Diagnostic Bacteriology.pptx

September 3, 2023 1
Collection, Storage & Transport of
Samples & Diagnosis of Microbial
Infection
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Collection, Storage & Transport of Samples &
Diagnosis of Microbial Infection
 Learning objectives
 Explain objectives of diagnostic microbiology
 Explain how different types of specimens are collected
 Memorize different microbiological test that is important for
identification and characterization of microorganisms
 Explain how antimicrobial susceptibility test is performed
and its importance in controlling drug resistance
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Steps in the laboratory investigation of an
infected patient
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The objectives of diagnostic microbiology are
 Isolate and identify pathogenic microorganisms from clinical
specimens rapidly
 Appropriate selection of antimicrobial agents for treating
patients
 Formulation of local antibiotic policy for empirical
treatment
 Design a strategy in the control and prevention of
infectious diseases
 Public health impact e.g. food handlers
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Sample collection
 The value and reliability of microbiological reports
are dependent
 The quality of the specimen received by the laboratory and
 The length of time between its collection and processing.
 The collection of specimens must have their own SOP
and given to individuals that collect sample.
 The right result at the right time, on the right
specimen, from the right patient, with interpretation
based on correct reference data.
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Guidelines for sample collection, storage and transport
 Guidelines must emphasize on the important aspects:
 Collection of the specimen before the administration of
antimicrobial agents
 Prevention of contamination of the specimen
 The specimen should be collected in appropriate containers
and forwarded promptly to the clinical laboratory
Sample collection
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 The general rules for collection and transportation
of specimens are summarized as:
 Apply strict aseptic techniques throughout the procedure
 Wash hands before and after the collection
 Collect the specimen at the appropriate phase of disease
 Representative specimen of the infectious process
 Collect or place the specimen aseptically in a sterile and/or
appropriate container (leak proof)
Sample collection
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 Ensure that the outside of the specimen container is clean and
uncontaminated
 Close the container tightly so that its contents do not leak
during transportation
 Label, write date and time on the container appropriately
 Arrange for immediate transportation of the specimen to the
laboratory
 preserve and transport in ideal medium
Sample collection
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Patient-Collected Specimens
 In certain situations, patients are asked to collect the specimen
themselves.
 Medical personnel should provide patients with thorough instructions
on how to collect the sample.
 It should not be assumed that the patient knows how to collect a particular
type of specimen.
 Provide verbal and written instructions.
 It may be necessary to read the instructions to the patient.
 The instructions should be written using simple language and pictures to
help the patient understand the procedure
 The specimens commonly obtained by the patient are urine, sputum, and
stool.
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 Criteria for rejection of specimens
 Criteria should be developed by a laboratory as in the
following some examples:
 Missing, Mismatch or inadequate identification
 Short forms are dangerous
 Insufficient quantity
 Specimen collected in an inappropriate container
 Contamination suspected
 Inappropriate transport or storage
 Unknown time delay
Sample collection
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Specimens containing dangerous pathogens
Sample collection
 Specimens which should be marked as HIGH RISK include:
 Sputum likely to contain M. tuberculosis
 Fecal specimen that may contain V. cholera and S. typhi
 Fluid from ulcers pustules that may contain Bacillus anthracis
or Treponema species
 Specimen from patients suspected HIV infection, hepatitis,
viral hemorrhagic fever, Ebola
 Label with warning symbol such as red dot, star, or triangle
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Specimens collected for diagnosis of
septicemia, bacteremia and meningitis
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Sample collection procedures, storage and transport
A. Blood
 Whole blood is required for bacteriological examination
 The goal in blood collection is avoiding the contamination
 Skin antisepsis is extremely important. E.g. iodine
 Collect blood during the early stages of disease
 Small children usually have higher number of bacteria in their blood
as compared to adults
 Less quantity of blood needs to be collected from
 Adults 10-20ml, Infants 1-2ml, children: 2-5 ml blood (aseptically
from vein)
 if suspect bacterial endocarditis: 3 sets of blood culture
 Do not collect from existing or indwelling catheters
Septicemia….
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Anticoagulants
 Organisms bound up in clotted material are difficult to isolate.
 The type and concentration are important because some
anticoagulants have antimicrobial properties.
 Sodium polyanethol sulfonate (SPS) is the most common
anticoagulant used for microbiology specimens.
 concentration of SPS must not exceed 0.025% (wt/vol) because some
Neisseria and certain anaerobes are inhibited by higher concentrations.
 Heparin is another acceptable anticoagulant and is often used for
viral cultures and for isolation of Mycobacterium spp. from blood.
 Citrate and ethylenediamine tetraacetic acid (EDTA) should not
be used for microbiology specimens.

