot water to avoid losing the volatile oil and to simulate the traditional use of thyme extracts.
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Finally, the effect of thyme aqueous extract of sample 4 on LE antimicrobial activity was estimated using a broth microdilution assay (for this combination). The MIC of LE alone for the MDR isolates was with 125µg/ml median, in the presence of thyme aqueous extract the MIC median dropped to 62.5µg/ml. In addition, a synergistic effect was reported as 2 fold, 4 fold and 83.3 fold decrease in the MIC in 4, 3 and one isolates(out of 13 MDR), respectively. Surprisingly, 2 isolates showed increased MIC i.e. antagonism. This may be contributed to the high concentration of the thyme extract with LE (150 mg/ml). Also, Gislene et al., (2000) mentioned that plant extracts were active against antibiotic resistant bacteria under very low concentration. Therefore, the high concentration (150 mg/ml) used with these isolates may be the reason for this unusual antagaonism. Synergistic effect between thyme alchoholic extracts or oil with different antibiotics was also reported by other authors. Gislene et al., (2000) studied the association of antibiotics with thyme alcoholic extracts and showed synergistic antibacterial activity against antibiotic-resistant bacteria, especially with with P. aeruginosa. (149) Allam et al, (2015) reported that the combination of thyme oil and ciprofloxacin gave synergistic activity, which proved to be more effective in inhibiting the growth of ulcer-forming Shigella flexneri. (152)
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SUMMARY AND CONCLUSION Nowadays, MDR bacteria have widely spread and increasing by time causing death. This may be because of the overuse or misuse of antibiotics. With the increase in antibiotic-resistant bacteria and the lack of new antibiotics being brought onto the market, alternative strategies need to be found to cope with infections resulting from drug-resistant bacteria. A possible solution may be to combine existing antibiotics with plant extracts or phytochemicals to enhance the efficacy of antibiotics. The aim of this work was to estimate the effect of aqueous extract of thyme as antibacterial agent on MDR isolates and also to test its effect on the LE antibacterial activity against MDR isolates. Identification by biochemical methods was employed on the 36 isolates. Gm. staining, IMVC, urease, catalase, oxidase tests were done. Antibiotic sensitivity testing using Disk Diffusion Susceptibility Testing (Kirby-Bauer Method) was carried on Amoxicillin (AMX) , Ceftriaxone (CTR) , Vancomycin (VA) , Meropenem (MRP) , Aztreonam (AT) , Co-trimoxazole (COT) , Levofloxacin (LE) , Amikacin (AK) in addition to Erythromycin (E) antibiotic disks. MIC of LE using broth micro dilution test was determined. First, LE was tested against standard strains (E.coli ATCC 25922 and S.aureus ATCC 25923) for validation. Second, LE was tested against the 13 MDR. Then, the 4 samples of thyme were tested against the two standard strains. Finally, MIC of the combination of both LE and thyme aqueous extract for the MDR isolates. Microscopical examination was done for the four thyme samples and revealed that all the samples were contained thyme but only sample 4 was the least contaminated. In sample 4, the labiacious glandular hair appeared orange in color indicating the high content of volatile oil in the sample. The questionnaires were carried out to assess the misuse of antibiotics and awareness of antibacterial effect of thyme on a population of 155 one. The results of this study revealed the following: 1) This study includes 36 isolates of different clinical samples. 2) The causative bacteria isolated from the samples were 12 E.coli, 7 K.pneumonae, 3 P.aeurginsoa, 14 S.aureus (2 MRSA). 3) The highest resistance among the isolates was for amoxicillin and aztreonam and highest sensitivity was for amikacin. 4) 36.11% of isolates were MDR. 5) The range of MIC of LE for MDR isolates was 15.5- >250µg/ml with the median 125µg/ml. 6) After microscopical examination 3 Thyme samples were contaminated and sample 4 was the least contaminated. 7) Aqueous extract of sample 1 and 4 showed the best MIC for S.aureus and E-coli standard strains and hence their MIC was estimated against the 13 MDR strains.
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8) Aqueous extract of sample 1 showed MIC less than 150 mg/ml in 38.5% of MDR isolates compared to sample 4 that showed MIC less than 150 mg/ml in 53.8% of MDR isolates. 9) MIC of LE to MDR isolates was with median 125µg/ml compared to 62.5µg/ml when was estimated in combination with aqueous extract of thyme sample 4. 10) The thyme aqueous extract of sample 4 caused 2 fold, 4 fold, & 83.3 fold decreases in 30.7%, 23.08% & 7.69% of MDR isolates respectively.
From this study we conclude that: 1-There is high incidence of MDR isolates from miscellaneous sources in which K. pneumonia isolates had the highest percentage. 2- Thyme aqueous extract corresponded different antimicrobial activity depended on degree of contamination of the herb. 3-Thyme sample of different source are contaminated (according to microscopial examination). 4-Thyme aqueous extract has antibacterial effect against both MDR and non MDR isolates. 5- Thyme aqueous extract is mainly synergistic when used with LE against MDR bacteria.
74
RECOMMENDATION 1- The continued emergence MDR bacteria is of grave concern and urgently calls for clinicians to wisely use antimicrobial agents for the treatment of bacterial infections. 2- Health-care professionals should be more interested to provide awareness for the society for the proper use of different antimicrobial agents. 3- Thyme aqueous extracts were active against MDR isolates and showed synergistic effect with levofloxacin, so further research is needed especially against different herbal aqueous extracts that can be easily applied producing alternative strategies to cope with infections of MDR bacteria 4- As the microscopical examination matched with the antimicrobial testing, therefore microscopical examination is strongly needed before the investigation of herbal antimicrobial activity.
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