Infection Control

Biosafety in a Microbiology Laboratory

Five Most Frequently Acquired Laboratory Infection

1. Shigellosis
2. Salmonellosis
3. Tuberculosis
4. Brucellosis
5. Hepatitis

Disposal of Hazardous Waste

● All materials contaminated with potentially
infectious agents must be
DECONTAMINATED before disposal
● Infectious waste may be decontaminated
by use of AUTOCLAVE, INCINERATOR or
alternative waste-treatment methods
● Infectious waste (agar plates, tubes,
reagent bottles) should be placed into
leak-proof plastic bags (double bagging)
● Pipettes, swabs, & other glass objects
should be placed into rigid cardboard
containers before disposal
● Sharp objects (needles & scalpels) are
placed in SHARP CONTAINERS which are
autoclaved or incinerated
Biological Safety Cabinet (BSC)
● A device that encloses a workplace in such
way as to protect workers from aerosol
exposure to infectious disease agents
● Air containing the infectious material is
sterilized either by heat, UV light or passage
thru HEPA filter
● Most clinical lab use class II cabinets
Classes of BSC
CLASS I – allows room (unsterilized) air to pass
into cabinet & around the area and materials
within, sterilizing only the air to be exhausted
CLASS II – sterilize air that flows over infectious
material, as well as air exhausted
CLASS III – provide the most protection to the
worker; air coming & going out of the cabinet is
filter sterilized & the infectious material within is
handled w/ rubber gloves that are attached &
sealed to the cabinet
Classification of Biologic agent based in
Hazard:
1. BIOSAFETY LEVEL 1 AGENTS
– Include those that have no known
potential for infecting healthy people
– Used in laboratory teaching exercises of
students
– Bacillus subtilis, Mycobacterium gordonae
2. BIOSAFETY LEVEL 2 AGENTS
– the most commonly being sought in
clinical specimens
– They include all the common agents of
infectious diseases
– HIV, Bacillus anthracis, Yersinia pestis
3. BIOSAFETY LEVEL 3 AGENTS
– unlikely encountered viruses in routine
clinical lab
– Mycobacterium tuberculosis, systemic
fungi, Francisella tularensis, Brucella
Spp.
4. BIOSAFETY LEVEL 4 AGENTS
– require maximum level containment
facilities that decontaminates personnel
& materials before leaving the facility
– arbovirus, arenavirus, filovirus, small pox
virus
Antimicrobial Agent And Chemotherapy
Chemotherapy
• CHEMOTHERAPY is the treatment of disease
with chemical agents
• CHEMOTHERAPEUTIC AGENTS are chemical
substances synthesized in the laboratory which can
kill (bactericidal) or inhibit (bacteriostatic) the
growth of the organisms
• RESISTANCE:
– Bacteria that are not susceptible to a particular
antibiotic are said to be resistant to that antibiotic
• CROSS RESISTANCE:
– Bacteria that are resistant to one antibiotic and
also demonstrate resistance to another different
antibiotic (normally present in antibiotics with
similar structures)

Antimicrobial Agent (Antibiotic) • SPECTRUM :

• ANTIBACTERIAL/ANTIMICROBIAL AGENT is
the
drug used against microorganisms
• ANTIBIOTICS are chemical substances derived
from microorganisms which inhibit the growth of
or kills other microorganisms
– Examples: Penicillin and it’s derivatives
Ampicillin, Amoxycillin, Cephalosporins
Terminologies
• BACTERIOSTATIC AGENTS are antimicrobials
that inhibit the growth or multiplication of bacteria
but do not kill; reversible
– Examples: chloramphenicol, dapsone,
erythromycin, clindamycin, isoniazid (INH),
rifampicin, sulfonamides, tetracycline
– The range of bacteria against which an antibiotic
is effective is called the antibacterial spectrum of
that antibiotic
• PROPHYLAXIS
– use of a drug of the prevent imminent infection to
a person at risk
Features for Successful Effect:
a. ACTIVE FORM – ensure thru pharmacodynamic
design of the drug
b. ACHIEVE SUFFICIENT CONCENTRATION AT
THE INFECTION SITE – higher than the pathogens
MIC to be effective
c. SELECTIVE TOXICITY

• BACTERICIDAL AGENTS are antimicrobials that

kill bacteria usually when they are in the process of
multiplication; irreversible
– Examples: aminoglycosides, beta-lactams,
glycopeptides, INH, quinolones, bacitracin,
metronidazole

• MINIMUM INHIBITORY CONCENTRATION (MIC)

is the lowest drug concentration (smallest amount
of antibiotic) required to completely inhibit or
suppress the growth of the bacteria

