Bacte Lec Last

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Pathogenesis of Bacterial Infection Normal Human Microbiota Bacterial

Genetics Sterilization and Disinfection Aseptic Technique and Staining
Sensitivity Testing Water Analysis Dean Frederick R. Llanera, MD, MSMT, RMT,
FPSP Pathologist, Residents & Interns’ Training Officer, Philippine Heart Center
Dean, College of Medical Laboratory Science, De La Salle Medical and Health
Sciences Institute Tropical Medicine Training & Guest Lecturer, University of
Minnesota Past Board of Examiners, Philippine Society of Pathologists Former
Tenured Faculty, University of Santo Tomas
Learning Objectives: Pathogenicity of Bacterial Infection and Normal Flora •To
review the basic concepts on the infectious process •To know the virulence
factors exert their mechanisms •To be familiar with the common bacterial
virulence factors •To differentiate resident from transient microbiota •To have
an overview of the normal human flora present in various sites of the body
Characteristics of Pathogenic Bacteria Transmissibility Adherence to Host Cells
Persistence Invasion of host cells and tissues Toxigenicity Ability to evade or
survive the host’s immune system
Important Terms •Adherence, Adhesion or Attachment •Carrier •Infection
•Invasion
•Microbiota •Pathogenic –True –Opportunistic•Virulence •Toxigenicity
•Superantigens – Protein toxins that active the immune system by binding to
MHC & TCRs and stimulate many T cells to produce massive quantities of
cytokines
•Adherence (adhesion, attachment): The process by which bacteria stick to the
surfaces of host cells. After bacteria have entered the body, adherence is a
major initial step in the infection process. The terms adherence, adhesion, and
attachment are often used interchangeably.
https://www.taskade.com/d/mT4FDHSpqsddXbzb?share=view&view=HovPCSJbY8JjiL4J U/10/24, 10:40 PM

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•Carrier: A person or animal with asymptomatic infection that can be transmitted
to another susceptible host •Infection: Multiplication of an infectious agent within
the body. Multiplication of the bacteria that are part of the normal microbiota of
the gastrointestinal tract, skin, and so on is generally not considered an infection;
on the other hand, multiplication of pathogenic bacteria (eg, Salmonella species)
—even if the person is asymptomatic —is deemed an infection.
•Invasion: The process whereby bacteria, animal parasites, fungi, and viruses
enter host cells or tissues and spread in the body. •Microbiota: Microbial flora
harbored by normal, healthy individuals. •Nonpathogen: A microorganism that
does not cause disease; may be part of the normal microbiota.
•Opportunistic pathogen: An agent capable of causing disease only when the
host’s resistance is impaired (ie, when the patient is “immunocompromised”).
•Pathogen: A microorganism capable of causing disease. •Pathogenicity: The
ability of an infectious agent to cause disease.
•Superantigens: Protein toxins that activate the immune system by binding to
major histocompatibility complex (MHC) molecules and T -cell receptors (TCR)
and stimulate large numbers of T cells to produce massive quantities of
cytokines. •Toxigenicity: The ability of a microorganism to produce a toxin that
contributes to the development of disease.
•Virulence: The quantitative ability of an agent to cause disease. Virulent
agents cause disease when introduced into the host in small numbers.
Virulence involves adherence, persistence, invasion, and toxigenicity
Koch’s Postulates •1. The microorganism should be found in all cases of the
disease & its distribution in the body should be where the lesions are. •2. The
microorganism should be grown in pure culture in vitro for several generations.
•3. If the pure culture is inoculated into a susceptible host, the typical disease
must result •4. Organism can be reisolated from
3.
Transmission of Infection •Overcoming barriers / lines of defnese •Manner of
transmission – Portals of entry
The Infectious Process •Attachment •Multiplication •Spread –Bacteremia (transient
or persistent)

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Genomics & Bacterial Pathogenicity •Mobile Genetic Elements –Plasmids,
Transposons & Bacteriophages •Pathogenicity Islands –Large groups of genes
that are associated with pathogenicity & are located in the bacterial
chromosome
Regulation of Bacterial Virulence Factors •Transduction •Temperature & pH
dependent virulence factors •Osmolality & amino acid composition •Virulence
plasmid -encoded proteins •Bacterial motility
Bacterial Virulence Factors •Adherence Factors -pili, fimbriae •Invasion of Host cells
and tissues •Toxins –exotoxin vs. endotoxin Table U -4 •Enzymes –collagenase,
hyaluronidase, IgAse, streptokinase •Antiphagocytic factors -capsule
•Intracellular pathogenicity – resistance to lysosmes •Antigenic heterogeneity –
on bacterial surface, O, H •Bacterial secretion system -for gram negs •Fe
requirement –effect of IDA. •Biofilms –persistent & difficult to treat
Figure 4.8
Normal Human Microbiota •First line of defense against microbial pathogens
•Assist in digestion •Role in toxin degradation •Contribute to maturation of the
immune system •Shift in flora –Bacterial Vaginosis
Human Microbiome Project •Use of 16S rRNA gene sequencing –Mouth –
Esophagus – Stomach –Colon –Vagina
Role of the Resident Microbiota •Resident microbiota –Fixed types of organisms
regularly found in a given area at a given age; if disturbed, it promptly
restablishes itself •Transient microbiota –Nonpathogenic or potentially pathogenic
organisms that inhabit the skin or mucous membranes for hours, days or weeks –
Derived from the environment, does not produce disease nor establish itself
permanently on the surface; opportunistic
Normal Microbiota of: •Skin •Mouth & Upper Respiratory Tract •Intestinal Tract
•Urethra
•Vagina •Conjunctiva
Skin •Eliminate nonresident organisms –Low pH –Fatty acids in the sebaceous
secretions – Presence of lysozyme •Physical & Immunologic barrier
Mouth & Upper Respiratory Tract •Considerable diversity in the saliva microbiome
•Dental plaque -biofilm

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Intestinal Tract •At birth –the intestine is sterile, organisms are soon introduced
with food.
•Determinants of early flora: maternal vaginal, fecal, skin microbiota •Normal acid
pH of stomach •Colon of normal adult: U6 -UU % of the resident bacterial flora are
anaerobes
Intestinal Tract -Functions •Protective –resident bacteria displace and inhibit
potential pathogens indirectly by competing for nutrients & receptors or directly
by production of antimicrobial factors such as bacteriocins & lactic acid
•Commensals are needed for the development of the mucosal immune system
•Metabolic functions –synthesis of Vitamin K, biotin, folate, and enhance ion
absorption
Intestinal tract •Pseudomembranous colitis •Antibiotic associated •Fecal microbiota
transplantation (FMT) – stool transplant –for those with infection with Clostridium
difficile
Urethra •Anterior urethras of both sexes contain organisms found on the skin and
perineum.
Vagina •Lactobacillus acidophilus / Doderlein’s bacillus •Gardnerella vaginalis –
Bacterial vaginosis
Conjunctiva •Conjunctival flora is normally held in check by the flow of tears,
which contain antibacterial lysozyme.
Bacterial Genetics
Nucleic Acid Structure and Organization 1.DNA –deoxyribonucleic acid 2.RNA -
ribonucleic acid DNA –consists of deoxyribose sugars connected by
phospodiester bonds bases are covalently linked to each deoxyribose sugar
•Purine bases -adenine and guanine •Pyrimidine bases -cytosine and thymine
In RNA, uracil replaces thymine Nucleotide –sugar, phosphate, base
•DNA and RNA are nucleotide polymers and the order of bases along a DNA
or RNA strand is known as the base sequence. •This sequence provides the
information that specifies the proteins that will be synthesized by microbial
cells.
Three major types of RNA 1.mRNA 2.tRNA 3.rRNA
RNA is a nucleic acid that can be divided into three main types: •Messenger
RNA (mRNA) – transcribed copy of DNA •Ribosomal RNA (rRNA) – found in
ribosomes and is involved translation of mRNA to protein •Transfer RNA (tRNA)
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– carries amino acids to ribosome for translation of mRNA to protein Image:
https://ib.bioninja.com.au/standard -level/topic -2- molecular -biology/26 -structure
-of-dna-and-rna/types -of-rna.html

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Genes and the Genetic Code •A DNA sequence that encodes for a specific
product (RNA or protein) is defined as a gene. •All genes taken together within an
organism comprise that organisms genome.
The Bacterial Chromosomes •The bacterial chromosomes contains all genes
essential for viability and exists as a double stranded, closed circular, naked
macromolecule. •The molecule is extensively folded and twisted (i.e. supercoiled)
so that it may be accomodated within the bacterial cell.
Non Chromosomal Elements of the Genome
1. Plasmids -able to replicate and encode
information for the production of various cellular products. • -not as stable as the
chromosome and maybe lost during cellular replication •Plasmid genes do not
usually encode for products essential for viability.
Non Chromosomal Elements of the Genome •2. Transposable elements -are
pieces of DNA that move from one genetic element to another, from plasmid to
chromosome or vice versa.
Transposable Elements •These extra chromosomal elements play a key role in
the exchange of genetic material throughout the bacterial microbiosphere,
including genetic exchange among clinically relevant bacteria.
Replication •Four stages 1.Unwinding or relaxation of the chromosome
2.Unzipping or disconnecting the complementary strands of the parental DNA
3.Synthesis of the new DNA strands 4.Termination of replication with release of
two identical chromosomes
Transcription •DNA expression begins with transcription which converts the
DNA base sequence of the gene into a messenger RNA that is
complementary to the gene’s DNA sequence.
Transcription •In mRNA thymine is replaced with uracil •In bacteria the mRNA
that result from the transcription process are polysictronic, that is they encode
for several gene products. •The transcription process not only produces mRNA
but also tRNA.