B. Cerebrospinal fluid (CSF)
 Needed for the diagnosis of any patient
with evidence of meningeal irritation or
affected cerebrum
 If delay is anticipated leave at Room
Temperature (Refrigerator kills H.
Influenza)
 Almost 3-10 ml of CSF is collected and
part of it is used for
 Biochemical, Immunological
 Microscopic examination
 Bacteriological or fungal examination.
 CSF is a precious specimen, handle it
carefully and economically. It may not be
possible to get a repeat specimen
Septicemia….
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Specimens collected for diagnosis of
Upper Respiratory Infections
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Upper Respiratory Infections
 The commonest respiratory infections are localized in
Oropharynx, Nasopharynx, and nasal cavity
 Causes Sore throat, nasal discharge and often fever.
 Infect larynx, otitis media, sinusitis, conjunctivitis or
keratitis.
 May present with serious diseases whooping cough,
influenza, measles and infectious mononucleosis.
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Etiological agents in Upper Respiratory Infections
 In most cases the primary infections are caused by
virus, difficult to isolate.
 But many infections are caused by concomitant
carriage or secondary infection with
 Pneumococcus, Haemophilus influenza,
 Staphylococcus aureus, and Streptococcus pyogenes.
 Drug resistant coliform bacilli or yeasts may dominate
the throat flora in patients receiving antibiotics.
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Upper Respiratory Infections…
 Throat Swabs
 Collect throat exudates from
 Tonsils, posterior pharyngeal wall and other
area that is inflamed or bears exudates
 If it cannot be transported immediately to
laboratory
 It should be placed in a refrigerator at 4ºc until
delivery or
 Preferably submitted in a tube of transport
medium
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 Nasal specimens
 A deep nasal swab generally yields
the same information as throat
swab.
 Nasal swabs are taken to detect
healthy carriers than diagnose
deep infection
 Deep nasal are taken to diagnose S.
pyogenes and Diphtheria bacillus.
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Nasopharyngeal swab
 Tilt head backwards
 Insert flexible fine-
shafted polyester swab into
nostril and back to
nasopharynx
 Leave in place a few seconds
Withdraw slowly; rotating
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 Nasopharyngeal aspirate
 Tilt head slightly backward
 Instill 1-1.5 ml of VTM
/sterile normal saline into one
nostril
 Use aspiration mucus trap
 Insert silicon catheter in
nostril and aspirate the
secretion gently by suction in
each nostril
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 Specimens in sinusitis
 Pus collected or aspirated
from sinus, or
 A saline wash out should be
examined in a Gram film and
 Culture on aerobic and
anaerobic blood agar plates.
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Collection of Ear Swabs
 Acute otitis Media
 As long as eardrum remains
intact, none of the infected
exudates can be collected on an
ear swab
 But, culture of the throat swab
may give a provisional indication
of casual organism
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Collection of Ear Swabs….
 Chronic suppurative
otitis media
 Swabs of the discharge in the
external meatus should be cultured.
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Collection of Ear Swabs….
Otitis externa
 A swab should be taken from
the meatus and cultured
aerobically on blood agar and
MacConkey agar plates for
the bacteria.
 All specimens should also
cultured on Sabouraud’s agar
plate with Nystatin 50 units
for Candida and Aspergillus.
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Eye Swabs
 Obtaining adequate specimen
is difficult.
 It is best to make smears and
seed culture plates beside the
patient immediately after
collecting the material from
the eye.
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Eye Swabs….
 It is ideal to pick up the
material with
 A loop or
 on the smoothly rounded tip
of a thin glass rod or
 on the thin serum coated
swab
 Clinical material from
Conjunctiva, i.e. from
inverted eyelid,
 The margin of the eyelid
should be avoided.
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Specimens for diagnosis of Lower Respiratory
Infections
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Specimens for Lower Respiratory Infections
C. Sputum
 For investigation of bacterial and fungal infections of the
lower respiratory tract
 Avoid saliva or postnasal discharge
 It is of utmost importance in the diagnosis of pulmonary
tuberculosis
 Select a good wide-mouthed sputum container, which is
preferably disposable, made of clear thin plastic, unbreakable
and leak proof material
 Give the patient a sputum container with the laboratory serial
number written on it
 Show the patient how to open and close the container
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Lower Respiratory….
 Sputum…
 Instruct the patient to inhale deeply 2-3 times, cough up
deeply from the chest and spit in the sputum container by
bringing it closer to the mouth
 Make sure the sputum sample is of good quality
 A good sputum sample is thick and sufficient in amount (2-
3 ml)
 Give the patient an additional container for an early
morning specimen
Explain to the patient to rinse his/her mouth with plain
water before bringing up the sputum
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 Sputum…
 Several specimens should
be collected before coming
to negative conclusions
 Specimen should be
collected with biosafety
precautions
Lower Respiratory….
To ensure the survival of pathogens such as S. pneumoniae and H.
influenzae, transfer a purulent part of the sputum to a cotton-wool
swab, and insert it in a container of Amies transport medium
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Specimens for diagnosis of Urinary Tract
Infections
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Urinary Tract….
D. Urine
 Under normal circumstances urine is sterile
 The lower part of the urethra and the genitalia are normally
colonized by bacteria, many of which may also cause urinary
tract infection
 Since urine is a good growth medium for all sorts of bacteria,
proper and aseptic collection assumes greater importance
 Collect clean catch Mid stream specimens of Urine (Do not
collect spontaneously passed urine)
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 All collected specimens of urine to be transported to
laboratory with out delay
 Delay of 1 – 2 hour deter the quality of diagnostic