• MINIMUM BACTERICIDAL Classification of Chemotherapeutic Agents

CONCENTRATION(MBC) is the lowest drug
concentration (smallest amount of antibiotic)
required to destroy or kill 99.9% bacteria

• THERAPEUTIC INDEX is the ratio of the toxic

dose to the therapeutic dose
– The higher the index, the more effective the
chemotherapeutic agent
Classification - Source Spectrum of Activity

FUNGI BACTERIA
● Penicillin
● Cephalosporin
● Griseofulvin

BACTERIA
● Polymyxin B
● Colistin
● Bacitracin
Classification -Spectrum of Activity/ Target
● Tyrothricin
Microorganism
● Aztreonam

1. ANTIBACTERIAL: Pencillins, Aminoglycosides,

Erythromycin
2. ANTIFUNGAL: Griseofulvin, Amphotericin B,
Ketoconazole
3. ANTIVIRAL: Idoxuridine, Acyclovir,
Amantadine, Zidovudine
4. ANTIPROTOZOAL: Chloroquine,
Pyrimethamine, Metronidazole, Diloxanide
5. ANTHELMINTIC: Mebendazole, Piperazine,
Pyrantel pamoate

Classification o- Type of Action

PRIMARILY BACTERIOSTATIC
Sulfonamides
Tetracycline
Chloramphenicol
Erythromycin
Ethambutol

PRIMARILY BACTERICIDAL
Penicillins
Classification: Spectrum Cephalosporins
Aminoglycosides
1. NARROW-SPECTRUM – effective against a Vancomycin
limited Polypeptides Nalidixic
number of pathogens acid
– Examples: bacitracin, clindamycin, dapsone, Rifampin
erythromycin, gentamycin, isoniazid, penicillin, Ciprofloxacin
polymyxin B, vancomycin Cotrimoxazole Isoniazid
2. BROAD-SPECTRUM – destroy different kinds of
organisms
– Examples: ampicillin, cephalosporins, rifampicin,
chloramphenicol, ciprofloxacin, sulfonamides,
trimetoprim, tetracycline
Classification - Chemical Structure Combination therapy is necessary in patients with
1. β-LACTAM ANTIBIOTICS : Penicillins, mixed or life-threatening infections
Cephalosporins, Monobactams,
Carbapenems Why are Antibiotics Combined?
2. TETRACYCLINES : Oxytetracycline,
Doxycycline ● To achieve an additive or SYNERGISTIC
3. AMINOGLYCOSIDES : Streptomycin, EFFECT against a single organism
Gentamicin, Neomycin ● In mixed infections with bacteria are
4. MACROLIDE ANTIBIOTICS : Erythromycin, sensitive to different drugs
Oleandomycin, Roxithromycin ● To delay the development of resistance
5. QUINOLONES : Nalidixic acid, Norfloxacin, ● To decrease the adverse reactions of an
Ciprofloxacin, Pefloxacin individual drug - so that the dose of a drug
6. IMIDAZOLE DERIVATIVES : Miconazole, can be reduced
Ketoconazole, Clotrimazole, Fluconazole ● When the infection is severe and the body
defense is poor
Classification - Mechanism of Action
1. INHIBIT CELL WALL SYNTHESIS : Penicillins,
Cephalosporins, Cycloserine, Vancomycin,
Bacitracin
2. INHIBIT PROTEIN SYNTHESIS : Tetracyclines,
Chloramphenicol, Erythromycin, Clindamycin
3. INTERFERE WITH DNA FUNCTION : Rifampin,
Norfloxacin, Metronidazole
4. CAUSING LEAKAGE FROM CELL
MEMBRANES : Amphotericin B, Nystatin

Selection of Antimicrobial Agent

a. Clinical evaluation of probable etiology
b. Identification of causative microorganism & Beta-lactam Antibiotics
antimicrobial drugs • The discovery of Penicillin by Alexander Fleming
c. Nature of drug in his London Hospital Laboratory in 1928 was a
d. Severity of disease and general condition milestone in the battle between human & microbes
of patient • Penicillins, Cephalosporins – A boon to mankind
e. Risk of toxicity of drug
f. Patient’s Age
g. Pregnancy and neonatal period
h. Possibility of drug resistance
i. History of previous allergic reactions
j. Cost of therapy

Tissue and Cell Penetration

• Success of antimicrobial therapy depends on
the concentration of antibiotics that reaches the
site of infections
• Tissue Penetration is the amount of drug
found in tissue cells and interstitial fluid