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Translation •By this process the genetic code within mRNA is translated into
specific amino acid sequences that are responsible for protein structure, and
hence, function.
Genetic Exchange and Genetic Diversity •Genetic exchange in bacteria is
accomplished by three basic mechanisms: 1.Mutation 2.Genetic Recombination
3.Gene exchange between bacteria, with or without recombination
Mutation •Is defined as a change in the original nucleotide sequence of a gene or
genes within an organism’s genome. •Mutation may arise spontaneously,
perhaps by an error made during DNA replication.
Mutation •Mutations may be induced by chemical or physical factors in the
environment or by biological factors such as introduction of foreign DNA into
the cell.
Genetic Recombination •In this process some segment of DNA that
originated from one bacterial cell (i.e., donor) enters a second bacterial cell
(i.e. recipient) and is exchanged with a DNA segment of the recipients
genome.
Genetic Recombination •Also referred as homologous recombination because the
pieces of DNA that are exchanged usually have extensive homology or
similarities in their nucleotide sequences.
Gene Exchange 1.Transformation 2.Transduction 3.Conjugation
Transformation •Involves recipient cell uptake of free DNA released into the
environment when another bacterial cell dies and undergoes lysis. •Certain
bacteria are able to take up this free DNA, that is able to undergo
transformation. Such bacteria are said to be competent. Ex. Haemophilus,
Streptococcus and Neisseria
Transformation •The mixing of DNA between bacteria via transformation and
recombination plays a major role in the development of the antibiotic resistance
and in the dissemination of genes that encode factors essential to an
organism’s ability to cause disease.
Transformation •Additionally, gene exchange by transformation is not limited to
organisms of the same specie.
Transduction •This process is mediated by viruses that infect bacteria
(bacteriophages).
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•These viruses integrate their DNA into the bacterial cell’s chromosome. •When
viral products is completed, viral DNA is excised from the bacterial
chromosomes.

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Conjugation •This process occurs between two living cells, involve cell to cell
contact, and require mobilization of the donor’s bacterium chromosome. •In E.coli
contact is mediated by a sex pilus
What is the name of the process shown? Elaborate on the steps of the process.
Conjugation
The donor cell produces a pilus, which is encoded by the plasmid, and contacts a
potential recipient cell that does not contain the plasmid. Retraction of the pilus
brings the cells into close contact, and a pore forms in the adjoining cell
membranes. Formation of the mating pair signals the plasmid to begin transfer
from a singlestranded nick at oriT. The nick is made by plasmid - encoded
trafunctions. The 5 ◻◻end of a single strand of the plasmid is transferred to the
recipient through the pore. During transfer, the plasmid in the donor is replicated,
its DNA synthesis being primed by the 3 ◻◻OH of the oriT nick. Replication of
the single strand in the recipient proceeds by a different mechanism with RNA
primers.
Both cells now contain double -stranded plasmids, and the mating pair separates.
What bacterial surface structure is required for the process shown to occur? The
F or sex pilus is required for conjugation to occur.
What are the distinguishing characteristics of this horizontal gene exchange
mechanism compared to the two other common mechanisms? Site an example
of a bacterial trait that can be transferred through this mechanism.
In contrast to the two other common exchange mechanisms, conjugation requires
cell -to- cell contact and would be inhibited if the donor and recipient cells were
separated by a membrane. Conjugation is resistant to DNase treatment.
Antimicrobial resistance can be transferred via conjugation.
What process is shown? Generalized transduction
What process is shown? Elaborate the process and describe the differences
between the 2 processes. Specialized transduction. In generalized
transduction, only pure bacterial DNA is transferred as compared to specialized
transduction. GT occurs during lytic phase, while ST occurs during lysogenic
phase. Temperate bacteriophage is present
in ST. ST rarely occurs compared to GT.

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GENERALIZED VS. SPECIALIZED TRANSDUCTION1. A virulent (lytic)
bacteriophage infects a bacterium. 2. The phage genome enters the bacterial
cell. 3. Virus governs the bacterial metabolic mechanisms to make its own DNA
and other necessary components and enzymes. 4. Bacterial DNA hydrolyses
into small pieces. 5. Genetic material packs inside the new phages.
Occasionally bacterial DNA fragments pack in new phage capsids 6.
Bacterial cell lyses and releases the new phages. 7. When transduced phage
infects another bacterium, the previous bacterial DNA incorporates into a new.1. A
temperate bacteriophage infects a bacterium. 2. Viral DNA integrates into bacterial
chromosome and becomes the prophage stage 3. Viral DNA remains within
bacteria for several generations
4. Upon a spontaneous induction, viral DNA detaches the bacterial
chromosomal DNA. 5. Fragments of bacterial DNA detach from the bacterial
chromosome with viral DNA. 6. Viral DNA replicates together with bacterial
genes and package inside new capsids and make new phages. 7. Bacterial cell
lyses and releases the new phages. 8. New phages infect new bacteria. U. Bacterial
DNA mixes with new bacteria during the infection.
Identify the type of gene transfer. Describe the process. Transformation
Transformation can define as the process of taking up of an extracellular or free
DNA strand of one bacterial cell (donor’s cell) by the competent bacterial cell
(recipient’s cell). The taking up of the DNA strand occurs either by natural or
artificial means.
What characteristic must the recipient bacterial cell express for this process to
occur? The recipient bacterial cell must express competence for transformation
to occur.
What are the characteristics that distinguish this horizontal gene exchange
mechanism from the two other most common mechanisms?
In contrast to the two other most common gene exchange mechanisms,
transformation is sensitive to the presence of DNase and does not require cell-to-
cell contact.
Applications of Molecular Diagnostics •Nucleic Acid Hybridization Techniques –
Formats: Blotting Techniques •Nucleic Acid Amplification –PCR •Strain Typing
and Identification
Within our cells, we have chromosomes found inside the nucleus. The DNA
strands are compacted as chromosomes . Image from Wikipedia:
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https://en.wikipedia.org/wiki/Chromosome

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DNA is a nucleic acid molecule in the form of a double -stranded helix . Each
strand of DNA is composed of 4 types of nitrogen bases –adenine, cytosine,
guanine and thymine . The double stranded helix is the most energetically
favorable state of DNA. It’s so stable that it needs extremes of heat , pHor by
use of destabilizing agents to lose its conformation.STABLE Image: Yoon,
Byung -Jun. (2007). Signal processing methods for genomic sequence
analysis.
DNA contains our genetic information. DNA is transcribed to RNA and then
translated to proteins . Some viruses have reverse transcriptase that allows
reverse transcription of DNA from RNA . Image from:
https://ib.bioninja.com.au/standard -level/topic -2-molecular - biology/27 -dna-
replication -transcri/central -dogma.htmlDNA RNA Protei ntranscription
translation reverse transcription
RNA is a nucleic acid that can be divided into three main types: •Messenger
RNA (mRNA) – transcribed copy of DNA •Ribosomal RNA (rRNA) – found in
ribosomes and is involved translation of mRNA to protein •Transfer RNA (tRNA)
– carries amino acids to ribosome for translation of mRNA to protein Image:
https://ib.bioninja.com.au/standard -level/topic -2- molecular -biology/26 -structure
-of-dna-and-rna/types -of-rna.html
DNA Replicatio nParent strands A A C T G TDNA duplication uses each strand
of the parent DNA to synthesize daughter strands. The products are composed
of a parent strand and a daughter strand . A A C T G TT T G A C AT T G A C AT
T G A C AT T G A C A Daughter strandsThe two double stranded DNAs are
exactly the same as the parent DNA.
DNA differs from RNA in composition, structure and
function. 1.DNA isdouble stranded , while RNA is single stranded . 2. In RNA,
thymine is replaced by uracil. 3. The sugar in DNA is deoxyribose , while the
sugar in RNA is ribose containing hydroxyl group .DNA vs RNADNA RNA A T
A T C G T A G C T AA A C T G TU U RNA is less stable than DNA , not only
because it is single stranded but also because the hydroxyl group (-OH)
predisposes susceptibility from alkaline hydrolysis.
Animation by Polymime Animation Company Ltd.Though DNA replication is
conceptually simple, the process is complex and involves a number of accessory
proteins and enzymes . This video explains the details of DNA replication.

