This drug plays a role in empiric therapy because it is active against β-lactamase–producing gram-positive

and gram-negative organisms, anaerobes, and P. aeruginosa.

Meropenem and doripenem have antibacterial activity similar to that of Imipenem. However, ertapenem is

not an alternative for P. aeruginosa coverage because most strains exhibit resistance.

Ertapenem also lacks coverage against Enterococcus species and Acinetobacter species.

Trimethoprim-sulfamethoxazole

A combination called co-trimoxazole shows greater antimicrobial activity than equivalent quantities of

either drug used alone. The synergistic antimicrobial activity of Co-trimoxazole results from its inhibition of

32

 Arranged by Sarah Mohssen

Section I– Microbiology By Dr. Mohammed Ayad

two sequential steps in the synthesis of tetrahydrofolic acid: sulfamethoxazole inhibits incorporation of

PABA into folic acid, and trimethoprim prevents reduction of dihydrofolate to tetrahydrofolate. It is

effective in treating urinary tract infections and respiratory tract infections as well as in Pneumocystis

jiroveci pneumonia and ampicillin- and chloramphenicol-resistant systemic Salmonella infections.

It has activity versus methicilin-resistant S. aureus and can be particularly useful for community acquired

skin and soft tissue infections caused by this organism.

Drug Resistance

Intrinsic and acquired drug resistance modes:

In some species antimicrobial resistance is an intrinsic or innate property. For example, E. coli is

intrinsically resistant to Vancomycin because Vancomycin is too large to pass though porin channels in their

outer membrane.

Gram-positive bacteria, on the other hand, do not possess an outer membrane and thus are not intrinsically

resistant to Vancomycin. Bacteria also can acquire resistance to antimicrobial agents by genetic events

such as mutation, conjugation, transformation, transduction and transposition.

Mutation: Chromosomal resistance develops as a result of spontaneous mutation in a locus that controls

susceptibility to a given antimicrobial agent. Spontaneous mutation occurs at a relatively low frequency but,

when the bacteria are exposed to the antibiotic, only the mutant cell survives. It then multiplies and gives

rise to a resistant population. Spontaneous mutations may also occur in plasmids. For example, mutations in

plasmids containing genes for beta-lactamase enzymes can result in altered beta-lactamases often with

extended activity.

Conjugation: Bacteria often contain extrachromosomal genetic elements called plasmids, many of which

carry genes for antimicrobial resistance. When two bacterial cells are in close proximity, a bridge-like

structure known as a pilus forms between them. This allows a copy of the plasmid as it is replicated, to be

transferred to another cell. The result is a bacterium that expresses the antimicrobial resistance encoded in

the plasmid.

Transformation: Bacteria may encounter naked fragments of DNA that carry antimicrobial resistance

genes. These fragments are taken into the cell by a process called transformation. The DNA fragment is

incorporated into the host cell chromosome by recombination and the resulting cell is resistant.

Transduction: When bacterial viruses (bacteriophage) are multiplying in the cytoplasm of a bacterium,

fragments of DNA from plasmids or chromosomes may by chance be packaged in a viral coat and enter

another host cell. When the fragments contain genes for resistance to an antimicrobial agent they can confer

resistance in the new host cell.

Transposition: Specialized genetic sequences known as transposons are “mobile” sequences that have the

capability of moving from one area of the bacterial chromosome to another or between the chromosome and

plasmid or bacteriophage DNA. Since transposon DNA can carry genes for antimicrobial resistance they

have contributed to the development of plasmids encoding genes for multiple antibiotic resistances. Some

transposons are capable of moving from one bacterium to another without becoming incorporated into a

chromosome, a plasmid or a bacteriophage.

33

 Arranged by Sarah Mohssen

Section I– Microbiology By Dr. Mohammed Ayad

NOTES:

 Penicillin G resistance of S. aureus from 3% to > 90%

 Multidrug-resistant S. aureus = MRSA

 Vancomycin-resistance

 Evolution of drug resistance:

 Vertical evolution due to spontaneous mutation

 Horizontal evolution due to gene transfer

A variety of mutations can lead to antibiotic resistance

1. Enzymatic destruction of drug

2. Prevention of penetration of drug

3. Alteration of drug's target site

4. Rapid ejection of the drug

Resistance genes are often on plasmids or transposons that can be transferred between bacteria.

