PROCESSES OF ANTIMICROBIAL RESISTANCE

PROCESSES OF ANTIMICROBIAL RESISTANCE


Over eighty years ago Alexander Flemming discovered penicillin, and since then a multitude of natural and synthetic agents have been developed in our humankind fight against microorganisms, which comprise three main groups: bacteria, fungi and viruses. Antibiotic or antibacterial agents are selective poisons with activity only against bacteria, not viruses or fungi, since other specific agents kill these microbes selectively.

Antibiotics are the naturally occurring entities such as penicillins, while the term antimicrobial encompasses a range of synthetic agents like quinolon, as well as naturally derived ones. The key consideration of antimicrobial agents is their mode of action against bacteria. A typical bacterial cell antimicrobial and identify which sites certain antimicrobial agents act upon. Irrespective of their shape or size, this fundamental cellular structure permits certain generalizations.

Additionally the structure and composition of the cell wall, i.e. whether it is gram positive or gram negative, has a bearing on how effectively an antimicrobial agents can penetrate the bacterial cell. One way of viewing the role of antimicrobial agents and their selective responses to bacteria is by way of comparison to battlefield. In essence there are :
(1) The bacterial soft spots, or targets; key targets are cell wall, protein and DNA synthesis, substituent biosynthetic pathways.
(2) Our weapons, or antimicrobial agents; the various antimicrobial agents attack the different key target sites.
(3) The enemy’s response, or the ways of bacterial defense.

What processes of antimicrobial resistance are expressed by these gene sequences ? Basically there are four main mechanisms by which these processes occur; 1. Drug inactivation (enzyme inactivation), 2. Cellular access (decreased permeability), 3. Site modification (altered target site), 4. Biochemical Feedback (by pass). The main mechanisms of antimicrobial resistance processes are illustrated on the figure below; modified from Schentag JJ, 1999, (Operation Resistance 2000; The Terrain, Dynamics and Defense of Antimicrobial Resistance, Sheffield Dawson Publisher Ltd, 3-16).


A. DRUG INACTIVATION (enzyme inactivation)
The mechanism is a process by which bacterial enzymes either completely destroy the antimicrobial, or modify the drug by adding a molecule to it and rendering it incapable of specific activity. Examples of these two activities are β-lactamase; which destroy the β-lactam ring, the acetylation of chloramphenicol, the modification of aminoglycoside by acetylation or other additions.

B. CELLULAR ACCESS (decreased permeability)
The mechanism is controlled in terms of allowing entry to the bacterial cell, or an active process of ejecting drugs via an efflux pump. Coincidental with these processes is intrinsic resistance due to physical barriers – e.q. Gram-negative outer membrane provides resistance to some β-lactams.
Efflux pump mechanisms are increasingly recognized as a common method by which bacteria can remove a wide range of antimicrobials, from tetracyclines to quinolones.

C. SITE MODIFICATION (altered target site)
Site modification – involves alteration of the target site of an antimicrobial agent so that the fit is no longer sufficient to exert activity. Analogous to a lock and key situation, wherein a small change in the lock can render the key useless; a good example would be the alteration of the 23s ribosome to prevent macrolides, such as clarithromycin, from binding to the ribosome.

D. BIOCHEMICAL FEEDBACK (by pass)
Biochemical feedback – via target hyperproduction is best represented by the folic acids pathway in which an organism may deliberately over-produce an enzyme so as to saturate all the sulfonamide or trimethoprim present and still be able to catalyze the biosynthetic pathway.


See other contains :
1. Antimicrobial Resistance
2. Resistance Mechanism and Their Genetic Bases
3. Factors That Encourage the Spread of (Antimicrobial) Resistance

RESISTANCE MECHANISM AND THEIR GENETIC BASES

Resistance Mechanism and Their Genetic Bases

Genetic of Resistance
Bacteria posses a remarkable number of genetic mechanisms for resistance to microbials. They can undergo chromosomal mutations, express a latent chromosomal resistance gene, or acquire new genetic resistance material through direct exchange of DNA (by conjugation), through a bacteriophage (transduction), through extrachromosomal plasmid DNA via transformation. The information encoded in this genetic material enables a bacterium to develop resistance through three major mechanisms: production of an enzyme that will inactivate or destroy the antibiotic; alteration of the antibiotic target site to evade action of the antibiotic; or prevention of antibiotic access to the target site.