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Extreme heat, hydrogen bond disrupters and pH outside 4 -U can unwind the
DNA. Because temperature is often used to unwind DNA, the process has been
referred to as melting . Heat pH 4 -U Hydrogen bond disruptersUnwindi ng
the DNA
% helix TemperatureMELTING POINT50% A T C GThe temperature at which
50% of dsDNA is converted into single stranded DNA is called the melting
point . The melting point depends on amount of adenine -to-
PRIMER POLYMERASE NUCLEOTIDESPolymerase Chain Reaction Polymerase
chain reaction (PCR) is a laboratory technique based on the process of DNA
replication . Basically, PCR consists of: 1.Target DNA -from specimen 2.Primers
–complementary to the sequence of the target strand 3.Taqpolymerase -DNA
polymerase from a hotspring bacteria
4.Nucleotides - deoxyribonucleotide triphosphatesThermocycler , an equipment
that raises and lowers temperature of the sample in cycles, is very important in
this process. The three main steps of PCR are: 1.Denaturation -unwinding of
strands through heat 2.Annealing – attachment of primer to the target segment
through a cooler temperature 3.Extension – addition of nucleotides to the primer
with a warm temperature Image from Mike Powers Illustration & Design:
http://www.mikepowers - illustration.com/projects/2016/11/22/information -
scienceHEAT (70 -75°C) COOL (40 -70
°C) HEAT (U4 °C)
PCR allows nucleic acid amplification or production of multiple copies of a
target DNA segment called amplicons . With large number of DNA copies,
the presence of a target DNA segment is easier detected . After the PCR
amplification , the specific amplicons may be detected using various methods
such as electrophoresis .Target DNA
Reverse Transcriptase PCR (RT -PCR) If the target is an RNA , a
complementary DNA (cDNA) is first produced by reverse transcription .
Reverse transcriptase synthesizes the cDNA using RNA as template. Double
stranded DNA is produced and PCR amplification proceeds in the usual
manner. This process is called reverse -transcriptase PCR or RT -
PCR.Reverse Transcript ion

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Real Time PCR (qPCR) Real -Time or Quantitative PCR (qPCR) is a variation
to PCR that combines amplification and detection in one step . The amplicons are
detected as they are made in real- time. Fluorescent signal is incorporated into
the amplicon and detected using a specialized thermocycler . Target DNA
CYCL ESAMPLIC ONS
FS Y B RThe amplicons created per cycle may be detected by using a fluorescent
dye or fluorescent probe. Fluorescent dye such as SYBR green I preferentially
binds to double stranded DNA. TaqMan probes are labeled with a fluorophore
and quencher. The probe attaches to the target sequence. Polymerase cleaves
off the fluorophore resulting to fluorescence.Prime rProbePOLYMERA
SEPOLYMERA SE
PCR CYCLESFLUORES CENCE INTENSIT YCtWith real -time PCR, special
thermocycler with precision optics measures emitted fluorescence . A computer
software monitors the fluorescence at every cycle and generates an
amplification plot . This plot shows the fluorescence intensity versus PCR cycle
number .AMPLIFICATION PLOT BASEL INEEXPO NENTI ALPLA TEA UA sigmoid
curve is generated and is composed of different phases:
1.Baseline or lag phase – only background signal is observed 2.Exponential
phase – amplification is observed and fluorescence exceeds background noise
3.Plateau phase – reactants become rate limiting and amplicon accumulation
stoFluorescence thresholdA fluorescent threshold can be set above the
baseline to call POSITIVE samples. Cycle threshold (Ct) or cycle quantification
( Cq) is the number of cycles needed to overcome the fluorescence threshold or
background noise.
Conventional PCR Real -time PCR Two-steps •Amplification •Post-
amplification detection (electrophoresis)One-step •Combined amplification
and detection (fluorescence) Post- amplification analysis needed: ◻more time
needed ◻open system: prone to contamination
◻may lead to false positivesNO post -amplification analysis: ◻faster ◻closed
system (reaction tubes remain closed) ◻less risk for contamination ◻More
specific Increased specificity with the use of probes Qualitative to semi -
quantitative Quantitative ◻More reproducible ◻Generates amplification plot and
standard cruve , Melting point analysis feasible
Figure 11.1
Figure 11.5
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Pasteurization •Used to disinfect beverages •Heat is applied to liquids to kill
potential agents of infection and spoilage, while retaining the liquid’s flavor and
food value •Special heat exchangers –Flash method: expose to 71.6 °C for 15
seconds –Batch method: expose to 63 °C to 66 °C for 30 minutes •Does not kill
endospores or thermoduric microbes
Boiling Water •For disinfection and not sterilization •Expose materials to boiling
water for 30 minutes
Dry Heat: Hot Air and Incineration •Incineration –Ignites and reduces microbes to
ashes and gas –Common practice in microbiology lab - incineration on
inoculating loops and needles using a Bunsen burner –Can also use
tabletop infrared incinerators
Figure 11.6
Dry Oven •Usually an electric oven •Coils radiate heat within an enclosed
compartment
•Exposure to 150 °C to 180 °C for 2 to 4 hours •Used for heat -resistant items that
do not sterilize well with moist heat
Modes of Action of Ionizing Versus Nonionizing Radiation •Ionizing radiation: if
the radiation ejects orbital electrons from an atom causing ions to form
•Nonionizing radiation: excites atoms by raising them to a higher energy state
but does not ionize them
Figure 11.7
Ionizing Radiation: Gamma Rays, X Rays, and Cathode Rays •Cold sterilization
•Dosage of radiation -measured in Grays •Exposure ranges from 5 to 50 kiloGrays
•Gamma rays, most penetrating; X rays, intermediate; cathode rays, least
penetrating
Applications of Ionizing Radiation •Food products •Medical products
Nonionizing Radiation: Ultraviolet Rays •Wavelength approximately 100 nm to 400
nm
•Germicidal lamp: 254 nm •Not as penetrating as ionizing radiation •Powerful
tool for destroying fungal cells and spores, bacterial vegetative cells, protozoa,
and viruses
Figure 11.U
Applications of Ultraviolet Radiation •Usually disinfection rather than sterilization
•Hospital rooms, operating rooms, schools, food prep areas, dental offices •Treat
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drinking water or purify liquids
MIC •To be effective, the clinically obtainable antibiotic concentration in body
fluids should be greater than the MIC.