evaluations.
 continuous growth of bacteria in vitro altering the actual
concentration of organisms.
 If the delay is anticipated the specimens are preserved
at 40c
 In field conditions Boric acid can be added at a
concentration of 10 g/l (1%w/v)
Urinary Tract….
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Specimen for diagnosis of Genital
Tract Infections
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Genital Infections in women
 Genital infections:
 Urethritis
 vaginitis
 genital ulceration
 cervicitis
 uterine sepsis
 salphingits
 oophoritis, and
 pelvic inflammatory disease.
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Genital infections….
 Collection of specimens
 Vaginal swab
 The specimen commonly
collected for the diagnosis of
vaginitis's, vaginosis or uterine
sepsis is high vaginal swab
 The swab is inserted into
upper part of the vagina and
rotated there before
withdrawing it.
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Specimen collection in Gonorrhea
 endocervical swab
 for gonococci.
 A vaginal speculum must
be used to provide a clear
sight of the cervix and
 Swab is rubbed in and
around the introitus of the
cervix and withdrawn
without contamination from
vaginal wall.
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Specimens from other genital areas
 Other swabs should be collected
from any exudate discharged
from the meatus of the urethra
or a Bartholin's gland.
 Rectal or pharyngeal swabs
should be considered depends
on sexual habits of the patient
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Specimen collection in Men
The infection in men are mostly caused by the same
organism as in women.
 Urethritis is commonest presentation may be caused by
Gonococci or Non-gonococcal.
 May present with Genital Ulcers.
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Gonococcus infection in Men
 Urethral discharge
 Collected by milking the urethra and
 Urethral discharge is smeared on slides and inoculated on
warmed plates of heated blood agar or selective medium
for isolation of Gonococci
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Other Genital infections
 When prostatitis is
suspected and there is no
spontaneous discharge
from urethra, massage of
the prostate per rectum
may express some
exudate for examination,
and culture.
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Collection of specimens in Chancres
The examination of chancre requires the careful
collection of exudates and its preparation for
dark ground microscopy.
Many patients need clotted blood for specific
serological investigation
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Transportation of specimens
 All the swabs to be promptly transported to
laboratory, in cases of delay or in cases of delicate
microbes to be transported in Amie's transport
medium.
 If possible two swabs to be collected and submitted
for each site.
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Specimen for diagnosis of
Wound, Skin, abscess, and
Deep Sepsis
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Wound sample
 Should be collected before an
antiseptic dressing is applied
 Pus or exudates is often submitted
on a swab for laboratory
investigation.
 The swabs are inefficient sampling
device and tends to desiccate the
specimen and trap the bacteria
which are then not released on to
culture plate
Wound, Skin, and Deep Sepsis…
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Ideal samples from wounds
 The ideal sample is pus or
exudates should be submitted in a
small screw- capped bottle in
firmly stoppered tube or syringe or
a sealed capillary tube.
 Fragments of excised tissue
removed at wound toilet or
curettings from infected sinuses
and other tissues should be sent in
a sterile container.
 transport using Amies transport
medium
Wound, Skin, and Deep Sepsis…
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Specimen for diagnosis of
Gastrointestinal Infections
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E. Stool
 Collect fecal specimens for the etiological diagnosis of acute
infectious diarrheas
 in the early stage of illness (within 48 hours of onset )
 prior to treatment with antimicrobials
 The feces specimen should not be contaminated with urine
 1 to 2 gm quantity is sufficient
 If possible, submit more than one specimen on different days
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 Storage
 refrigerate at 4°C; do not freeze
 store at -15°C - for Ag detection,
polymerase chain reaction (PCR)
 Transport
 4°C (do not freeze); dry ice for (Ag
detection and PCR)
 Sent with Cary-Blair medium if
delay is more than 1 hr
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Generally Specimen collection
 Type of specimen
 The correct type of specimen to collect will depend on the pathogens to
be isolated
 Time of collection
 Urine and sputum are best collected soon after a patient wakes
(organisms multiply over several hours).
 Blood for culture is collected when a patient’s temperature begins to rise.
 The time of collection for most other specimens will depend on
 the condition of the patient, and
 The times agreed between the medical, nursing, and laboratory staff
 Important: Every effort must be made to collect specimens for
microbiological investigation before antimicrobial treatment
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 Collection & transportation of Good quality
specimen for microbiological examination is crucial
“Collecting the appropriate specimen is neglected part
of Diagnostic Microbiology, although it ends up with
enormous errors”
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Biosafety Precaution
All the
Technical staff should
follow the Universal
and other Biosafety
Precautions while
handling and
Disposing the
Microbiology
Specimens
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Microbiological laboratory techniques
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Identification of bacteria (Diagnostic methods)
 Microscopic examination
 Wet mount (unstained) methods
Saline
hanging drop method
Dark field microscope
Staining
Negative
Positive
 Culture
 Biochemical test
 Immunological test
 Molecular tests
 Antimicrobial
susceptibility test
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MICROBIOLOGY
Information derived from the results has impact
on :
 Diagnosis of infectious diseases
 Antibiotic prescribing
 Formulation of local antibiotic policy
 Public health impact e.g. food handlers
 Infection Control measures e.g. MRSA,
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Quality Assurance

