The document discusses various methods for controlling microbial growth, including moist heat sterilization methods like pasteurization, boiling, steaming, and tyndalization. It also discusses dry heat sterilization and chemical methods using agents like phenol, halogens, alcohols, heavy metals, soaps/detergents, aldehydes, ethylene oxide, and food preservatives. The document then discusses mechanisms of antibiotic resistance in bacteria, including antibiotic inactivation, target modification, efflux pumps, and changes to outer membrane permeability.

1. CONTROL OF MICROBIAL GROWTH

3. Moist heat sterilization:
1- At normal pressure (at or below 100°C)
a- Pasteurization:
It is the use of mild heat to reduce the number of microorganisms in a
product or food. In the case of pasteurization of milk, the time and temperature
depend on killing potential pathogens that are transmitted in milk. Pasteurization
kills many spoilage organisms, as well, and therefore increasesthe shelf life of
milk especially at refrigeration temperatures (2°C). It is not a method of
sterilization, rather a method for preventing spoilage of food.
Milk is usually pasteurized by heating, typically at 62°C for 30 minutes,
then cooling to 7°C (holding process) or at 72°C for 15 seconds then rapid
cooling to 4°C (flash process), to kill bacteria and extend the milk's usable life.

4. b- Boiling in water:
100o for 30 minutes. Kills everything except some endospores. It
also inactivates viruses. For the purposes of purifying drinking water, 100o
for five minutes is a "standard".
c- Steaming:
The process is carried out in a double walled box with the inner one
perforated to allow the flow of steam (Koch sterilizer)
d- Tyndalization:
Three 30-minute intervals of boiling, followed by periods of cooling. Kills
bacterial endospores. First heat treatment kills vegetative cells and during
incubation spores germinate which are in turn killed by the second and third
heat treatment.

5. 2- At high pressure (above 100°C)
To obtain temperature over 100°C, water must boil under pressure,
where temperature is directly proportional to pressure. Sterilization by moist
heat usually involves the use of steam at a temperature between 115-134°C.
a- Pressure cooker:
Used in emergency for sterilization of surgical tools in minor
operations. Heat treatment is usually at 115-121°C for 20- 25 min.
b- Autoclave:
Autoclaving is the most effective and most efficient means of
sterilization. All autoclaves operate on a time/temperature relationship. These
two variables are extremely important. Higher temperatures ensure more
rapid killing. The usual standard temperature/pressure employed is 121ºC for
15 minutes.

6. Dry heat sterilization
1. Incineration: Burning of inoculating needles, blades, forceps etc.
2. Hot air oven: 1700 C for around 2 hr.

12. Chemical agents are generally not intended to achieve
sterilization. Most reduce the microbial populations to safe levels or
remove pathogens from objects. An ideal disinfectant or antiseptic
(chemical agent) kills microorganisms in the shortest possible time without
damaging the material treated.
CHEMICAL METHODS OF CONTROL

13. Phenol.
One of the first chemicals to be used for disinfection
was phenol. First used by Joseph Lister in the 1860s, it is the standard for
most other antiseptics and disinfectants. Phenol derivatives
called phenolics contain altered molecules of phenol useful as antiseptics
and disinfectants. The phenolics damage cell membranes and inactivate
enzymes of microorganisms, while denaturing their proteins. They
includecresols, such as Lysol, as well as several bisphenols, such as
hexachlorophene, which is particularly effective against staphylococci

14. Halogens.
Among the halogen antiseptics and disinfectants are chlorine and
iodine.Iodine is used as a tincture of iodine, an alcohol solution. Combinations
of iodine and organic molecules are called iodophors. They include Betadine
and Isodyne, both of which contain a surface active agent called povidone.
Iodine combines with microbial proteins and inhibits their function.
Chlorine also combines with microbial proteins. It is used as sodium
hypochlorite (bleach). As calcium hypochlorite, chlorine is available to
disinfect equipment in dairies, slaughterhouses, and restaurants. The
chloramines contain chlorine together with ammonia. They are used to sanitize
glassware and eating utensils and are effective in the presence of organic
matter. Chlorine is also used as a gas to maintain a low microbial count in
drinking water.