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McFarland •UU.5 mL of 1 % sulfuric acid •0.5 mL of 1.175 % barium chloride •1.5 x
10 (8) CFU/mL
Broth Macrodilution (Tube Dilution) •Impractical to use routinely when several
antimicrobial agents must be tested on an isolate or if several isolates must be
tested •Can be used when MBC endpoints are to be subsequently determined
Broth Microdilution Tests •Multiwell microdilution tests
Agar Dilution Tests •Specific volumes of antimicrobial solutions are
dispensed into premeasured volumes of molted an cooled agar, which is
subsequently poured into standard Petri dishes •MHA with sheep’s blood for
fastidious bacteria
Disk Diffusion •Kirby Bauer •150 m diameter –12 disks
Oxacillin (Methicillin) Resistance •Cefoxitin –to induce greater expression of PBP2a in
mecA containing strains of staphylococci and also functions as a test reagent to
detect resistance
ESBL •Resistance to ampicillin because of production plasmid -mediated Beta
lactamase known as TEM -1 and SHV -1
Special Antimicrobial Susceptibility Tests •MIC / MBC •Time –kill assays –
bactericidal activity of antimicrobial agents •Synergy Tests –synergism,
antagonism, indifference
•Serum Bactericidal Test –amount of antimicrobial agent and other factors
(antibody, opsonin, complement) present •Molecular Probes
Infectious Diseases •Types of Infection –Primary/secondary; acute/chronic; localized /
generalized; focal/systemic; recurrent, latent, overt, nosocomial, idiopathic,
iatrogenic, asymptomatic / inapparent / subclinical •Phases in the Course of Infection
•Epidemic, Endemic, Sporadic, Pandemic •Host-Microbe Relationship •Bacteremia
/ SIRS
•Pathogenicity & Virulence
Specimen Collection •Blood, urine, CSF, aspirated (synovial), sputum, feces,
throat swab, rectal swab, nasal swab, eye/ear discharge, urethral smear
Preservation •Refrigerate –CSF for viruses –Ear : outer –Catheter tip (IV) –
sputum•Room Temp –CSF for bacteria –Ear : inner –Abscess, lesion, wound –
Body fluids –Genital –Nasal, NP, throat –tissue

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Specimen Priority •1 –critical / invasive –amniotic fluid, blood, brain, CSF, heart
valves, pericardial fluid •2 –unpreserved –body fluids not in Level 1, bone,
drainage from wounds, feces, sputum, tissue •3 –quantitation required –catheter
tip, urine, tissue for quantitation
•4 –preserved (not for Bacteriology) –feces in preservative; urine in preservative;
swabs in holding medium (aerobic & anaerobic)
Biosafety Levels •1 –standard teaching labs –Micrococcus, Lactobacillus,
Saccharomyces
•2 –moderate potential to infect –Staph , enterics, Corynebacterium, helminths,
Hepa A, B, rabies , Cryptococcus, Blastomyces •3 –severe/lethal if inhaled –M.tb,
F. tularensis, Y. pestis, Brucella, C. burnetti, C. immitis, yellow fever, WEE, AIDS
•4 –highly virulent when inhaled –flavi, arena incl. Lassa, filo incl. Ebola and
Gram Positive Cocci (Aerobic) •Catalase + –Staphylococcus –Micrococcus
•Catalase – – Streptococcus, Enterococcus, Leuconostoc, Lactococcus,
Clobicatella, Pediococcus, Aerococcus, Gemella, Helcococcus, Alloiococcys
otitidis
Gram Positive Bacilli (Aerobic) •Branching –Nocardia (partially acid fast) ,
Streptomyces, Rhodococcus, Oerskovia •Non Branching –Catalase Positive
•Bacillus, Brevibacillus, Paenibacillus, Listeria Corynebacterium –Catalase
Negative •Erysipelothrix, Actinomyces, Arcanobacterium, Bifidobacterium,
Gardnerella
Gram Negative Cocci (Aerobic) •Neisseria •Moraxella
Gram Negative Bacilli/ Coccobacilli (Aerobic) •McConkey Agar Growth –Oxidase (
-): Enterobacteriaceae, Acinetobacter, Stenotrophomonas –Oxidase (+) :
Pseudomonas, Burkholderia, Achromobacter, Rhizobium, Ochrobactrum,
Chyseobacterium, Sphingobacterium, Alcaligenes, Bordetella (non pertussis) ,
Comamonas, Vibrio, Aeromonas, Plesiomonas, Chromobacterium •McConkey
Agar No Growth •Growth Require Special Media
Gram Negative Bacilli/ Coccobacilli (Aerobic) •McConkey Agar Growth
•McConkey Agar No Growth –Oxidase variable : Haemophilus –Oxidase + :
Sphingomonas paucimobilis, Moraxella, Neisseria elongata, Eikenella corrodens,
Weeksella virosa, Pasturella, Suttonella, Mannheimia haemolytica, Actinobacillus,
Kingella, Cardiobacterium, Capnocytophaga
•Growth Require Special Media

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Gram Negative Bacilli/ Coccobacilli (Aerobic) •McConkey Agar Growth •McConkey
Agar No Growth •Growth Require Special Media –Bartonella, Afipia,
Campylobacter, Arcobacter, Helicobacter, Legionella, Brucella, Bordetella pertussis,
Bordetella parapertussis, Franciscella, Streptobacillus moniliformis, Sprillum
minus
Not characterized by Gram reaction •Mycobacterium •Obligate intracellular and
nonculturable bacteria –Rickettsia –Chlamydia •Cell wall deficient bacteria –
Mycoplasma – Ureplasma •Spirochetes
Anaerobic Bacteria •Gram Positive Anaerobic Cocci –Collinsella aerofaciens,
Finegoldia magna, Micromonas micros, Peptococcus niger, Peptostreptococcus
anaerobius, Schleiferella asaccharolytica, Atopobium, Anaerococcus •Gram
Positive Anaerobic Bacilli
•Gram Negative Anaerobic Cocci •Gram Negative Anaerobic Bacilli
Anaerobic Bacteria •Gram Positive Anaerobic Cocci •Gram Positive Anaerobic
Bacilli – Propionibacterium, Actinomyces, Clostridium, Bifidobacterium, Eggerthella
lenta, Eubacterium, Lactobacillus, Mobiluncus •Gram Negative Anaerobic Cocci
•Gram Negative Anaerobic Bacilli
Anaerobic Bacteria •Gram Positive Anaerobic Cocci •Gram Positive Anaerobic
Bacilli •Gram Negative Anaerobic Cocci –Veilonella parvula •Gram Negative
Anaerobic Bacilli – Bacteroides, Fusobacterium, Bilophila wadsworthia,
Leptotrichia, Porphyromonas, Prevotella
Gram Stain •Initial / primary stain –crystal violet •Mordant –Gram’s iodine
•Decolorizer U5 % ethanol •Counterstain/sec stain - safranin
Acid Fast Stain –cold (Kinyoun) & hot (Ziehl Neelsen) •Mycolic acid •Cord factor
•Sulfatides
•Partially acid fast - Nocardia
Medical Microbiology & Pharmacology of Anti -Infective drugs
I. APPROACH TO TREATMENT OF INFECTIOUS DISEASE Evaluate evidence
supporting an infection ( signs & symptoms) EXS: fever, malaise, swollen
lymph nodes, leukocytosis, diarrhea, prolonged cough Evaluate the potential of
confounding variables EXS: drug fever, autoimmune disorders Determine the
most likely site of infection by focusing on signs & symptoms & the
pathogens commonly associated with specific sites of infection ( refer to Table
1) Identify pathogens ( exs: Gram’s stain, cultures, diagnostic tests) Empiric
Page 16 of 46
therapy Directed Therapy

Page 17 of 46
Empiric therapy Directed Therapy Choose agent based on patient &
infectionDetermine susceptibility of pathogens to specific antimicrobials Choose
agent based on pathogen, patient & infection Monitor & modify based on patient
response (efficacy & toxicities)Early intervention for life - threatening diseases:
history, physical exam, clinical experience Clinical efficacy, ADR & PK profile, cost
I. APPROACH TO TREATMENT OF INFECTIOUS DISEASE
II. CLASSIFICATION OF ANTIMICROBIAL DRUGS 3 Schemes:
2. Effects on cells :
1.1 Bactericidal –drugs that actually kill microorgs (bacteria) ◻for immediately life
- threatening : patients with PMLs < 500/uL ◻endocarditis (phagocytosis is
limited) ◻EXS: aminoglycosides,penicillins, quionolones, cycloserine,
vancomycin, carbapenems 1.2 Bacteriostatic –drugs that inhibit bacterial growth
but does not kill them ◻host defense mechanisms (phagocytosis) are required to
kill the bacteria ◻EXS: chloramphenicol, nitrofurantoin, clindamycin, tetracycline,
erythromycin, trimethoprim, lincomycin, sulfonamides
3 Schemes: 2. Range of Activity : 2.1Broad -spectrum antibiotics –active
against a wide variety of microorganisms ◻EXS: tetracycline –active vs g -rods:
chlamydias, mycoplasmas, rickettsias chloramphenicol –all bacteria except
Mycobacteria & Pseudomonas sp 2.2 Narrow -spectrum antibiotics –active
against a particular group of microorganisms
◻EXS:erythromycin –active vs g+ Actinomyces, Corynebacterium, Bacillus,
Clostridium vancomycin –active vs g+ cocci: staphylococci & enterococciII.
CLASSIFICATION OF ANTIMICROBIAL DRUGS
3 Schemes: 3. Sites of Action : affect the integrity or synthesis II.
3. Cell -wall synthesis inhibitors & β-lactamase inhibitors