Examples of organisms that are known to possess resistance mechanisms of the various types are shown in tables, together with the genetic mechanism for the resistance. It is not unusual for a single bacterial strain found in a hospital to possess several of these resistance mechanisms simultaneously.




Modified from : Neu HC. The Crisis in antibiotic resistance. Science 1992;257

Factors That Encourage the Spread of (Antimocrobial) Resistance

Factors That Encourage the Spread of (Antimicrobial) Resistance



The emergence and spread of antimicrobial resistance are complex problems driven by numerous interconnected factors, many of which are linked to the misuse of antimicrobials and thus amenable to change. In turn, antimicrobial use is influenced by an interplay of the knowledge, expectations, and interactions of prescribers and patients, economic incentives, characteristics of a country's health system, and the regulatory environment.

Patient-related factors are major drivers of inappropriate antimicrobial use. For example, many patients believe that new and expensive medications are more efficacious than older agents. In addition to causing unnecessary health care expenditure, this perception encourages the selection of resistance to these newer agents as well as to older agents in their class.

Self-medication with antimicrobials is another major factor contributing to resistance. Self-medicated antimicrobials may be unnecessary, are often inadequately dosed, or may not contain adequate amounts of active drug, especially if they are counterfeit drugs. In many developing countries, antimicrobials are purchased in single doses and taken only until the patient feels better, which may occur before the pathogen has been eliminated. Inappropriate demand can also be stimulated by marketing practices. Direct-to-consumer advertising allows pharmaceutical manufacturers to market medicines directly to the public via television, radio, print media, and the Internet. In particular, advertising on the Internet is gaining market penetration, yet it is difficult to control with legislation due to poor enforceability.

Prescribers' perceptions regarding patient expectations and demands substantially influence prescribing practice. Physicians can be pressured by patient expectations to prescribe antimicrobials even in the absence of appropriate indications. In some cultural settings, antimicrobials given by injection are considered more efficacious than oral formulations. Such perceptions tend to be associated with the over-prescribing of broad-spectrum injectable agents when a narrow-spectrum oral agent would be more appropriate. Prescribing “just to be on the safe side" increases when there is diagnostic uncertainty, lack of prescriber knowledge regarding optimal diagnostic approaches, lack of opportunity for patient follow-up, or fear of possible litigation. In many countries, antimicrobials can be easily obtained in pharmacies and markets without a prescription.

Patient compliance with recommended treatment is another major problem. Patients forget to take medication, interrupt their treatment when they begin to feel better, or may be unable to afford a full course, thereby creating an ideal environment for microbes to adapt rather than be killed. In some countries, low quality antibiotics (poorly formulated or manufactured, counterfeited or expired) are still sold and used for self-medication or prophylaxis.

Hospitals are a critical component of the antimicrobial resistance problem worldwide. The combination of highly susceptible patients, intensive and prolonged antimicrobial use, and cross-infection has resulted in nosocomial infections with highly resistant bacterial pathogens. Resistant hospital-acquired infections are expensive to control and extremely difficult to eradicate. Failure to implement simple infection control practices, such as handwashing and changing gloves before and after contact with patients, is a common cause of infection spread in hospitals throughout the world. Hospitals are also the eventual site of treatment for many patients with severe infections due to resistant pathogens acquired in the community. In the wake of the AIDS epidemic, the prevalence of such infections can be expected to increase.

Veterinary prescription of antimicrobials also contributes to the problem of resistance. In North America and Europe, an estimated 50% in tonnage of all antimicrobial production is used in food-producing animals and poultry. The largest quantities are used as regular supplements for prophylaxis or growth promotion, thus exposing a large number of animals, irrespective of their health status, to frequently subtherapeutic concentrations of antimicrobials. Such widespread use of antimicrobials for disease control and growth promotion in animals has been paralleled by an increase in resistance in those bacteria (such as Salmonella and Campylobacter) that can spread from animals, often through food, to cause infections in humans.