Figure shows Resistance Modes to Antibiotics

Misuse of antibiotics selects for resistance mutants; Misuse includes

 Using outdated or weakened antibiotics

34

 Arranged by Sarah Mohssen

Section I– Microbiology By Dr. Mohammed Ayad

 Using antibiotics for the common cold and other inappropriate conditions

 Using antibiotics in animal feed

 Failing complete the prescribed regimen

 Using someone else's leftover prescription

Classification of the Antibiotics

I- β-Lactam antibiotics which includes:

First: Penicillin which includes:-

1- Benzylpenicillins like

Penicillin G (benzylpenicillin sodium, procaine benzylpenicillin, benzathine penicillin)

2- Phenoxy-penicillins (oral penicillins) like Penicillin V , Propicillin

3- Penicillinase resistant penicillins (anti-staphylococcal penicillins) like Oxacillin, Dicloxacillin,

Flucloxacillin

4- Amino benzyl penicillins like Ampicillin, Amoxicillin

5- Ureidopenicillins (broad-spectrum penicillins) like Mezlocillin, Piperacillin

6- β-Lactam inhibitors like Ampicillin with or without sulbactam, Amoxicillin with or without

clavulanate, Piperacillin with or without tazobactam

Second: Cephalosporins which includes:-

1- (First generation) Cephalosporins like

 Cefazolin

 Cefalexin (oral)

 Cefadroxil (oral)

2- (Second generation) Cephalosporins like

 Cefuroxime

 Cefotiam

 Cefuroxime axetil

 Cefaclor (oral)

 Loracarbef

3- (third and fourth generation)

 Cefotaxime

 Ceftriaxone

 Ceftazidime

 Cefepime

 Cefixime (oral)

 Cefpodoxime

35

 Arranged by Sarah Mohssen

Section I– Microbiology By Dr. Mohammed Ayad

 Proxetil (oral)

 Ceftibuten (oral)

Third: other like

 Monobactams like Aztreonam

 Carbapenems like Imipenem, Meropenem, Ertapenem, and Doripenem

 β-Lactamase inhibitors like Clavulanic acid, Sulbactam, Tazobactam

II- Other drugs un related to β–lactam ring drugs like:

1- Aminoglycosides like Streptomycin, Gentamicin, Tobramycin, Netilmicin, and Amikacin

2- Tetracyclines like Tetracycline, long acting Doxycycline, oxytetracycline, and Minocycline

3- Quinolones or Fluoroquinolones like

Group I: Norfloxacin

Group II: Enoxacin, Ofloxacin, Ciprofloxacin

Group III: Levofloxacin

Group IV: Moxifloxacin

The usage of Quinolones groups in clinical situations:

I: Indications essentially limited to UTI

II: Widely indicated

III: Improved activity against Gram-positive and atypical pathogens

IV: Further enhanced activity against Gram-positive and atypical pathogens, also against anaerobic bacteria

4- Lincosamides like Clindamycin

5- Azol derivatives like Miconazole, Ketoconazole, Fluconazole, Itraconazole, Voriconazole, and

Posaconazole

6- Nitroimidazoles like Metronidazole

Glycopeptides antibiotics like Vancomycin, Teicoplanin, and Telavancin

Macrolides like Erythromycin, Spiramycin, Roxithromycin, Clarithromycin, and Azithromycin

7- Polyenes like Amphotericin B, Nystatin

8- Glycylcyclines like Tigecycline

9- Echinocandins like Caspofungin, Anidulafungin, Micafungin

10- Streptogramines like Quinupristin / dalfopristin

11- Ketolides like Telithromycin

12- Oxazolidinones like Linezolid

13- Lipopeptides like Daptomycin

14- Epoxides like Fosfomycin

15- Polymyxins like Colistin (polymyxin E), Polymyxin B

16- Ansamycins like Rifampicin

36

 Arranged by Sarah Mohssen

Section I– Microbiology By Dr. Mohammed Ayad

Figure shows antibiotics discs response against isolates in Mueller-Hinton agar plate

Antibiotic Assays to Guide Chemotherapy

Resistance to antimicrobial agents has resulted in morbidity and mortality from treatment failures and

increased health care costs.

Although defining the precise public health risk and estimating the increase in costs is not a simple

undertaking, there is little doubt that emergent antibiotic resistance is a serious global problem.

Appropriate antimicrobial drug use has unquestionable benefit, but physicians and the public frequently use

these agents inappropriately. Inappropriate use results from physicians providing antimicrobial drugs to treat

viral infections, using inadequate criteria for diagnosis of infections that potentially have a bacterial

etiology, unnecessarily prescribing expensive, broad-spectrum agents, and not following established

recommendations for using chemo prophylaxis.

Widespread antibiotic usage exerts a selective pressure that acts as a driving force in the development of

antibiotic resistance. The association between increased rates of antimicrobial use and resistance has been

documented for nosocomial infections as well as for resistant community acquired infections.