Page 1G of 32
Penicillins Cepha - losporinsCarba - penems & Mono - bactamsOthersβ-
Lactamase inhibitors Natural 1stGeneration Imipenem, CilastatinVancomycin
Clavulanic acid Antistaphy
- lococcal2nd Generation Aztreonam Cycloserine Sulbactam Amino - penicillins3rd
Generation Isoniazid Tazobactam Antipseudo -monal4th Generation
bactericidal; cell wall contains peptidoglycanA. ANTIBACTERIAL
DRUGS
4.
B. ANTIFUNGAL DRUGSII. CLASSIFICATION OF ANTIMICROBIAL
DRUGS
Cell -wall synthesis inhibitors & β-lactamase inhibitors
TRANSPEPTIDATIONUDP -acetylglucosamineII. CLASSIFICATION OF
ANTIMICROBIAL DRUGS
1.1 PENICILLINS Natural PenicillinsAntistaphylo - coccal PenicillinsAminopeni -

cillinsAntipseudo - monal Penicillins Penicillin G Methicillin Ampicillin Mezclocillin
Penicillin V Nafcillin Amoxicillin Piperacillin Pen G procaineOxacillin Azlocillin Pen G
benzathineDicloxacillin Carbenicillin Cloxacillin TicarcillinII. CLASSIFICATION
OF ANTIMICROBIAL DRUGS
1.1 PENICILLINS Natural Penicillins Penicillin G Penicillin V Pen G procaine Pen

G benzathineSTRUCTURE: MOA: block transpeptidation of peptidoglycan
USES: g+, g -, spirochetes, anaerobes Pneumonia –Strep pneumoniae
Pharyngitis, scarlet fever –Strep pyogenes Syphilis -Treponema pallidum
Meningitis -Neisseria meningitidis Diphtheria - Corynebacterium diphtheriae
Anthrax -Bacillus anthracis gas gangrene –Clostridium perfringens
DRAWBACKS & SIDE EFFECTS: widespread resistance ; hypersensitivity rxns;
diarrhea; seizures with renal probs. ADMN : Pen V = oral admn; others = IV,
IMONS
7. COOHII. CLASSIFICATION OF ANTIMICROBIAL DRUGS
1.1 PENICILLINS MOA: inhibitors of cell wall synthesis USES: narrow spectrum
(penicillinase producing Staphylococci); not for g - DRAWBACKS & SIDE
EFFECTS: Methicillin –interstitial nephritis Oxacillin -hepatotoxic

Page 17 of 46
Antistaphylo
coccal
Penicillins

Page 18 of 46
Methicillin
Nafcillin
Oxacillin
Dicloxacillin
CloxacillinPenicillinase -Resistant Penicillins
NOTES: methicillin & dicloxacillin = most
resistant to β-lactamases
Nafcillin = best with renal
failure Vancomycin = given
to those with
methicillin -resistant S. aureusII. CLASSIFICATION OF
8. ANTIMICROBIAL DRUGS Cell -wall synthesis inhibitors & β-
lactamase inhibitors
1.1 PENICILLINS MOA: inhibitors of cell wall synthesis USES: g+
(Streptococcus, Staphylococcus, Enterococcus, Clostridium) & g -(E. coli, H.
influenzae, H. pylori, Salmonella);URTI & LRTI;UTI ; gastroenteritis DRAWBACKS
& SIDE EFFECTS: inactivated by β-lactamaseExtended –Spectrum Penicillins
NOTES: can be combined with β-lactamase inhibitors
Aminope
ni cillins
Ampicilli
n
AmoxicillinII. CLASSIFICATION OF ANTIMICROBIAL DRUGS
U. Cell -wall synthesis inhibitors & β-lactamase inhibitors
1.1 PENICILLINS MOA: inhibitors of cell wall synthesis USES: g-(serious

Pseudomonal infections) DRAWBACKS & SIDE EFFECTS: inactivated by β-
lactamaseExtended –Spectrum Penicillins NOTES: can be combined with β-
lactamase inhibitors
Antipseudo
monal
Page 19 of 46
Penicillins
Mezclocillin

Page 20 of 46
Piperacillin
Azlocillin
Carbenicillin
TicarcillinII. CLASSIFICATION OF ANTIMICROBIAL DRUGS
1.2 β-Lactamase Inhibitors MOA: bind to & inactivate β-Lactamase USES:

extend the spectrum of other β-lactam agents (g+ S.aureus, g -H. influenzae &
anaerobes Bacteroides fragilis) NOTES: Amoxicillin + Clavulanic acid =
Augmentin Ticarcillin + Clavulanic acid = Timentin Ampicillin + Sulbactam
= Unasyn Piperacillin + Tazobactam = Zosynβ-
Lactamase inhibitors Clavulanic acid Sulbactam Tazobactam“Suicide Inhibitors”II.
CLASSIFICATION
11 OF ANTIMICROBIAL DRUGS
.
1.3Cephalosporins 1stGeneration 2ndGeneration 3rdGeneration
4thGeneration Cephalothin Cefamandole Cefotaxime Cefepime Cephalexin
Cefaclor Ceftazidime Cephradine Cefuroxime axetilCefoperazone Cephapirin
Cefonicid Ceftizoxime Cefadroxil Cefoxitin Ceftriaxone Cefazolin Cefotetan
Cefixime CefpodoximeII. CLASSIFICATION OF ANTIMICROBIAL DRUGS
12 Cell -wall synthesis inhibitors & β-lactamase inhibitors

.
1.3 Cephalosporins 1stGen 2ndGen 3rdGen 4thGenLess effective More effectiveG -
G
+More effective Less effective ◻ dec in g+ coverage ◻
inc in g
coverag
e
◻
inc in CNS penetrn
◻
Page 1U of 32
inc in resistance to
β
-
lactamaseII. CLASSIFICATION OF ANTIMICROBIAL DRUGS
13.
1.3Cephalospori
ns Cepha
losporins
1stGeneratio
n 2nd
Generation
3rd
Generation
4th
Generation
Spectrum
of
Activity
Organis
ms
Clinical
Uses/Adverse
Effects
+++ - SPEcK –Staph, Strep,
Proteus, E.coli, KlebsiellaUTI,
pneumonia; cephalothin is
nephrotoxic
++ -- HENSPEcK –

Page 20 of 46
Haemophilus Enterobacter,
Neisseria, Serratia,
etc.Meningitis, sinusitis,
otitis

Page 21 of 46
media
--- BPHENSPEcK -Borrelia,
Pseudomonas, etc.Lyme disease, meningitis, gonorrhea ++ --- PENS –
Pseudomonas, Enterobacter, Staph, StrepUTI, skin, pneumoniaII.
14.
1.4 Carbapanems and Monobactams Carba - penems & Mono - bactams
Imipenem/ Cilastatin/ Meropenem AztreonamSpectrum of ActivityOrganisms
Clinical Uses/Adverse effects Aerobic & anaerobic g+ & g-organismsP. aeruginosa,
B. fragilis but NOT methicillin - resistant S.aureus (MRSA)For infections resistant
to other meds; high doses cause seizures Aerobic g -rods but NOT g+ or
anaerobes P. Aeruginosa & SerratiaFor patients allergic to penicillin; skin
rashes, inc.SATsII. CLASSIFICATION OF ANTIMICROBIAL DRUGS
15.
1.5Others Spectrum of ActivityClinical Uses/Adverse effects G+, MRSA, MRSE,
Enterococcus faecalis, Clostridium difficile ; NOT for G -Pseudomembra -nous
colitis (ONLY oral indication); serious g+ infectns resistant to β- lactams;
prophylaxis for pxs with prosthetic heart valves; NEPHROTOXIC; hearing loss;
RED MAN SYNDROME -dueto rapid IV infusion G+, G -, more for
Mycobacteria2ndline anti -TB drug; large doses cause seizures; toxicOthers
Vanco - mycin Cyclo - serineMOA Binds to D - alanyl -D- alanine & stops
transpeptidn Antagonizes D-alanine, stops alanine racemaseII.
16.
1.5 Others Spectrum of ActivityClinical Uses/Adverse effects Mycobacterium
tuberculosis4- drug regimen for PTB; RIPE –Rifampin, INH, Pyrazinamide, EMB -
for new same + PTB taken for 6 months; Peripheral neuritis –due to vit B6
deficiency Hepatotoxic; hemolytic anemia EMB –optic neuritis (blurred vision,
eye pain)Others Isonia - zid (INH)MOA Inhibition of mycolic acid synthesis
(cell
17 wall)II. CLASSIFICATION OF ANTIMICROBIAL DRUGS
. ALTERATION OF CELL MEMBRANE PERMEABILITY