Source : WHO Media Center (November 17 2009)

Antimicrobial Resistance

ANTIMICROBIAL RESISTANCE

Antimicrobial resistance results in increased morbidity, mortality, and cost of health care. Prevention of the emergence of resistance and the dissemination of resistant microorganisms will reduce these adverse effects and their attendant costs. Appropriate antimicrobial stewardship that includes optimal selection, dose, and duration of treatment, as well as control of antibiotic use, will prevent or slow the emergence of resistance among microorganisms. A comprehensively applied infection control program will interdict the dissemination of resistant strains.

Stuart Levy, MD proposed a provocative hypothesis: the intensity of antibiotic use in a population may be the most important factor in selection of resistance. Moreover, there may be a “threshold” for such selection that may differ for an individual, as compared to a population, and from one population to another. This may explain why, in intensive-care units, where there is usually a small population undergoing intensive antibiotic therapy or prophylaxis, resistance tend to be more common, pathogens are more often multiply resistant, and spread within the population is more likely. The same concept might explain resistance problems in the poultry manufacturing industry and in other setting where antibiotic use is intensive within a small and confined population. [1]

The Prevention of Antimicrobial Resistance In Hospital

The 1990s is the era of multidrug resistance. Some bacteria causing several kinds of human infectious diseases are resistant to multiple antibiotics and are continuing to increase. Resistant infections confront and thwart the treatment of some patients in the community as well as in the hospital. Major resistant hospital organisms include Staphylococcus aureus, enterococcus, Klebsiella, Enterobacter, Escherichia coli, Pseudomonas and more recently Acinetobacter. Multidrug resistant bacteria causing community acquired infections include pneumococcus, gonococcus, Mycobacterium tuberculosis, group A streptococci and E. coli. [See : NFID]

It therefore is recommended that hospitals, large and small, with and without perceived problems of bacterial resistance to antimicrobials, do the following :

1. Establish a system for monitoring bacterial resistance and antibiotic usage.
2. Establish practice guidelines and other institutional policies to control the use of antibiotics, and respond to data from the monitoring system.
3. Adopt the recommendations of the Centers for Disease Control and Prevention’s (CDC) “guidelines for Isolation Precautions in Hospitals” as concerns the isolation of patients colonized or infected with resistant microorganisms.
4. Utilize hospital committees to develop local policies and to evaluate and adopt, as appropriate, guidelines from state advisory boards and national societies.
5. Recognize that the financial well-being of the institution and the health of its patients are at stake be accountable for the implementation and enforcement of policies adopted by hospital committees.
6. By measuring outcomes, evaluate the effectiveness of the policies that are put in place.

The Joint Commission on the Accreditation of Healthcare Organizations, or a similar review organization skilled in oversight functions, should take into account the priority hospitals give to antimicrobial resistance; policies, procedures, and measurements hospitals put into place; and evidence of ongoing review of data to judge the effectiveness of the plan.

Recommendations for future studies to examine. Means to prevent and reduce the development and dissemination of antimicrobial resistance :

1. The development and testing of protocols for measuring the effect of a variety of antimicrobial usage controls is recommended for use in multiple hospitals to determine the most effective ways to prevent and reduce antimicrobial resistance in specific species to specific antimicrobial.
2. Pharmaceutical industry and governmental support for such studies is recommended and encouraged.
3. It is recommended that educational methods, including those that are interactive and computer-based, be developed to improve the appropriateness of antimicrobial prescribing.
4. It is recommended that protocols to evaluate antimicrobial resistance include the ability to relate resistance rates to the “defined drug density” (the amount of antimicrobial used per geographyc area per unit time).
5. The transfer of resistance determinants in situ in a patient population is very poorly understood. First, the genetics of resistance transfer, the construction of composite transposons, and the actual mechanism of dissemination of these elements in situ, especially intergeneric transfer within the gram-positive bacteria, all should be studied further.
6. Method for interdicting transfer of resistance requires further study, especially in the behavioral area. Novel approaches to this area are needed.
7. The efficacy of various levels of infection control precaution should be documented by controlled trials.
8. Controlled studies of behavior modification, including novel approaches, to permit the efficient application of recommended guidelines within hospitals are recommended.
9. The efficacy of quality improvement approaches to control resistance should be studied.