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Learning Objectives (1)
At the end of this session participants should be able to:
 Enumerate the components of a quality assurance (QA) program
 Explain the principles of quality control (QC), quality improvement
& proficiency testing (or external quality assessment – EQA)
 Summarize the positive aspects of a QA program
 Enumerate QC parameters in the microbiology laboratory
 Explain why QC is necessary
 Develop standard SOP format
 Maintain patient reports, QC records, instrument/equipment
maintenance, & other documents

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Content Overview
 Definition
 Components of a QA program
QC, Quality Improvement, Proficiency Testing
 QC Parameters
 Culture Media QC

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Quality Assurance
 A system designed to continuously improve
the reliability, efficiency, & use of laboratory
services
 A continuous system of QC needs to be
established in order to achieve the required
technical quality in laboratory diagnosis

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Components of a Quality Assurance
Program
Quality control
Quality improvement
Proficiency testing

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 A process of effective & systematic monitoring of the
performance of bench work in the laboratory against
established limits of acceptable performance
 QC ensures that the information generated by the
laboratory is accurate, reliable, & reproducible
 Serves as a mechanism by which laboratories can
validate the competency of their diagnostic services
Quality Control

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Quality Improvement
 A process by which the components of the
laboratory services are analysed continuously to
improve their reliability, efficiency, & utilization
 Data collection, data analysis, & creative problem
solving are the key components of this process
 Involves continuous monitoring, identification of
defects, followed by remedial action to prevent
recurrence of problems

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 Also called external quality assessment (EQA) by
WHO standards
 A system of retrospectively & objectively compared
results from different laboratories by means of
programmes organized by an external agency, such
as a reference laboratory
 To establish between-laboratory comparability, in
agreement with a reference standard
Proficiency Testing

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Proficiency Testing Process (1)
 Materials for testing may be prepared in the central
or reference laboratory & distributed to lower level
laboratories
 The recipients perform the necessary procedures &
report their results to the central or reference
laboratory which then assess proficiency
 Detection of deficiencies through this indirect
system will then determine the need for quality
improvement

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Proficiency Testing Process (2)
Internal proficiency testing may also be considered:
 Simulated specimen with unknown organisms
 Reprocess specimens for analysis by different
personnel
 Send a portion of the specimen to a reference
laboratory

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 Potential problems in isolation & identification
procedures can be greatly reduced by monitoring media
and reagents before using them on clinical specimens
 Serious & costly breakdowns of equipment may be
minimized by routine monitoring & maintenance
 Laboratory reports can be more accurate & expeditious
as the use of inadequate media, equipment & technique
is minimized
Positive Aspects of QA Programs (1)

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Positive Aspects of QA Programs (2)
 The QA program can be a learning exercise,
enabling the recognition & identification of
problem areas that might have been overlooked
 A good QA program will enhance the credibility
of the laboratory to outside clients

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General Quality Control Parameters
 Specimen collection & transport
 Procedure manual
 Personnel
 QC Records
 Patient Reports

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 Provide instructions for collection & transport
 Establish criteria for acceptable specimens
 Establish criteria for unacceptable specimens
 Include screening tests among the criteria for
acceptable specimens
 Do not test unacceptable specimens
General QC Parameters:
Specimen Collection & Transport

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 Write in standard format (ISO, CLSI)
 Group all procedures, when applicable, as pre-
analytical, analytical, & post-analytical
 Review & initial annually
 Approve and date all changes
 Make available in work area
 Retain obsolete procedures for 2 years
Procedure Manual

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 Employ sufficient number of personnel for volume
& complexity of work
 Document continuing education activities
 Provide employees with written performance
standards
 Evaluate employees annually
 Hold regular scheduled meetings
Personnel

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 Record all QC results on QC forms or in computer
 Report all out-of-control results to supervisor and
note corrective action(s) taken on QC forms
 Hold at least monthly review of QC record with
supervisor
 Retain QC records for a minimum of 2 years
QC Records

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 Report results only to authorized personnel
 Notify test requester of “panic” values immediately
 Review hardcopies of all patient reports; use computer flags
for unusual reports
 Provide normal ranges, when appropriate
 When rendering verbal reports, record name of individual
notified, date & time
 Correct errors in patient reports in a timely fashion
 Retain records for a minimum of two years Note: time may
vary in different countries
Patient Reports

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Specific Quality Parameters
Instrument & equipment performance
Media
Stains, reagents & antisera
Commercial kits

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 Perform as frequently as necessary to ensure proper
function or as specified by manufacturer
 Document function checks of equipment
 Document routine preventive maintenance
 Retain maintenance records for life of equipment
Specific QC Parameters:
Instrument or Equipment Performance

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 Record amount prepared, lot number, preparation
date, pH, expiration date, & name of preparer
 Test medium with QC organisms of known
physiological & biochemical properties
 Test each batch of prepared media for sterility,
ability to support growth, & ability to produce
appropriate biochemical reactions
Culture Media