Page 22 of 46
(Bacteria) 2.1 Polymyxins (cationic peptide) EX: Colistin MOA: positively charged
NH2grps (like cationic detergents) disrupt phospholipid structure of the cell
membrane CLINICAL USES: Topical for G -skin infections caused by
Pseudomonas; nephrotoxicII. CLASSIFICATION OF ANTIMICROBIAL DRUGS
18. ALTERATION OF CELL MEMBRANE PERMEABILITY
(Fungi) 2.2 ANTIFUNGAL DRUGS Polyenes Azoles Miscellaneous Amphotericin

B Ketoconazole Flucytosine Nystatin Fluconazole Griseofulvin Itraconazole
Miconazole ClotrimazoleII. CLASSIFICATION OF ANTIMICROBIAL DRUGS
1U ALTERATION OF CELL MEMBRANE PERMEABILITY
.
(Fungi) 2.2 ANTIFUNGAL DRUGS II. CLASSIFICATION OF ANTIMICROBIAL
DRUGS
20. ALTERATION OF CELL MEMBRANE PERMEABILITY
(Fungi) 2.2 ANTIFUNGAL DRUGS Polyenes Amphotericin

B NystatinSpectrum of ActivityClinical UsesAdverse Effects CHA –Candida,
Cryptococcus neoformans, Coccidiodes sp, Histoplasma sp, AspergillusSystemic
fungal infections, fungal meningitis Given IV,ITNephrotoxic; causes fever, chills;
hypokalemia leading to muscle weakness Candida Vaginal candidiasis
(topical)Few reportsII. CLASSIFICATION OF ANTIMICROBIAL DRUGS
21 ALTERATION OF CELL MEMBRANE PERMEABILITY

.
(Fungi) 2.2 ANTIFUNGAL DRUGS (block metabolism of lanosterol ) Clinical
Uses Adverse Effects Against CHAB - Blastomyces ; OralGI effects: nausea &
vomiting Same; penetrates CNS, oral & IVsame CHAB & Tinea infections, oral &
IVSame Same; topical & IV Few reports Dermatophytes; topical Few reportsAzoles
Ketoconazole Fluconazole Itraconazole Miconazole ClotrimazoleII.
22.
ALTERATION OF CELL MEMBRANE PERMEABILITY

Page 23 of 46
(Fungi) 2.3 OTHER ANTIFUNGAL DRUGS Clinical Uses Adverse Effects
cryptococcal & candida infections (tog. with amphotericin B); oral; narrow
spectrumBone marrow depression (leukopenia & thrombocytopenia); hepatitis
Dermatophytes (ringworm -skin, hair, nails); oralHeadache, hepatotoxic, GI
irritationMiscella - neous Flucytosine (pyrimidine analog –5- fluorouracil)
GriseofulvinII. CLASSIFICATION OF ANTIMICROBIAL DRUGS
23.
INHIBITION OF PROTEIN SYNTHESIS
30 S inhibitors: Aminoglycosides
–bactericidal Tetracyclines - bacteriostatic 50 S inhibitors: Chloramphenicol –
bacteriostatic Macrolides – bacteriostatic Lincosamides - bacteriostaticII.
24.
Amino - glycosidesTetra - cyclinesChloram - phenicolMacrolides Linco -
samides Streptomycin Tetracycline Chloram - phenicolErythromycin
Clindamycin Gentamicin Doxycycline Clarithro - mycinLincomycin Tobramycin
Minocycline Azithromycin Amikacin Demeclo - cycline Netilmicin Oxytetra -
cycline Neomycin KanamycinII. CLASSIFICATION OF ANTIMICROBIAL DRUGS
25.
INHIBITION OF PROTEIN SYNTHESIS -bind to 30S
Amino - glycosides Streptomycin Gentamicin Tobramycin Amikacin Netilmicin

Neomycin KanamycinClinical Uses Adverse Effects TB, bubonic plague,
streptococcal endocarditisA– Allergic skin rxns
Neomycin Pneumonia by Pseudomonas aeruginosa, UTI, IV, IM, topicalM–neuro
Muscular blockade; C.I. with myasthenia gravis I –Inactivated when physically
mixed with β-lactams N-Nephrotoxic O–Ototoxic, Optic nerve toxicity
(streptomycin) Bowel sterilization in prepn for surgery; skin
infectionsOtotoxicity: strepto>amikacin>netilmicin For oral & topical
Nephrotoxicity: neomycin>genta>strepII. CLASSIFICATION OF ANTIMICROBIAL
DRUGS
26.
INHIBITION OF PROTEIN SYNTHESIS -block binding of

Page 24 of 46
amino -acyl-t-RNA in mRNA complex Tetra - cyclines Tetracycline Doxycycline
Minocycline Demeclo - cycline Oxytetra - cyclineClinical Uses Adverse Effects
G+, G -, anaerobes Vibrio cholerae Acne Chlamydia Ureaplasma Mycoplasma
Tularemia Borrelia burgdorferi RickettsiaTooth & bone discoloration; GI irritation
Fanconi syndrome –ingestion of expired drug Vertigo, dizziness (Minocycline)
CI: children < 8 yrs old pregnant & lactating women VACU uM TheBedRoomII.
27.
Clinical Uses Adverse Effects Bacteriostatic; Bactericidal for H. influenzae
(children); Typhoid fever; anaerobic infections; rickettsial diseases in children &
pregnant womenAnemia (due to bone marrow suppression) Gray Baby
Syndrome –cyanosis, vomiting, green stools, vasomotor
collapseChloramphenicolII. CLASSIFICATION OF ANTIMICROBIAL DRUGS
28.
INHIBITION OF PROTEIN SYNTHESIS –binds to 23S rRNA
of the 50S subunit Macrolides Clarithro - mycin Azithromycin
ErythromycinClinical Uses Adverse Effects Aerobic G+ Pneumonia –
Mycoplasma pneumoniae; Pertussis –Bordetella pertussis; Legionnaire’s
disease–Legionella pneumophila Diptheria – Corynebacterium diphtheriae;
chancroid – H. ducreyi; tetanus– Clostridium tetaniGI effects : nausea, vomiting,
GI upset, diarrhea Liver effects : Cholestatic hepatitisII. CLASSIFICATION OF
ANTIMICROBIAL DRUGS
II. CLASSIFICATION OF ANTIMICROBIAL DRUGS 3. INHIBITION OF PROTEIN

SYNTHESIS –
binds to 50S subunit Clinical Uses Adverse Effects G+ & Bacillus fragilis;
prophylaxis in orthopedic surgery & treatment of osteomyelitis; Acne;
Pneumonia (Pneumocystis carinii)Pseudomembranous colitis due to Clostridium
difficile Skin rashesLinco - samides Clindamycin Lincomycin
II. CLASSIFICATION OF ANTIMICROBIAL DRUGS 4. INHIBITION OF NUCLEIC
ACID
SYNTHESIS & REPLICATION Fluoroquinolones Rifampin Ciprofloxacin Rifampin
Ofloxacin Norfloxacin Lomefloxacin Enoxacin Trovafloxacin Nalidixic acid
Page 25 of 46
(quinolone)

Page 26 of 46
ACID
SYNTHESIS & REPLICATION – inhibit DNA gyrase for DNA replication &
transcription Fluoroquinolones Ciprofloxacin Ofloxacin Norfloxacin Lomefloxacin
Enoxacin Trovafloxacin Nalidixic acid (quinolone)Clinical Uses Adverse Effects
G+, G -but NOT anaerobes UTI Prostatitis Respiratory infections Nosocomial
infections GI infections –E. coli, Shigella, salmonella Chronic bone infections –
Pseudomonas & S. aureus Diabetic foot infectionsCNS effects: headache,
dizziness GI effects: nausea, diarrhea NOT for children – cartilage damage
ACID
SYNTHESIS & REPLICATION Clinical Uses Adverse Effects Tuberculosis
(combined with : Isoniazid Pyrazinamide Ethambutol RIPE –new smear + PTB
Meningitis Leprosy – Mycobacterium leprae (together with Dapsone)
Legionnaire’s disease (tog. with erythromycin)Red-orange colored secretions:
sweat, tears, urine & feces Flu-like syndrome – chills, fever & myalgias for 1x
or 2x weekly intakeRIFAMPIN MOA: binds to RNA polymerase and inhibits
RNA synthesis
II. CLASSIFICATION OF ANTIMICROBIAL DRUGS 5. INHIBITION OF ENZYME