Microbiologic Diagnostic Tests
[Minimum Bactericidal Concentration (MBC) and Minimum Inhibitory Concentration (MIC)]
Descriptions :
Varying concentrations of an antimicrobial are prepared in liquid growth medium or on solid medium. A standard number of organisms are added to each test tube or agar plate in incubated. After the appropriate incubation period, the test tubes or agar plate that contains the lowest concentration of antibiotic that prevents visible growth is considered the MIC. The tubes that demonstrate no growth are then placed onto an antibiotic free agar an incubated for 24 hours. The MBC is the lowest concentration of drug that results in a 99,9 % reductions in the initial bacterial count.

The MIC is quantitative measure of particular drug’s activity against identified bacteria. This allows the comparison of antibiotics in order to choose the antibiotic with the lowest MICs for likely eradication of infection by use of usual doses of an antimicrobial agent. The MBC can determine if the antibiotic will kill the organism (i.e., bactericidal).

Clinical implication :

1. Although useful in selecting the antimicrobial agent, MICs are not reliable predictors of success or failure of drug therapy.
2. Susceptible organism are those with the lowest MICs that will be effectively treated by the antimicrobial.
3. Moderately susceptible organism are those less likely to be effectively treated and should therefore be treated with maximum doses of the antimicrobial.
4. Resistant organism are those with high MICs to the tested antimicrobial suggesting probable failure of treatment.

Guidelines for Testing Bacterial Pathogens for Antimicrobial Resistance

A major role of the clinical microbiology laboratory is to provide antimicrobial susceptibility testing data on bacterial isolates to guide clinicians in their choice of anti-infective therapy. Susceptibility testing data can serve both as guide to therapy and, in some instances, as an initial means of strain typing for investigations of potential outbreaks of infection.
The guidelines for testing bacterial pathogens for antimicrobial resistance are shown in table. This guide is adapted from The National Committee for Clinical Laboratory Standards Document M100S6 for initial susceptibility testing and reporting of antimicrobial agents for several bacterial pathogen groups . (Not all drugs should be reported routinely).


)*Laboratories may screen for penicillin resistance in pneumococci by using a 1 μg oxacillin disk. Organisms with zone diameters of ≥20 mm are considered susceptible to all β-lactam drugs. Isolates with zona diameters of ≤19mm should be tested by minimum inhibitory concentration method against both penicillin and cefotaxime or ceftriaxone, especially if the organism is causing invasive disease.

by Andi Surya Amal,SSi,Apt,MKes
e.mail : suryaamal88@gmail.com

Reference :

1. Shlaes DM. et al. “Society for Healthcare Epidemiology of America and Infectious Diseases Society of America Joint Committee on the Prevention of Antimicrobial Resistance: Guidelines for the Prevention of Antimicrobial Resistance in Hospitals”. Infection Control and Hospital Epidemiology, Vol. 18 No. 4, 1997, 275-291
2. Boh LE. Clinical Clerkship Manual. Applied Therapeutics, Inc, Vancouver, Washington, 1996, 5-57,
3. National Committee for Clinical Laboratory Standards Document M100S6 for initial susceptibility testing and reporting of antimicrobial agents for several bacterial pathogen groups.
4. National Foundation for Infectious Diseases

(Please see on the video below; why antimicrobial resistance is a serious problem in both the hospital and community settings. And then, do we have any solution ?)

Clinical Laboratory Test of Lipoprotein Panel

CLINICAL LABORATORY TEST OF LIPOPROTEIN PANEL

Include cholesterol, low density lipoprotein (LDL) cholesterol, trigliserides, and hight density lipoprotein (HDL) cholesterol.

CHOLESTEROL

Normal Values :
Desirable : 0 – 199 mg/dL [SI = 0-5.17 mmol/L]
Borderline : 200 – 239 mg/dL [SI = 5.2-6.21 mmol/L]
High Risk : ≥ 240 mg/dL [SI = ≥6.22 mmol/L]

Description :
Cholesterol exists in muscle, RBCs, and cell membranes. It is used by the body to form steroid hormones, bile acids, and cell membranes. Elevated cholesterol concentrations are associated with atherosclerosis and an increased risk of coronary artery disease.