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 Should be derived from well-characterized
strains
 American Type Culture Collection (ATCC)
reference strains
 Commercial sources (ATCC strains also from
commercial suppliers)
 Proficiency testing programs (EQA)
Sources of QC Organisms

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Label in-house prepared reagents as to:
 Contents
 Concentration
 Date prepared
 Expiration date
 Storage requirements
 Date in-use
Stains, Reagents, & Antisera (1)

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 Label commercial reagents with:
 Date received
 Date opened
 Store according to manufacturer’s recommendations
 Test reagents with positive & negative controls prior to
use with each:
 Batch
 Lot number
 Shipment

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 Test Gram stain reagents with control organisms with
each batch, lot number, shipment, & weekly thereafter
 Check each lot number & shipment of antiserum
when opened & once every 6 months with positive &
negative controls
 Discard outdated materials & reagents that fail
performance standards

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 Notify the supervisor
 Repeat the test
 If the test remains out of acceptable range, repeat the test
with new lots of reagent and/or fresh isolate from the
stock
 If QC testing results remain outside of acceptable ranges,
do not test patient specimens until the problem has been
resolved
 Record all pertinent actions
When QC Result is Out of Range

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Quality control is the responsibility
of all laboratory workers

Thank You
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Pathogenic Gram Positive Cocci
Staphylococcus
Streptococcus
Enterococcus
Micrococcus
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The Staphylococci
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General Characteristics
 Gram positive cocci arranged in grape like clusters
 Possess slime layer
 Catalase positive
 It is a part of normal bacterial flora present in human body
(skin, ear, mucous membranes like nasal cavity, vaginal
mucosa etc.)
 Normally non pathogenic in these areas of the body
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 Cause nosocomial infection
 S. aureus is an important nosocomial pathogen
 Carrier may harbor S. aureus in their nasal canal are called
nasal carriers
 Methicillin resistant Staphylococcus aureus (MRSA) is the
challenge of public health
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Medical Important species
• S. aureus –important pathogen( coagulase positive)
• S. saprophyticus –coagulase -ve
• S. epidermidis –coagulase -ve
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Staphylococcus Aureus
 The important pathogen with many virulence factor
 incubation on blood agar produces white colonies that
tend to golden color with time
 Beta hemolytic in blood agar
 Grows in medias that contain salts( Manitol salt agar)
 Produce of coagulase, binds to prothrombin, forming a
complex that initiates the polymerization of fibrin
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Attributes of Pathogenicity
A. Structural Features
 Slime layer /Capsule – Inhibits phagocytosis
 Peptidoglycan – endotoxin like activity (pyrogenic)
 Teichoic acid – binds to fibronectin
 Protein-A – antiphagocytic, Binds to Fc portion of IgG
(Inhibits antibody mediated clearance)
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B. Enzymes
 Catalase – catalyzes removal of hydrogen peroxide
 Coagulase (Clumping factor)- converts fibrinogen to fibrin
 Hyaluronidase- Hydrolyzes hyaluronic acids in connective
tissue, thus promoting the spread of S.aureus in tissues
 Fibrinolysin - dissolves fibrin clots
 Lipases -Hydrolyzes lipids
 Nucleases - Hydrolyzes DNA
 Penicillinases - Hydrolyzes penicillins
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C. Exotoxins
 Cytolytic toxins – cytotoxins ( α,β,γ)
 Leucocidins – Destroys WBCs
 Exfoliative toxins – damages epidermis of skin
responsible for scalded skin syndrome
 Enterotoxins – (A-E, G-I) – Responsible for food
poisoning – Heat stable toxin (stable at 1000C / 30mins) –
Enterotoxin A-D are important
 Toxic shock syndrome toxin-1 (TSST-1) – responsible for
toxic shock syndrome (TSS)
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Clinical Diseases
It is classified into three categories
1. Skin infections
2. Toxin mediated disease
3. Other Infections
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1. Skin Infections
• It include mainly pyogenic (pus forming) infections
Impetigo: localized cutaneous infection characterized by pus
filled vesicle on face and limbs
Folliculitis: impetigo involving hair follicules
Furuncles (boils): large, painful pus-filled cutaneous nodule
Carbuncle : coalescence of furuncles with extension into the
subcutaneous tissue and evidence of systemic disease (fever
chills bacteremia)
surgical wound infections, burn infections
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Pustular Impetigo
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2. Toxin Mediated disease
• Toxic shock syndrome (TSS)
• Scalded skin syndrome (SSS)
• Food Poisoning
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Toxic shock syndrome(TSS)
• TSST-1 toxin is responsible for this syndrome
• This is normally seen in menstruating women
• uses of tampon for a prolonged period, S aureus present in vaginal
mucosa multiplies and secretes TSST-1 toxin
• TSST-1 enters blood stream through minor abrasions leading to fever,
hypotensive shock – low blood pressure, vomiting, diarrhea, sore
• throat, and muscle pain ,erythematous rash followed by multi organ
failure (liver, kidney, heart, lungs etc) leading to death
• TSS may occur in men and children also
• If S .aureus infects wounds or burns, it may secrete TSST-1 toxin leading
to TSS 101