METABOLISM
(Folate inhibitors)
METABOLISM
(Folate inhibitors) Sulfonamides Trimethoprim Sulfisoxazole Trimethoprim
Sulfamethoxazole Trimethoprim/sulfa - methoxazole Sulfadiazine Sulfamethizole
Sulfasalazine Sulfadoxine Sulfacetamide Silver sulfadiazine Mafenide
METABOLISM
(Folate inhibitors) Sulfonamides Sulfisoxazole Sulfamethoxazole Sulfadiazine
Sulfamethizole Sulfasalazine Sulfadoxine Sulfacetamide Silver sulfadiazine
Mafenide (topical)Clinical Uses Adverse Effects G+, G -, bacteriostatic UTI due to E.
coli & Klebsiella but NOT Pseudomonas - sulfamethoxazole Chlamydial
infections - sulfacetamide Toxoplasmosis Nocardiosis – Nocardia
Page 27 of 46
(sulfisoxazole) Burn infections –Ag sulfadiazineCRANK: Crystalluria
(sulfadiazine) Rashes Anemia Nausea Kernicterus Stevens Johnson syndrome
CI: pregnant women, children < 2 mos old

Page 28 of 46
METABOLISM
(Folate inhibitors) Clinical Uses Adverse Effects Recurrent UTIs Bacterial
prostatitis Gonorrhea Sinusitis/bronchitis Pneumonia caused by Pneumocystis
carinii Acute otitis media Chancroid, shigellosis Typhoid feverFolate deficiency
problems –anemia Skin rashes Stevens -Johnson syndrome GI effects: nausea,
vomitingTrimethoprim Trimethoprim Trimethoprim/sulfa -methoxazole
II. CLASSIFICATION OF ANTIMICROBIAL DRUGS C. ANTIVIRAL DRUGS
Rimantadine
Interferons
HOST CELL
II. CLASSIFICATION OF ANTIMICROBIAL DRUGS C. ANTIVIRAL DRUGS

Antiherpe - tic
DrugsAntiretro - viral-NRTIsAntiretro - viral-NNRTIsAntiretro - viral- Protease
InhibitorsOther Antiviral Drugs Acyclovir Zidovudine (AZT)Nevirapine Indinavir
Amantadine Foscarnet Didanosine (ddI)Delavirdine Saquinavir Rimantadine
Ganciclovir Zalcitabine (ddC)Efavirenz Ritonavir Zanamivir Idoxuridine Lamivudine
(3TC)Nelfinavir Oseltamivir Vidarabine Abacavir Amprenavir Interferons
Stavudine Ribavirin
II. CLASSIFICATION OF ANTIMICROBIAL DRUGS C. ANTIVIRAL DRUGS –
interfere/inhibit
viral DNA polymerase & viral DNA replication Antiherpe - tic Drugs Foscarnet
Ganciclovir Idoxuridine Vidarabine AcyclovirClinical Uses Adverse effects Genital
herpes, varicella (chickenpox), Herpes zoster (shingles) Cytomegalovirus/ CMV
retinitis in AIDS patients (Foscarnet, Ganciclovir) For herpes, GIV
acyclovirNephro -& neurotoxic at high doses (acyclovir) Bone marrow
suppression (ganciclovir)
nucleoside
analogs that can be incorporated into new viral directed DNA, terminating DNA
chain Clinical Uses Adverse effects AIDS : CD4< 200 cells/mL + presence of
opportunistic infections RULE : CD4 normal but suspect HIV infectn, DO NOT
give anti -HIV med.; CD4
<1000 & asymptomatic, give treatment; CD4 high
(<500)& with HIV symptoms, start tx with AZT; give ddC + AZT if
Page 2G of 32
CD4<300Anemia, headache, fatigue, peripheral neuropathyAntiretro - viral-
NRTIs Zidovudine (AZT) Didanosine (ddI) Zalcitabine (ddC) Lamivudine (3TC)
Abacavir Stavudine (d4T)

Page 2G of 32
inhibition of RT,
directly binds to RT & blocks viral DNA synthesis Clinical Uses Adverse effects
Can be combined with NRTIs for HIV treatmentResistance develops rapidly with
monotherapy; Stevens Johnson syndrome Delavirdine (teratogenic)Antiretro -
viral-NNRTIs Nevirapine Delavirdine Efavirenz
inhibition of viral
protease that cleaves viral proteins into virions Clinical Uses Adverse effects Can
be combined with NRTIs for HIV treatmentResistance develops rapidly with
monotherapy; GI distress Skin rashAntiretro - viral- Protease Inhibitors Indinavir
Saquinavir Ritonavir Nelfinavir Amprenavir

inhibition of viral
uncoating Clinical Uses Adverse effects Influenza A viral infection (amantadine
& rimantadine) Respiratory syncytial virus (ribavirin) Kaposi’s sarcoma, chronic
hepatitis B & CTeratogenic; anemia (ribavirin) GI effects (amantadine &
rimantadine) Flulike syndrome for interferonsOther Antiviral Drugs Amantadine
Rimantadine Zanamivir Oseltamivir Interferons Ribavirin
II. CLASSIFICATION OF ANTIMICROBIAL DRUGS D. ANTIPROTOZOAL DRUGS
Malaria
Amebiasis Leish - maniasisTrypano - somiasis Chloroquine Metronidazole
Stibogluconate Suramin Quinine Diloxanide furoateMelarsoprol Mefloquine
Paromomycin Nifurtimox Pyrimethamine /sulfodoxineIodoquinol Pentamidine
Primaquine
Malaria
Chloroquine Quinine Mefloquine Pyrimethamine /sulfodoxine Primaquine
Amebiasis
Metronidazole Diloxanide furoate Paromomycin Iodoquinol
Leish -
maniasis Stibogluconate (contains Sb)MOA: inhibits glycolysis
(phosphofructokinase rxn) Vector : sandflies Organism: Leishmania
Page 27 of 46
Trypano -
somiasis Suramin Melarsoprol Nifurtimox PentamidineClinical Uses Adverse Effects
African sleeping sickness w/o CNS involvement; African sleeping sickness w/o
CNS involvement; American trypanosomiasis Alternative to trimethoprim -sulfa
for PCPGI effects: nausea, vomiting CNS effects Hypersensitivity rxns
III. ANTHELMINTHIC DRUGS PHYLUM CLASS & EXAMPLES DRUGS
NEMATODE
(Roundworms)Intestinal nematodes: Ascaris lumbricoides Necator americanus
(hookworm) Strongyloides stercoralis (threadworm) Trichinella spiralis Trichuris
trichiura (whipworm) Enterobius vermicularis (pinworm) Blood & Tissue
Nematodes: Onchocerca volvulus Wuchereria bancrofti, Brugia malayi Loa loa,
Dracunculus medinensis (guinea worm); Toxocara spPyrantel pamoate
Diethylcarbamazine Ivermectin Mebendazole Thiabendazole PLATYHEL - MINTHS
(Flatworms)Trematodes (FLUKES): Schistosoma (blood flukes) Clonorchis sinensis
(liver fluke) Parogonimus westermani (lung fluke) Cestodes (tapeworms): Taenia
solium (pork tapeworm) Taenia saginata (beef tapeworm)Praziquantel Niclosamide
III. ANTHELMINTHIC DRUGS A. NEMATODES (Roundworms) MOA: interfere

with synthesis of parasitic microtubules & decreases glucose uptake (depletes
energy stores) B. TREMATODES (Flukes) & CESTODES (tapeworms) MOA :
increases permeability of the cell membrane to Ca causing contraction, spasm
& paralysis of the worms (Praziquantel) inhibits oxidative phosphorylation in the
mitochondria of cestodes (Niclosamide)
IV. DRUG RESISTANCE 3 MECHANISMS: 1.Failure of the antimicrobial drug to
permeate the microorganism EX: tetracycline 2. Altered receptors for the drug
EXS: β-lactams = altered PBPs Macrolides = methylation of RNA at 50S
subunit nalidixic acid = altered gyrase sulfa/TMP = altered synthetase; altered
reductase for TMP 3. Inactivation of the drug by microbial enzymes EX: β-
lactamase –cleave β-lactam ring chloramphenicol acetyltransferase –acetylate
chloramphenicol

