BACTERIOLOGY

PRINCIPLES OF
ANTIMICROBIAL ACTION
Gerard Andrew Ramos, RMT
South Seed LPDH College
OVERVIEW
Antibiotic
chemicals produced by microorganisms that inhibit the
growth of other microorganisms
Antimicrobial agents
collective term which refers to agents that destroy
microorganisms through inhibiting their development of
action. They may be obtained either from microorganisms
or synthetically in the laboratory
Antibacterial
Antiviral
Antifungal
Antiparasitic
OVERVIEW
Bactericidal agent
Kills target microorganism, resulting to cell lysis

Bacteriostatic agent
Inhibit bacterial growth but do not kill the organism
Rely on the host immune system to follow through to
destroy the organism


OVERVIEW
Narrow spectrum antibiotics
Antimicrobial agents with a limited spectrum of action
Example: Penicillin G is effective only against gram-
positive bacteria

Broad spectrum antibiotics
Have action against both gram-positive and gram-negative
bacteria, however disadvantage is the inhibition or
destruction of the normal flora of the patient.
Example: Tetracycline

SELECTION OF ANTIMICROBIAL AGENTS
Antimicrobial agent has strong activity against microorganism
Agent has low toxicity toward the host
Agent is least toxic toward the normal flora of host; narrow
spectrum antibiotics should be used when possible
Pharmacologic properties of antibiotic
Host immune status: use of steroids, prolonged antibiotic therapy
Host organ function, consideration toward renal function for drug
elimination and hepatic function for biotransformation
Underlying medical disease in host, including circulatory problems
Age of patient; certain agents are toxic to very young and old pts
Site of infection
Route of administration
Does the antibiotic cross the placenta which can be toxic to fetus?
Does the antibiotic cross the blood-brain barrier?
Can effective blood or tissue levels be achieved?
Is the host allergic or hypersensitive to the agent?

BACTERIAL RESISTANCE TO ANTIMICROBIAL AGENTS
Intrinsic Resistance
Results from the normal genetic, structural or physiologic
state of microorganism
This resistance is naturally and consistently inherited
characteristic, thus being predictable
Ex. Staphylococcus saprophyticus to novobiocin

Acquired Resistance
Results from altered cellular physiology and structure
caused by changes in a microorganisms genetic makeup
Could be a trait assoc. with only some strains of particular
microorganism, but not others, thus being unpredicatble
May result from chromosomal mutations or from plasmids

EXAMPLES OF INTRINSIC RESISTANCE

ACQUIRED RESISTANCE
In chromosomal mutations, the antibiotic exerts selective
pressure on the susceptible strain of the organism. The resistant
mutant strain overgrows the susceptible bacterial cells, and the
new population consists now of resistant cells
Plamids can also be a source of acquired resistance, wherein
they act independently from the chromosome, and the resistance
genes may be transferred from chromosome to plasmid, or vice
versa
Transposons known as jumping genes can insert pieces of
DNA or carry a part of plasmids to other bacteria.
Another form of acquired resistance is inactivation of the
antimicrobial agent, where bacterial enzymes convert the drug
into an inactive form.
Example: Beta-lactamases group of enzymes that convert
the beta-lactams into inactive forms
ANTIBIOTIC INTERACTIONS
Autonomous: Indifferent
Results obtained when two drugs is equal to result with most
effective drug by itself
Antagonistic
Result when two drugs is significantly less than the autonomous
result
Additive
Result when two drugs combined would result to the sum of the
drugs effects.
Synergistic
Result when two drugs is significantly greater than additive
response. The antibiotics may have different modes of action or
function at different sites.
Ex. Penicillin derivative + aminoglycoside shows synergistic
action to treat enterococcal endocarditis

BACTERIOLOGY
LABORATORY METHODS AND
STRATEGIES FOR ANTIMICROBIAL
SUSCEPTIBILITY TESTING
Gerard Andrew Ramos, RMT
South Seed LPDH College
ANTIMICROBIAL SUSCEPTIBILITY TESTING
Goal: Determine whether the bacterial etiology of concern is
capable of expressing RESISTANCE to the
antimicrobial agents that are potential choices of
therapy.
Testing for intrinsic resistance is not needed anymore
Components of antimicrobial susceptibility testing that are
STANDARDIZED include the following:
Bacterial inoculum size
Growth medium (Mueller-Hinton medium)
Incubation atmosphere
Incubation temperature
Incubation duration
Antimicrobial concentration tested



LIMITATIONS OF STANDARDIZATION
In vitro laboratory tests do not necessarily dictate what
would be the definite outcome of drug actions to in vivo
environment.
Factors that play roles in patient clinical outcome:
Antibiotic diffusion in tissues and host cells
Serum protein binding of antimicrobial agents
Drug interactions and interference
Status of patient defense and immune system
Multiple simultaneous illnesses
Virulence and pathogenicity of infecting bacterium
Site and severity of infection
But despite these limitations, susceptibility testing
provides valuable data that are used in conjunction with
other diagnostic information to guide therapy of patients.