Clinical Implication :
1.Increased levels of > 200 mg/dL are considered to be high and require a triglyceride evaluation. Associated conditions include cardiovascular disease, atherosclerosis, Type II familial hypercholesterolemia, and obstructive jaundice.
2.Decreased levels are associated with malabsorption, liver disease, sepsis, and pernicious anemia.
3.A patient must fast for 12 hours before blood is obtained to measure the serum concentration of cholesterol, and should maintain a “normal” diet for 7 days prior. Alcohol should not be consumed 24 hours before testing and all lipid-lowering drugs should be withheld.

LOW DENSITY LIPOPROTEINS (LDL)

Normal Values (Adult) :
Desirable : < 130 mg/dL [SI = <3 data-blogger-escaped-.36="" data-blogger-escaped-br="" data-blogger-escaped-mmol="">Borderline : 130-159 mg/dL [SI = 3.36-3.11 mmol/L]
High Risk : ≥ 160 mg/dL [SI = >4.13 mmol/L]

Description :
LDL are beta cholesterol esters

clinical Implication :
High LDL values are associated with coronary vascular disease or familial hyperlipidemia. Levels may also be elevated samples taken from non fasting subjects. Levels may also be elevated in types IIa and IIb hyperliproteinemia, diabetes mellitus, hypothyroidism, obstructive jaundice, nephrotic syndrome, familial and idiopathic hyperlipidemia, and with the use of estrogens or estrogens-containing oral contraceptives.
Decreased LDL levels may occur in patients with hypoproteinemia.

HIGH DENSITY LIPOPROTEINS (HDL)

Normal Values : Adult : 30-70 mg/dL [SI = 0.78-1.18 mmol/L]
Description : HDL are the products of liver and intestinal synthesis and triglyceride catabolism.

Clinical Implication :

1. There is an inverse relationship between HDL-cholesterol levels and the incidence of coronary artery disease.
2. Increased HDL cam occur in chronic alcoholism, primary biliary cirrhosis, and subsequent to exposure to industrial toxins or polychlorinated hydrocarbons. Patients taking clofibrate, estrogens, nicotinic acids, oral contraceptives, and phenytoin may have increased HDL levels.
3. Decreased HDL can occur in patients with cystic fibrosis, severe hepatic cirrhosis, diabetes mellitus, Hodgkin’s disease, nephritic syndrome, malaria, and some acute infections. Patients receiving probucal, hydrochlorothiazide, progestins, and prolonged parenteral nutrition may have decresed HDL levels.

TRIGLYCERIDES

Normal Values (desirable adult) :
Male : 40-160 mg/dL [SI = 0.44-1.88 mmol/L]
Female : 35-135 mg/dL [SI = 0.4-1.53 mmol/L]
Description :
Triglycerides are found in plasma lipids are chylomicrons and very low density lipoproteins (VLDL)

Clinical Implications :

1. Triglycerides are increased in patients with alcoholic cirrhosis, alcoholism, anorexia nervosa, biliary cirrhosis, biliary obstruction, cerebral thrombosis, chronic renal failure, diabetes mellitus, Down’s syndrome, hypertension, idiopathic hypercalcemia, hyperlipoproteinemia (type I, IIb, III, IV and V), gout, ischemic heart disease, hypothyroidism, pregnancy, acute intermittent porphyria, respiratory distress syndrome, thalassemia major, viral hepatitis, and Werner’s syndrome.
2. Cholestyramine, corticosteroids, estrogens, ethanol, high carbohydrate diets, intravenous miconazole, oral contraceptives, and spironolactone can increase triglycerides.
3. Decreased triglycerides may be seen with chronic obstructive lung disease, hyperparathyroidism, intestinal lymphangiectasia, severe parenchymal liver disease, malabsorption, and malnutrition.
4. Ascorbic acid, asparaginase, clofibrate, and heparin can decrease triglyceride serum concentrations.