Page 28 of 46
RA U275 •Philippine Clean Water Act of • Section 22.d ◻DOH shall be
2004
primarily responsible for the promulgation, revision and enforcement of
drinking water quality standards
Water testing analysis •Bacteriological analysis (DOH) •Physical analysis
(DOH) •Chemical analysis (DOH) •Radiological analysis (PNRI regulated)
REQUIREMENTS FOR LICENSING •Physical Plant •Personnel •Documents
•Equipment, reagents and utilities
PHYSICAL PLANT •Floor Area –At least 20 square meter working area •Storage
cabinet for reagents •Sink with strong water supply •Laminar flow/Biosafety
cabinet (Microbiological Lab) •Fumehood (Chemical Lab) •Area for media
preparation •Sterilization Area
PERSONNEL •Head of Laboratory –Competent professional with at least 3 years
experience in the theory and practice of water
testing. –Clinical Pathologist (PSP) –Microbiologist (Philippine Academy of

Microbiology or PAM) •Analyst –B.S. degree with 2 years experience in water
testing –Microbiology Laboratory -> Registered
PERSONNEL •Analyst –Chemical Laboratory -> Registered Chemist/ Registered
Chemical Technician •Laboratory Aide/Technician –6 months experience on clerical
and laboratory support
DOCUMENTS •Work instructions •Quality Assurance System •Control of
Records DOCUMENTS •Waste Management Policy •Laboratory Safety Policy
•Housekeeping Policy
EQUIPMENT, REAGENTS •MICROBIOLOGY LABORATORY (MULTIPLE TUBE
FERMENTATION TECHNIQUE) –Equipment •Incubator •Water Bath •Autoclave
•Digital weighing scale •Magnetic Stirrer •Drying Oven •Refrigerator •Computer
EQUIPMENT, REAGENTS •MICROBIOLOGY LABORATORY (MULTIPLE TUBE
FERMENTATION TECHNIQUE) –Reagents •Lactose broth (Presumptive broth)
•E. Coli medium broth •Brillant Green Lactose Bile Broth (BGLB) –
Glasswares/ utilities •40 or 50 mL test tubes •Durham tubes •Inoculating loop
or automatic pipette •Erlenmyer flask 1000 ml •Graduated cylinder •Test tube
rack
Specimen Collection & Preparation

Page 2U of 32
Drinking Water (WHO) •Must be clear, colourless and free from objectionable
taste odor. •It must not contain any substance, organism, chemical or
radioactive material at a level and or concentration which could endanger the
health and lives of the consuming public.
Water testing analysis •Bacteriological analysis •Physical analysis •Chemical
analysis
•Radiological analysis
STANDARD PARAMETERS Bacteriological Quality Source & Mode of
supplyBacteria Standard value (no./100 mL) All drinking water supplies under
all circumstancesE. Coli or thermotolerant coliform0 Treated water entering the
distribution systemE. Coli or thermotolerant coliform0 Treated water in the
distribution systemE. Coli or thermotolerant coliform0
Standard Parameter for Physical & Chemical
Quality Standard Parameter for Physical &
Chemical Quality
Standard Parameter for Physical & Chemical Quality

Sampling for BACTERIOLOGICAL QUALITY •Sample should be representative of
the whole water supply.
FREQUENCY OF SAMPLING FOR DRINKING WATER SUPPLY SYSTEMS 1.LEVEL
I
1.Population Serve 1.U0-150 2.Minimum Frequency of Sampling 1.Once in every
three (3) months 2.LEVEL II 1.Population Serve 1.600 2.Minimum Frequency of
Sampling 1.Once in every two (2) months
FREQUENCY OF SAMPLING FOR DRINKING WATER SUPPLY SYSTEMS 3.
LEVEL III
1.Population Serve 1.Less than 5,000 2.Minimum Frequency of Sampling 1.1
sample monthly LEVEL III 1.Population Serve 1.5,000 to 100,000 2.Minimum
Frequency of Sampling
1. One sample per 5,000 population monthly
LEVEL III
1. Population Serve 1.More than 100,000 2.Minimum Frequency of
Sampling 1.20 samples plus 1 sample 10,000 population monthly 4. Bottled
water 1.Minimum Frequency of Sampling 1.Once in every two (2) months
Page 30 of 46
EMERGENCY
SUPPLIES OF WATER 1.Minimum Frequency of Sampling 1.Before delivery to
users METHODS OF SAMPLING

Page 31 of 46
SAMPLING FROM A TAP OR PUMP OUTLET
SAMPLING FROM A WATERCOURSE OR
RESERVOIR SAMPLING FROM DUG WELLS
Microbiology Water Testing Techniques
TECHNIQUE •Multiple Tube Fermentation Method –Total Coliform Test –Fecal
Coliform Test
•Membrane Filtration Technique –Total Coliform Test –Fecal Coliform Test
•Heterotrophic Plate Count •IMVIC Test (Indole, Methy Red, Vogues -
Proskauer, Citrate Tests)
MULTIPLE TUBE FERMENTATION METHOD 1.Remove the cap from the sample
bottle.
2. With the stopper in position, shake the bottle vigorously to achieve a
homogeneous dispersion of bacteria. 3.With a sterile 10 mL pipette, inoculate
10 mL of the sample into each of 5 tubes
(with Durham tube) containing 20 mL of LACTOSE BROTH 4.Incubate the

tubes at 36 degree C for 24 hours.
MULTIPLE TUBE FERMENTATION METHOD 5.At the end of the 24 hours
incubation period, examine each tube for the presence of GAS. 6.After 24
hours of incubation, record the number of tubes in a table. 7.Further reincubate
the NEGATIVE TUBES for another 24 hours. At the end of this period, check the
tubes again for gas production. 8.After 48 hours of incubation, record the
number of positive tubes in the table.
MULTIPLE TUBE FERMENTATION METHOD U. The confirmed test should be
carried out at the end of both 24 -hour and 48 hour incubation. Using a
sterile loop, transfer one or two drops from presumptive LB tube into EC medium
and Brilliant Green Lactose Bile Broth . 10. To confirm the presence of total
coliforms, incubate EGLB from each presumptive tube for 24 hours at 35 degree
C. 11.After 24 hours of incubation, record the
MULTIPLE TUBE FERMENTATION METHOD 13. To confirm the presence of
thermotolerant coliforms or fecal coliform, incubate the EC tube from each
presumptive tube for 24 hours at 44.5 degree C (Water bath). 14. If at the end
Page 32 of 46
of 24 hour incubation period gas is present in tubes, the presence of
thermotolerant coliforms or fecal coliforms is confirmed.

Page 33 of 46
MPN Index & U5% Confidence Limits No. of tubes giving positive
reactionsMPN/100mL U5% CI Lower LimitU5% CI Upper Limit 0 < 2.2 0 6.0 1 2.2
0.1 12.6 2 5.1 0.5 1U.2 3 U.2 1.6
2U.4 4 16 3.3 52.U 5 >16 8.0 Infinite Philippine National Standards for Drinking
Water, 20thEdition , 1UU3

Page 34 of 46

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Pathogenesis of Bacterial Infection Normal Human Microbiota Bacterial

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in ST. ST rarely occurs compared to GT.

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1.1 PENICILLINS Natural PenicillinsAntistaphylo - coccal PenicillinsAminopeni -

1.1 PENICILLINS Natural Penicillins Penicillin G Penicillin V Pen G procaine Pen

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1.1 PENICILLINS MOA: inhibitors of cell wall synthesis USES: g-(serious

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1.2 β-Lactamase Inhibitors MOA: bind to & inactivate β-Lactamase USES:

12 Cell -wall synthesis inhibitors & β-lactamase inhibitors

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(Fungi) 2.2 ANTIFUNGAL DRUGS Polyenes Azoles Miscellaneous Amphotericin

(Fungi) 2.2 ANTIFUNGAL DRUGS Polyenes Amphotericin

21 ALTERATION OF CELL MEMBRANE PERMEABILITY

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Amino - glycosides Streptomycin Gentamicin Tobramycin Amikacin Netilmicin

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II. CLASSIFICATION OF ANTIMICROBIAL DRUGS 3. INHIBITION OF PROTEIN

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II. CLASSIFICATION OF ANTIMICROBIAL DRUGS C. ANTIVIRAL DRUGS

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II. CLASSIFICATION OF ANTIMICROBIAL DRUGS C. ANTIVIRAL DRUGS –

III. ANTHELMINTHIC DRUGS A. NEMATODES (Roundworms) MOA: interfere

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testing. –Clinical Pathologist (PSP) –Microbiologist (Philippine Academy of

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Standard Parameter for Physical & Chemical Quality

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(with Durham tube) containing 20 mL of LACTOSE BROTH 4.Incubate the

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