METHODS THAT DIRECTLY MEASURE
ANTIMICROBIAL ACTIVITY
These are methods that bring the antimicrobial agents of
interest and the infecting bacterium together in the same in
vitro environment to determine the impact of the drugs
presence (whether susceptible or resistant) on bacterial
growth.
Direct measurement include:
Conventional susceptibility testing methods
Broth dilution
Agar dilution
Disk diffusion
Commercial susceptibility testing system
Special screens and indicator tests

GENERAL CONSIDERATIONS ON
CONVENTIONAL TESTING METHODS
I. Inoculum preparation
Requirements to obtain properly prepared inocula:
Pure culture
Select 4 to 5 colonies of same morphology
Inoculate to broth or 0.85% saline solution and incubate for 16 to 24
hours
Standardized inoculum
Comparison with McFarland turbidity standards to obtain standardized
optical density
McFarland standard (1% H
2
SO
4
+ 1.175% BaCl
2
)
0.5 MacFarland standard
provides an optical density comparable to the density of a
bacterial suspension of 1.5 x 10
8
CFU / mL
II. Selection of Antimicrobial Agents for Testing
Antimicrobial Battery / Panel = drugs that are chosen to be tested against a
particular bacterial isolate
Drugs to be tested should come not only from the laboratory alone, but from the
medical staff (infectious disease specialists) and also pharmacy dept.

MACFARLAND STANDARD
CONVENTIONAL TEST METHOD: BROTH DILUTION
Involves challenging the organism of interest with
antimicrobial agents in a broth environment.
2 Types: Macrodilution and Microdilution

Each antimicrobial agent is tested using a range of
concentrations expressed as g of drug/ mL of broth

Usually concentrations tested for each antibiotic are a
series of doubling dilutions (16, 8, 4, 2, 1, 0.5, 0.25 g/mL)

Lowest antimicrobial concentration that COMPLETELY
INHIBITS visible bacterial growth as detected visually
(automated or semi-automated method) is recorded as the
= MINIMAL INHIBITORY CONCENTRATION

ANTIBIOTIC MICROBROTH DILUTION


READING AND INTERPRETATION OF RESULTS
After incubation, microdilution trays are examined for bacterial
growth. The first wells need to be observed are the CONTROL
WELLS.
Growth well (does not contain an antimicrobial agent)
Sterility well (well that was not inoculated)
Presence of growth is examined in each of the wells and is often
augmented through the use of light boxes or reflecting mirrors.
MIC is then determined, by identifying the microdilution well
containing the LOWEST drug concentration that completely
INHIBITED visible bacterial growth.
Once the MICs for the antimicrobials are obtained, they are
translated into Interpretive Categories of:
SUSCEPTIBLE
INTERMEDIATE
RESISTANT
ANTIBIOTIC MICROBROTH DILUTION


CONVENTIONAL TEST METHOD: AGAR DILUTION
Antimicrobial agents and microorganisms to be tested are
brought together on an agar-based medium (Mueller-
Hinton medium or MHA) rather than in broth.
Each dilution of a specific antimicrobial agent = 1 plate
Ex. 6 dilutions of an antimicrobial agent = 6 plates
Method also requires one positive growth control plate
After incubation, plates are examined for growth and MIC
is determined
MIC (LOWEST conc. of an antimicrobial agent in agar that
completely inhibits visible growth)
Results are also translated into interpretive categories of
Susceptible, Intermediate or Resistant
Labor intensive for use in clinical laboratories
Advantage: means of determining MIC of N. gonorrhoeae
which does not grow sufficiently in broth dilution methods

CONVENTIONAL TEST METHOD: DISK DIFFUSION
Developed thru the study made by Bauer, Kirby, Sherris
and Turck in 1966. They standardized and correlated the
used of ANTIBIOTIC-IMPREGNATED PAPER DISKS
with MICs using many bacterial strains.
Using this test, resistance is detected by challenging
bacterial isolates with antibiotic disks that are placed on
the SURFACE of an agar that has been seeded/inoculated
with a lawn of bacteria.
The agent from the disks DIFFUSES and establish a
concentration gradient around the paper disk.
Upon incubation, bacteria grow on the surface of plate
EXCEPT where the antibiotic concentration in the gradient
around the each disk is sufficiently high to inhibit growth
Following incubation, the DIAMETER of the zone of
inhibition around each disk is measured in millimeters.




Date Reported: July 27, 2013
DISK DIFFUSION (KIRBY BAUER
Mueller Hinton Agar
depth: 4mm
pH: 7.2 7.4
Steps:
Pick 4-5 colonies into TSB. Incubate at 37C for 2 5 hrs.
Compare turbidity w/ 0.5 McFarland standard
(BaSO
4
Standard)
99.5 mL of 1% H
2
SO
4

0.5 mL of 1.175% BaCl
2

1.5 x 10
8
CFU/mL = (inoculum compared to 0.5
McFarland standard)

DISK DIFFUSION (KIRBY BAUER
Inoculate MHA
Apply antibiotic disks
wait for 3-5 mins but not > 15 mins to allow diffusion of
antibiotics
150 mm agar can accommodate NOT MORE THAN 12
DISKS
Invert plates and incubate at 37C for 16 18 hrs. (not
more than 5 stacks high)
Measure zone of growth inhibition
Interpret susceptibility from standard chart zone

POSSIBLE SOURCE OF ERROR:
Use of mixed culture
Inocolum Too Light=LARGE ZONE OF INHIBITION (Falsely Sensitive)
Inoculum Too Heavy=SMALL ZONE OF INHIBITION (Falsely Resistant)
Too much moisture on agar = Small Zone
Very Dry agar surface (poor growth) = Large Zone
Improper storage of disks
indicator of improper storage: Penicillin and Methicillin
Reading and Clerical Error
Deterioration of Turbidity standard or control strains
Ca & Mg causes INCREASED RESISTANCE of P. aeruginosa to
Aminoglycosides

Susceptibility w/ Sulfonamides (2 concentric rings around the disk)
Measure the diameter of the OUTER ZONE