Toxic shock syndrome
102

Scalded skin syndrome (SSS)
• It is due to exfoliative toxin
• The disease is most common in neonates and children less than 5
years of age
• The face, axilla, and groin tend to be affected first,
• subsequent desquamation of epithelial sheets can spread to all parts
of the body
• The skin will pill out
• This toxin damages epidermis of skin
• Not killer and the skin become intact in 7-10 days after antibody
production 103

Scalded skin syndrome
104

Staphylococcal Food Poisoning
• Due to enterotoxins designated as A-E, G-I
• A-D are important
• These are toxins to intestinal mucosa
• food is contaminated with S aureus enterotoxin
• acute vomiting and diarrhea with no fever within 1
to 5 hours of ingestion
• Recovery is rapid
• Eg. Potato salad, ice cream
105

Other Infections
• Bacteremia
• endocarditis
• osteomyelitis (important infection)
• Pneumonia
• septic arthritis
106

Coagulase negative staphylococcus species
S.epidermidis
• Bacteremia
• endocarditis
• surgical wound infections
• UTI
• Opportunistic infections of
catheters
• prosthetic devices (infection
of artificial joints)
S. Saprophyticus
UTI, opportunistic
infections
107

Laboratory Diagnosis Staphylococcus
Specimens :
 Pus
 Nasal swab
 Blood
A. Smears
• Gram’s stain (Typical organisms)
• Not helps to distinguish among different Spp.
108

Gram positive cocci
grapelike clusters
109

B. Culture
 Blood agar plates give typical colonies in 18 hrs at 370C
 Media containing 7.5% NaCl inhibits the growth of other
bacteria, but not S. aureus
 S. aureus is distinguished by coagulase positivity
 S. aureus colonies tend to be yellow and β-hemolytic,
rather than gray and non hemolytic like the coagulase-
negative staphylococci
 S. aureus is also distinguished from most coagulase-
negative staphylococci by being manitol-positive and
gave yellow colony Manitol salt agar (MSA)
110

C. Catalase Test
– A drop of H202 (+) small amount of bacterial growth
on a slide
– Formation of bubbles (release of 02) ----- Positive
test
– Staphylococcus positive VS streptococcus negative
D. Coagulase Test
– Human / citrated rabbit / plasma diluted 1:5 and
equal volume of growth from colonies from broth
culture
– Incubated at 370C
• Positive test: If clots form in 1- 4 hrs
N.B. All coagulase positive staphylococci are considered
pathogenic to humans
111

Table: Biochemical characteristic of staphylococci
112

S.aureus on manitla salt agar
113

S aureus on Manitol salt agar
Staphylococcus aureus
114

Catalase test
115

Catalase test
Catalase
POS
Staph.
Catalase NEG
116

Coagulase test
S. aureus
117

E. Susceptibility Testing
– Disk diffusion susceptibility testing
– to penicillin detected by a pos. test for ß –lactamase
– Should be done for oxacillin and cefoxitine to detect
MRSA
F. Serological Testing
• Anti-bodies to teichoic acid (Chronic infections)
Treatment
– Tetracycline
– Amino glycosides
– Cephalosporins
– Erythromycins
– Vancomycine
118

Prevention & control
»Early detection & treatment
»Personal hygiene
»Proper storage of foods
»Carrier in hospital / food industry should
be treated, Effective sterilization,
119

The Streptococcus
120

• Gram-positive cocci arranged in chains
• Normal flora of the oropharynx
• Chains or pairs
• facultative anaerobes, fastidious, capnophilic
• Grows best in enriched medias
• Usually capsulated
• Non motile
• Non spore forming
• Catalase test – Negative [Staphylococci are catalase
positive]
122

Classification of Genus Streptococcus
• Streptococcus can be classified according to
A. Group specific cell wall carbohydrate : serological
classification based on the -- Lancefield grouping - 21
Lancefield ---groupings - A – H, K – M,O – V
e.g. Group A Streptococcus: S. pyogenes
Group B Streptococcus agalactiae
123

B. Hemolysis on Blood Agar
– -hemolysis
• Partial hemolysis
• Gray discoloration around the colonies
• e.g. non-groupable streptococci (S. pneumoniae & S.
viridans)
– -hemolysis
• Complete hemolysis
• Clear zone of hemolysis around the colonies
• e.g. Group A & B (S. pyogenes & S. agalactiae)
– -hemolysis
• No lysis
• e.g. Group D (Enterococcus spp.)
Streptococci
-hemolysis -hemolysis -hemolysis
124

Hemolysis on Blood agar
-hemolysis
-hemolysis
-hemolysis
125

C. Biochemical Test
 include sugar fermentation reactions, tests for the
presence of enzymes, and tests for susceptibility or
resistance to drugs
 used to classify streptococci after the colony growth and
hemolytic characteristics have been observed
 e.g. bile solubility and optochin susceptibility for S.
pneumoniae,
 Bacitracin susceptibility for S. pyogenes
 Fermentation of sugar Enterococcus spp
126