As resistance develops to "first-line" antibiotics, therapy with new, broader spectrum, more expensive

antibiotics increases, but is followed by development of resistance to the new class of drugs.

Resistance factors, particularly those carried on mobile elements, can spread rapidly within human and

animal populations. Multidrug-resistant pathogens travel not only locally but also globally, with newly

introduced pathogens spreading rapidly in susceptible hosts.

Antibiotic resistance patterns may vary locally and regionally, so surveillance data needs to be collected

from selected sentinel sources. Patterns can change rapidly and they need to be monitored closely because of

their implications for public health and as an indicator of appropriate or inappropriate antibiotic usage by

physicians in that area.

The results of in-vitro antibiotic susceptibility testing, guide clinicians in the appropriate selection of initial

empiric regimens and, drugs used for individual patients in specific situations. The selection of an antibiotic

37

 Arranged by Sarah Mohssen

Section I– Microbiology By Dr. Mohammed Ayad

panel for susceptibility testing is based on the commonly observed susceptibility patterns, and is revised

periodically.

Principle

The principles of determining the effectivity of a noxious agent to a bacterium were well enumerated by

Rideal ,Walker and others at the turn of the century, the discovery of antibiotics made these tests (or their

modification) too cumbersome for the large numbers of tests necessary to be put up as a routine. The ditch

plate method of agar diffusion used by Alexander Fleming was the forerunner of a variety of agar diffusion

methods devised by workers in this field.

The Oxford Group used these methods initially to assay the antibiotic contained in blood by allowing the

antibiotics to diffuse out of reservoirs in the medium in containers placed on the surface.

With the introduction of a variety of antimicrobials it became necessary to perform the antimicrobial

susceptibility test as a routine. For this, the antimicrobial contained in a reservoir was allowed to diffuse out

into the medium and interact in a plate freshly seeded with the test organisms.

Even now a variety of antimicrobial containing reservoirs are used but the antimicrobial impregnated

absorbent paper disc is by far the commonest type used. The disc diffusion method of AST is the most

practical method and is still the method of choice for the average laboratory.

Automation may force the method out of the diagnostic laboratory but in this country as well as in the

smaller laboratories of even advanced countries, it will certainly be the most commonly carried out

microbiological test for many years to come. It is, therefore, imperative that microbiologists understand the

principles of the test well and keep updating the information as and when necessary. All techniques involve

either diffusion of antimicrobial agent in agar or dilution of antibiotic in agar or broth. Even automated

techniques are variations of the above methods.

38

 Arranged by Sarah Mohssen

Section I– Microbiology By Dr. Mohammed Ayad

Factors Influencing Antimicrobial Susceptibility Testing like pH The pH of each batch of Mueller-Hinton

agar should be checked when the medium is prepared. The exact method used will depend largely on the

type of equipment available in the laboratory. The agar medium should have a pH between 7.2 and 7.4 at

room temperature after gelling. If the pH is too low, certain drugs will appear to lose potency (e.g.,

aminoglycosides, Quinolones, and macrolides). While other agents may appear to have excessive activity

(e.g., tetracyclines). If the pH is too high, the opposite effects can be expected.

Moisture

If excess surface moisture is present, the plates should be placed in an incubator (35°C) or a laminar flow

hood at room temperature with lids ajar until excess surface moisture is lost by evaporation (usually 10 to 30

minutes). The surface should be moist, but no droplets of moisture should be apparent on the surface of the

medium or on the Petri dish covers when the plates are inoculated.

Testing strains that fail to grow satisfactorily

Only aerobic or facultative bacteria that grow well on unsupplemented Mueller-Hinton agar should be tested

on that medium.

Certain fastidious bacteria such as Haemophilus spp., N. gonorrhoeae, S. pneumoniae, and viridans and ßhaemolytic streptococci do not grow sufficiently on unsupplemented Mueller-Hinton agar. These organisms

require supplements or different media to grow.

Methods of Antimicrobial Susceptibility Testing

Antimicrobial susceptibility testing methods are divided into types based on the principle applied in each

system; they include:

Diffusion Kirby-Bauer method

Dilution Minimum Inhibitory Concentration include Broth and Agar Dilution

Diffusion and Dilution E-Test method

39

 Arranged by Sarah Mohssen

Section I– Microbiology By Dr. Mohammed Ayad

Lecture Three

Gram-positive bacilli

Aerobic non-spore forming bacilli

Usual Corynebacterium

Unusual Arcanobacterium, Rothia

Acid-fast Rhodococcus, Nocardia, Gordonia

Aerotolerant anaerobes non-spore forming bacilli