15. Alcohols.
Alcohols are useful chemical agents when employed against bacteria and
fungi, but they have no effect on bacterial spores. The type of alcohol most widely
used is 70 percent ethyl alcohol (ethanol). Isopropyl alcohol (rubbing alcohol) is
also useful as an antiseptic and disinfectant. Because alcohols evaporate quickly,
they leave no residue and are useful in degerming the skin before injections
Heavy metals.
A number of heavy metals have antimicrobial ability. For
example,silver is used as silver nitrate in the eyes of newborns to guard against
infection byNeisseria gonorrheae. It is also used to cauterize wounds. Copper is
used as copper sulfate to retard the growth of algae in swimming pools, fish tanks,
and reservoirs. Zincis useful as zinc chloride in mouthwashes and as zinc oxide as
an antifungal agent in paints. The heavy metals are believed to act by combining
with sulfhydryl groups on cellular proteins.

16. Soaps and detergents.
Soaps and detergents decrease the surface tension between
microorganisms and surfaces, and thereby help cleanse the surface. Soaps emulsify
the oily film on the body surface, carrying the oils, debris, and microorganisms
away in a degerming action. The cationic detergents are quaternary ammonium
compounds.They solubilize the cell membranes of microorganisms. Among the
popular compounds are Zephiran (benzalkonium chloride) and Cepacol
(cetylpyridinium chloride)
Aldehydes.
Two aldehydes, formaldehyde and glutaraldehyde, inactivate microbial
proteins by crosslinking the functional groups in the proteins. Formaldehyde gas
is commonly used as formalin, a 37 percent solution of formaldehyde gas.
Glutaraldehyde is used as a liquid to sterilize hospital equipment. However,
several hours are required to destroy bacterial spores

17. Ethylene oxide.
Sterilization can be achieved with a chemical known as ethylene
oxide (ETO). This chemical denatures proteins and destroys all
microorganisms, including bacterial spores. It is used at warm temperatures in
an ethylene oxide chamber. Several hours are needed for exposure and flushing
out the gas, which can be toxic to humans. ETO is widely used for plastic
instruments such as Petri dishes, syringes, and artificial heart valves
Food preservatives.
Foods can be preserved by using a number of organic acids to
maintain a low microbial population. Sorbic acid is used in a number of acidic
foods, including cheese, to prevent microbial growth. Benzoic acid also
inhibits fungi and is used in acidic foods and soft drinks. Calcium propionic
acid prevents the growth of mold in breads and bakery products.

18. Understanding the mechanisms of resistance has become a significant
biochemical issue over the past several years and nowadays there is a large
pool of information about how bacteria can develop drug resistance.
Biochemical and genetic aspects of antibiotic resistance mechanisms in
bacteria are shown in Fig. 1.
DRUG RESISTANCE

20. 1. Antibiotic inactivation
The defence mechanisms within the category of antibiotic inactivation include
the production of enzymes that degrade or modify the drug itself. Biochemical
strategies are hydrolysis, group transfer, and redox mechanisms.
Antibiotic inactivation by hydrolysis
Many antibiotics have hydrolytically susceptible chemical bonds (e.g.
esters and amides). Several enzymes are known to destroy antibiotic activity by
targeting and cleaving these bonds. These enzymes can often be excreted by the
bacteria, inactivating antibiotics before they reach their target within the
bacteria. The classical hydrolytic amidases are the b-lactamases that cleave the
b-lactam ring of the penicillin and cephalosporin antibiotics. Many Gram-
negative and Gram-positive bacteriaproduce such enzymes, and more than 200
different b-lactamases have been identified.

21. Antibiotic inactivation by group transfer
The most diverse family of resistant enzymes is the group of
transferases. These enzymes inactivate antibiotics (aminoglycosides,
chloramphenicol, streptogramin, macrolides or rifampicin) by chemical
substitution (adenylyl, phosphoryl or acetyl groups are added to the periphery of
the antibiotic molecule). The modified antibiotics are affected in their binding to
a target.
Antibiotic inactivation by redox process
The oxidation or reduction of antibiotics has been infrequently exploited
by pathogenic bacteria. However, there are a few of examples of this. One is the
oxidation of tetracycline antibiotics by the TetX enzyme. Streptomyces virginiae,
producer of the type A streptogramin antibiotic virginiamycin M1, protects itself
from its own antibiotic by reducing a critical ketone group to an alcohol at
position 16.