Classification of streptococcus
127

Group A streptococcus [GAS] /Streptococcus pyogenes
General characteristics
• Gram positive cocci in chain
• GAS infections affect all ages peak incidence at 5-15 years of
age
• 90% of cases of bacterial pharyngitis
• GAS is β hemolytic production of clear zones of haemolysis
around colonies following overnight incubation on blood agar
• “Flesh eating bacteria”
• S .pyogenes is sensitive to antibiotics like bacitracin and
penicillin 128

Virulence Factor of Group A Streptococcus (GAS)
 Has numerous surface and extracellular factors that confer
virulence
 More than 40 virulence associated genes
 Evolved sophisticated strategies and complex mechanisms
overcome host defenses and successfully colonize host
 The cell surface M-protein is the main antigenic
determinant of GAS
 Used for adherence and evade phagocytosis
 There are different types virulence factors
129

Virulence Factors of GAS
Antiphagocytic Adherence to
epithelia cell
Internaliz
ation
Invasion Spread
through
tissues
Systemic
toxicity
M-protien
M-like protein
M-related
protein(Mrp)
Hyaluronic acid
capsule
C5a peptidase
Lipoteichoic
acid
Fibronectin
binding protein
M-protein
Hyaluronic acid
capsule
M-protein
Protien F1
M-protien
Hyaluronic
acid
capsule
Hyaluronidase
streptokinase
SPeB
DNAses A-D
SLO,SLS
Supra –Ag
exotoxin
130

Virulence Factors of GAS
(structural)
 Capsule – hyaluronic acid antiphagocytic
 Fimbriae – attachment
 Lipoteichoic acid – Binds to epithelial cells
 M-Protein – Adhesin, antiphagocytic property
 M-like protein – Binds to IgM, IgG then it prevents
antibody mediated clearance
 F-protein – Adherence to epithelial cells
131

Virulence Factors of S. pyogenes GAS
(Toxins)
 Streptococcal Pyrogenic exotoxins (A,B.C) – It is responsible for
pyrogenicity (fever)
 Hemolysins – two types of hemolysins - Streptolysin-S and
Streptolysin-O
 Streptolysin-S -- lysis of WBCs, RBC, platlets, responsible for the
β hemolysis on blood agar medium, non-immunogenic
 Streptolysin-O -is oxygen labile protein, lysis of WBC, RBC,
platlets, immunogenic, diagnostic marker of Streptococcal
infection
132

Virulence Factors of S. pyogenes
(Enzymes)
Streptokinase – lyses blood clots, facilitates spread
of bacteria in infected tissues
DNase – designated as A –D, depolymerises host
cell DNA
Hyaluronidase – it facilitates the spread in tissue
NADase –hydrolyses host cell NAD
133

M-protein as a Major Virulence Factor of GAS
 The major virulence factor
for GAS
 N-terminal region is
extremely variable region
and base for serotype
identity(>200 serotypes)
 They can be also calcified
into subtypes
 GAS strains are most
commonly discriminated
by identification of the M
protein (M typing) 134
Cell Membrane

 Serotyping of GAS was historically done by serological analysis of a
purified M protein fragment
 currently it is done emm typing sequence analysis targeting the that
codes antigenic region of M protein
 The two methods, serological and genetic, are extremely well
correlated
 The serotypes are different on geographical area and spectrum of the
disease they cause
 There is dynamicity in serotypes thought time (dynamic
epidemiology)
 M-protein is antiphagocytic, prevent complement activation
135

Clinical Diseases of GAS
1. Suppurative Infection
• Pus forming infection
• Infections may be mild to extremely severe
• pyogenic infections involving :
 mucous membranes
 tonsils
 skin
 deeper tissues, including pharyngitis, impetigo/pyoderma, erysipelas,
cellulitis, necrotizing fasciitis, toxic streptococcal syndrome, scarlet
fever, septicemia, pneumonia, and meningitis
2. Non-suppurative Infection
 immunological sequele( Acute rheumatic fever, acute glomerulo nephritis )
 It is due to molecular mimicry or Ag-Ab complex
137

Suppurative Infection of GAS
A. Pharyngitis/Strep throat
 GAS is the most common bacterial cause of pharyngitis
 usually self-limiting
 Transmitted by air droplets
 primarily affect school-age children 5 to 15 years of age
 susceptibility increased under crowded conditions like schools and
military facilities
 infection is seasonal (February to May in Ethiopia)
 acute sore throat
 Malaise
 fever
 headache
 tonsillar sweeling/exudate
138
No cough
Cervical lymphadenophaty

B. Scarlet fever
 it is due to the production streptococcal pyrogenic exotoxin
(A,B,C)
 ChXd by pharyngitis with rash & characteristic is the
strawberry colored tongue
 A yellowish white coating initially covers tongue and is
later shed revealing red raw surface beneath
139

C. Impetigo
 The primary lesion is a small vesicle surrounded by an area
of erythema
 The vesicle enlarges over a period of days, becomes
pustular, and eventually breaks to form a yellow crust
 Common 2-5-year-old children on exposed body surfaces,
typically the face and lower Extremities
 S.aureus usually isolated from such lesions
140