Combination of Antibiotics
❑ Normally, single antibiotics are used
Beta-lactam Antibiotics – Chemical Structure
MECHANISM OF ACTION OF PENICILLINS
Beta-lactam Antibiotics - Penicillins
1. Penicillin G(parenteral), Penicillin V (oral)
2. Penicillinase-resistant Penicillins =
Methicillin, Cloxacillin/Oxacillin, Nafcillin
3. Aminopenicillins = Ampicillin, Amoxycillin
4. Antipseudomonal Penicillins = Ticarcillin
Beta-lactam Antibiotics - Cephalosporins
• CEPHALOSPORINS appear to inhibit bacterial
cell wall synthesis in a manner similar to that of
penicillins. Structurally they are closely related to
penicillins and belong to the same class of β-lactam
antibiotics
1. First – Generation Cephalosporins: Cefzolin,
Cephalexin, Cefadroxil
– have good activity against gram-positive
bacteria but poor activity against
gram-negative bacteria
2. Second – Generation Cephalosporins:
Cefuroxime, Cefoxitin, Cefaclor
– have increased activity against
gram-negative microorganisms
3. Third Generation Cephalosporins: Cefotaxime,
Ceftazidime, Cefoperazone, Ceftizoxime,
Ceftriaxone, Cefpodoxime Cefixime Oral
– generally have increased potency and a
wider spectrum of activity against clinically
important gram-negative bacteria. But they
are less potent than the first generation
agents against gram-positive bacteria.
 b. AMINOGLYCOSIDES
􀀀 also bind to 30S ribosomal subunit and can block
protein synthesis in two different ways:
1. attach to the 30S subunit of the ribosome and
prevent the 30S subunit from attaching to
messenger RNA (mRNA).
2. presence of the aminoglycoside on the ribosome
may cause misreading of the mRNA that leads
to the:
a) insertion of the wrong amino acid into the
protein or
b) interference with the ability of amino
acids to connect with one another.
❑ often occur simultaneously; overall effect is
BACTERICIDAL

Inhibition of the PROTEIN SYNTHESIS

Interference with the CYTOPLASMIC MEMBRANE
BINDING TO 50S RIBOSOMAL SUBUNIT
POLYMYXIN
a. MACROLIDES (e.g. erythromycin, azithromycin
􀀀 molecules diffuse through the susceptible
and clarithromycin) & lincosamides (e.g.
cell’s outer membrane & cell wall to the
clindamycin) attach to the 50S ribosomal
cytoplasmic membrane
subunit causing
􀀀 then bind to the cytoplasmic membrane
􀀀 termination of the growing protein chain and
and disrupt & destabilize it
inhibition of protein synthesis
􀀀 this causes the cytoplasm to leak out of
• BACTERIOSTATIC
the cell resulting in cell death
b. CHLORAMPHENICOL
􀀀 BACTERICIDAL
• also binds to the 50S subunit of the ribosome
and interferes with binding of amino acids to
POLYMYXIN
the growing proteins
Examples:
🖑 BACTERIOSTATIC
a. Polymixin B - for GNR (P. aeruginosa)
b. Polymixin E - antibiotic ointments
Temporary/ permanent BM depression leading
Interference with the PROTEIN SYNTHESIS
to APLASTIC ANEMIA & LEUKOPENIA (toxic side
BINDING TO 30S RIBOSOMAL SUBUNIT
effect)
a. TETRACYCLINES (e.g. tetracycline, minocycline
and doxycycline)
Nucleic acid Synthesis Inhibitors
b. AMINOGLYCOSIDES (gentamycin, kanamycin,
1. RIFAMPICIN – inhibits RNA synthesis or
neomycin, streptomycin, tobramycin, amikacin)
transcription
c. SPECTINOMYCIN
2. QUINOLONES (cipro/nor/levofloxacin)
d. FUSIDIC ACID
–interfere w/ DNA gyrase & replication; highly
effective for enteric bacteria , ex: E. coli
3. METRONIDAZOLE – DNA disruption; effective
a. TETRACYCLINES
against anaerobic bacteria
􀀀 bind to the 30S subunit of the ribosome & block
the attachment of aminoacyl-transfer RNA (tRNA)
􀀀 Since new amino acids cannot be added to the
growing protein chain, protein synthesis is
Inhibited
🖑 BACTERIOSTATIC
Essential Metabolite Inhibitors:
1. SULFONAMIDES – inhibits folic acid
metabolism; high therapeutic index
2. TRIMETHOPRIM – blocks tetrahydrofolate
synthesis
3. DAPSONE – interferes w/ folic acid synthesis
4. ISONIAZID – inhibits synthesis of cord factor
(mycolic acid)