Source : Boh, L.E. 1996. Clinical Clerkship Manual. Applied Threpuetics, Inc. Washington, 5-33, 5-34, 5-36

Oral Glucose Tolerance Test (OGTT)

ORAL GLUCOSE TOLERANCE TEST (OGTT)


DESCRIPTION
A blood sample to determine the fasting (baseline) blood glucose for the patient is drawn first. Then, the patient drinks a highly concentrated glucose solution (75 gm/300 mL for nonpregnant adults and 100 gm/300 mL for pregnant women). Subsequently, a timed series of blood glucose tests is performed at 30, 60, 90, and 120 minutes for nonpregnant adults and 1,2, and 3 hours for pregnant women to determine the rate of removal of glucose from the bloodstream. This test is not performed if the fasting blood sugar is > 140 mg/dL since virtually all patients will have blood glucose determinations that meet or exceed the diagnostic criteria for diabetes mellitus.

PURPOSE
OGTT is used to diagnose [or rule (R/O)] overt diabetes, glucose intolerance, cushing’s syndrome, and acromegaly.

FINDINGS

A.Normal
Adult, Non-pregnant :
Fasting blood glucose 115 < mg/dL
After 75 gm of oral glucose : [30 min < 200 mg/dL], [60 min < 200 mg/dL], [90 min < 200 mg/dL], [120 min < 140 mg/dL]

B.Abnormal
Adult :

a.Diabetes Mellitus
Sustained elevated blood glucose levels during at least 2 OGTTs.
The 2 hour sample and at least one other between 0 and 2 hr > 200 mg/dL.

b.Impaired Glucose Tolerance :
[2 hr OGTT blood glucose level of 140 to 200 mg/dL],
[0 to 2 hr OGTT blood glucose level > 200 mg/dL]

c.Gestational Diabetes
This diagnosis may be made if 2 blood glucose values equal or exceed : [Fasting : 105 mg/dL], [1 hr : 190 mg/dL], [2 hr : 165 mg/dL], [3 hr : 145 mg/dL]

PHARMACY IMPLICATIONS

1. Patient should be instructed to fast overmight (12 hr)
2. 75 gm glucose (Glucola) is given to nonpregnant women on morning of the test.
3. Insulin or oral hypoglicemics should not be given until after test is completed.
4. The following drugs should be discontinued at least 3 days before the test: hormones (including oral contraceptives), alcohol, salicylates, indomethacin, diuretics (especially thiazide), guanathidine, hypoglycemic agents, propranolol, corticosteroids, MAOIs, lithium, nicotinic acid, phenothiazines, and ascorbic acid.

Source : Boh, L,E (ed), 1996, Clinical Clerkship Manual, Applied Therapeutics,Inc. Washington, 6-19

GENETIC ASPECT OF LGI CANCER

GENETIC ASPECT OF LGI CANCER


The development of colorectal cancer from normal ephithelium is a multistep process. The process had been proven to be a sequence of genetical changes including activation of protooncogens as well as inactivation of tumor suppressor genes. Numbers of these genes, which involved in complex process of colorectal tumorigenesis, are inactivation of APC gene on chromosome 5q (80% of colorectal cancer), activation of ras oncogene on chromosome 5q, 17p and 18q (50% of colon cancer), inactivation of p53 tumor suppressor gene on chromosome 17p, deleted in colon cancer (DCC) gene at chromosome 18q22 (70% of colorectal cancer) and the presence of microsatelite instability (MSI) on chromosome 2p, 2q, 3p, and 7p in Hereditary Nonpolyposis Colon Cancer (HNPCC).

A colon cancer progression model is initiated by mutation of APC gene and abnormal methylation of DNA which cause hyperproliperative epithelium followed by mutation on K-ras gene which cause the progress of the hyperproliperative epithelium to be adenoma and DC LOH and LOH on chromosome 18q which cause the progress of adenoma to be carcinoma. Finally, the mutation of p53 gene lead to the end of carcinogenesis process as invasive cancer. The model show a significant relation between molecular changes and morphological features of colorectal tomurigenesis.

(By Syarifuddin Wahid, Division of Clinical Pathology Department of Hasanuddin University of Medicines, Indonesia)