22. 2. Target modification
The second major resistance mechanism is the modification of the
antibiotic target site so that the antibiotic is unable to bind properly.
Peptidoglycan structure alteration
The peptidoglycan component of the bacterial cell wall provides an
excellent selective target for the antibiotics. It is essential for the growth and
survival of most bacteria. Consequently, enzymes involved in synthesis and
assembly of the peptidoglycan component of the bacterial cell wall provide
excellent targets for selective inhibition. The presence of mutations in the
penicillin-binding domain of penicillin-binding proteins (PBPs) results in
decreased affinity to b-lactam antibiotics. Alterations among PBPs result in
ampicillin resistance among Enterococcus faecium, and penicillin resistance
among Streptococcus pneumoniae

23. Vancomycin inhibit cell wall synthesis of Gram-positive bacteria by
binding C-terminal acyl-D-alanyl-D-alanine (acyl-D-Ala-D-Ala)-containing
residues in peptidoglycan precursors. Resistance is achieved by altering the
target site by changing the D- -Ala-D-Ala to D-alanyl-D-lactate (D-Ala-D-Lac)
or D-alanyl- -D-serine (D-Ala-D-Ser) at the C-terminus, which inhibits the
binding of vancomycin.

24. Protein synthesis interference
A wide range of antibiotics interfere with protein synthesis on different
levels of protein metabolism. The resistance to antibiotics that interfere with
protein synthesis (aminoglycosides, tetracyclines, macrolides, chloramphenicol,
fusidic acid, mupirocin, streptogramins, oxazolidinones) or transcription via RNA
polymerase(the rifamycins) is achieved by modification of the specific target.
Mutations in the 16S rRNA gene confer resistance to the
aminoglycosides. Chromosomally acquired streptomycin resistance in M.
tuberculosis is frequently due to mutations in the rpsL gene encoding the
ribosomal protein S12. Microorganisms that produce aminoglycosides have
developed mechanism of high level antibiotic resistance by posttranscriptional
methylation of 16S rRNA in the aminoglycoside binding site. This mechanism of
resistance has recently been reported in human pathogens from nosocomial
infections and animal isolates.

25. DNA synthesis interference
Fluoroquinolones interact with the DNA gyrase and topoisomerase IV
enzymes and prevent DNA replication and transcription. Resistance is conferred
by mutations in specific regions of the structural genes that sufficiently alter these
enzymes preventing the binding of antibiotics . The most common mutations in
this region cause resistance through decreased drug affinity for the altered
gyrase–DNA complex

26. 3. Efflux pumps
Efflux pumps affect all classes of antibiotics, especially the macrolides,
tetracyclines, and fluoroquinolones because these antibiotics inhibit
different aspects of protein and DNA biosynthesis and therefore must be
intracellular to exert their effect. Efflux pumps vary in both their
specificity and mechanism . Although some are drug-specific, many
efflux systems are multidrug transporters that are capable of expelling a
wide spectrum of structurally unrelated drugs thus contributing
significantly to bacterial multidrug resistance (MDR)

27. 4. Outer membrane (OM) permeability changes
Gram-negative bacteria possess an outer membrane consisting of an
inner layer containing phospholipids and an outer layer containing the lipid A
moiety of lipopolysaccharides (LPS). This composition of the outer membrane
(OM) slows down drug penetration, and transport across the OM is achieved by
porin proteins that form water-filled channels. Drug molecules can penetrate the
OM employing one of the following modes: by diffusion through porins, by
diffusion through the bilayer or by self-promoted uptake. Antibiotics such as b- -
lactams, chloramphenicol and fluoroquinolones enter the Gram-negative outer
membrane via porins. As such, changes in porin copy number, size or selectivity
will alter the rate of diffusion of these antibiotics