Approaches to Overcome β-lactamase Resistance

a. Development of an entirely new class of
antibiotics.
eg: Quinolones
b. Development of new beta-lactam antibiotics
resisting hydrolysis by beta-lactamase
eg: New Cephalosporins
c. Development of β-lactamase inhibitors
eg: Sulbactam and Clavulanic acid

Beta-lactamase Inhibitor
• Sulbactam
– Binds irreversibly to the beta-lactamase
enzymes
– Effective against Class A & C of
beta-lactamases
• Clavulanic Acid
Basic mechanisms by which microorganisms may – Binds irreversibly to the beta-lactamase
become resistant to beta-lactams includes: enzymes
1. Beta-lactamases production – Induces production of beta-lactamase in
2. Permeability Resistance some organisms
3. Changes in Penicillin Binding Proteins (PBP) – Effective against Class A of
4. Mixed Mechanism of Resistance beta-lactamases

Beta-lactamase Mediated Resistance Semisynthetic Penicillins

􀀀 Beta-lactamases are enzymes that recognize 1. Penicilinase-resistant penicillins
and attack β-lactam ring of the antibiotics ☞ Carbapenems: very broad spectrum
􀀀 Penicillins are hydrolysed into bacteriologically ☞ Monobactam: Gram-negative
inactive penicilloic acid 2. Extended-spectrum penicillins
􀀀 Intact antibiotic never reaches its target proteins 3. Penicillins + β-lactamase inhibitors
in the bacterial cell wall
❑ Ability to produce these enzymes is no Macrolides
widespread in bacteria • Macrolide antibiotics, include:
Inactive form – Azithromycin
– Clarithromycin
– Dirithromycin
– Erythromycin
– Roxithromycin
Summary of Targets a. ESBLs-extended spectrum beta-lactamases
▪ resistant to extended spectrum
cephalosporins, penicillins,
aztreonam
▪ Examples: E. coli, Klebsiella
b. Carbapenemases (CRE)
▪ Klebsiella pneumoniae- KPC- Class A
▪ Class B (NDM, VIM, IMP)- metallo
beta-lactamases
▪ resistant to penicillins, cephalosporins,
carbapenems, and aztreonam
c. Cephalosporinases
▪ AmpC enzyme
▪ inducible
▪ “SPACE” organisms

MRSA
▪ resistant to penicillin & semi-synthetic
penicillins
▪ acquired after prolonged hospital stain in ICU
or
BURN UNITS in close proximity to MRSA (+)
cases or after receiving broad spectrum Abx
▪ PREVENTION: proper isolation & rapid Idn of
bacteria, HW, Co & tx of (+) cases
▪ Rapid Tests: IDI-MRSA test & BD Ohm assay
from nasal swab specimens
▪ Chromogenic Tests: mauve colored colonies
w/n 24hrs & confirmed w/n 48hrs
THE SUPERBUGS
❑ organisms resistant to previously effective
CAUSES: RESISTANCE TO PENICILLINASE
drugs
RESISTANT PENICILLIN AMONG S. aureus
a. MRSA
▪ altered penicillin binding protein known as
▪ methicillin-resistant Staphylococcus
PBP2a or PBP2
aureus
– found in CW & encoded by mecA gene
▪ mecA gene codes for a PBP that does
– low affinity for binding with all B-lactam drugs
not bind betalactam
– altered form does not bind w/ Oxacillin,
antibiotics
rendering this drug ineffective
▪ Resistant to oxacillin (ORSA)
▪ isolates of oxacillin-resistant S. aureus
b. Vancomycin
(ORSA) are commonly referred to as MRSA
▪ VRE –Enterococcus species
for historical reason
▪ VISA/VRSA- Staphylococcus aureus
▪ resistance occur in up to: 80% - CoNS
50% - S. aureus
THE SUPERBUGS:
THE BETA-LACTAMASES
▪ GRAM-NEGATIVE RODS that have genes on
chromosomes that code
for enzymes against certain antimicrobials
MODIFICATIONS IN RESPONSES OF
STAPHYLOCOCCUS
(as a result of ® to penicillinase resistant
penicillin)
1. HETERORESISTANT strains – all cells in the
pop’n have genetic element (mecA gene) for
oxacillin ®, BUT not all expresses this
resistance by virtue of PBP2 production

2. BORDERLINE OXACILLIN-RESISTANT
isolates
(BORSA) – generally have MICs right above the
breakpoint of oxacillin susceptibility