D. Erysipelas
 Affect skin and subcutaneous tissues primarily the dermis
 chxd by a spreading area of erythema and edema with
rapidly advancing, well-demarcated edges, pain, and
systemic manifestations including fever and
lymphadenopathy
 Infection usually occurs on the face and limbs
141

E. Necrotizing Fasciitis
 severe, rapidly spreading infection destroying subcutaneous and deep
tissues and muscles
 It is invasive streptococcal infection “flesh-eating bacteria”
 infection producing the toxic syndrome may occur in muscle and
fascia
 follows mild trauma with entrance of GAS through the skin
 Due to Pyrogenic exotoxins(A,B,C) and superantigens such as
exotoxin F (mitogenic factor) and streptococcal superantigen (SSA)
 The features
 hypotension
 shock
 multiple organ failure
 systemic toxicity
 severe local pain,
 rapid necrosis of subcutaneous tissues and skin, and gangrene
142

Necrotizing Fasciitis
143

F. Streptococcal toxic shock syndrome and bacteremia
 Similar to staphylococcal toxic shock syndrome
 Due to pyrogenic exotoxins (A,B,C) causing massive, nonspecific T
cell activation
 Release of large amounts of interleukins and inflammatory cytokines
such as tumor necrosis factor and gamma interferon
 initially flulike symptoms, followed shortly by necrotizing soft tissue
infection, shock, adult respiratory distress syndrome, and renal failure
 characterized by hypotension and multiple organ failure
 Becteremia and sepsis can also caused by GAS
144

Non-suppurative Infections
1. Rheumatic fever
 It is a delayed sequel to GAS pharyngitis
 Systemic inflammation involving connective tissues, heart,
joints, central nervous system (CNS)
 The disease manifests as an inflammation of the joints
(arthritis), heart (carditis), central nervous system (chorea),
skin (erythema marginatum), and/or subcutaneous nodules
145

 Autoimmune disease
 Due to autoreactive antibodies and T cells shown to cross-
react with components of GAS and host tissues
 Molecular mimicry of M-protien of GAS with the host
skeletal myosin, tropomyosin, Vimentin, Laminin, and
Keratin
 it leads to RHEUMATIC HEART DISEASE(RHD)
serious cardiac involvement, with myocarditis or valvulitis
leading to death or valve replacement
 Common in school ages
146

2. Acute Poststreptococcal Glomerulonephritis
 primarily childhood disease that follows from skin or
pharyngitis
 Males are twice affected than females
 increased in over crowding, poor hygiene, and poverty
 acute inflammation of renal glomeruli with edema,
hypertension, hematuria, proteinuria
 progressive and irreversible damage to kidneys
147

 The pathogenic mechanism
Circulating immune complex :Ag-Ab complex
deposited in the glomurili capillaries leads to damages
(increased IgG and IgM)
Glomerular tissues damages by GAS products :such as
a proteinase or streptokinase, and direct complement activation
by GAS Ags deposited in the glomeruli
Molecular mimicry: Molecular mimicry between GAS
and renal antigens
Autoantibodies damage the glomerular capillaries
148

Laboratory Diagnosis GAS
 Skin infections – pus
 Pharyngitis -- throat swab
 Serum
Gram staining
Gram positive cocci in chain
 Cultured on blood agar medium incubated in anaerobic
condition
 β hemolytic
 sensitivity to 0.04U bacitracin
 penicillin sensitive
 negative catalase test
 PYR-test (L-pyrrolidonyl arylamidase test)
149

 Serological test
Lancefield Grouping
Anti-Streptolysin O(ASO), Anti-DNase B
 Rapid streptococcal test
 PCR
 M-Protien typing for Stereotyping
150

Treatment
• Penicillin is the drug of choice
• If penicillin is allergic to some patients, erythromycin /
cephalosporins can be given
Prevention
 Penicillin prophylaxis to prevent recurrences of ARF during
the most susceptible ages (5 to 15 years)
 Vaccines using epitopes of the M protein molecule( 26
valent and 30-Valent is under development in Europe and
north America
151

Streptococcus agalactiae /Group B
Streptococcus) GBS
 Gram positive cocci in chains, capsulated, Produce β-
hemolysis
 less distinct β-hemolysis than with GAS and may even be
absent
 Normal vaginal flora
 Catalase negative
 Possess group specific carbohydrate (B- antigen) in cell wall
 11 serotypes are recognized
152

Virulence Factors
 Thick peptidoglycan layer – permits survival on dry
surfaces
 Capsule – Antiphagocytic in nature
 Hydrolytic enzymes – tissue destruction and systemic
spread of bacteria
153

CLINICAL DISEASE OF DISEASES
1. Two forms of neonatal disease
 Early onset neonatal disease is acquired in utero or at birth develop during
first week of life. chxed by bacteremia, pneumonia, meningitis
 Late onset neonatal disease is acquired from an exogenous source (from
mother, another infant). It may lead to bacteremia with meningitis
2. Infections in pregnant women: Urinary tract infections may lead to
secondary complications like endocarditis, meningitis, osteomyelitis
3. wound infections, endometritis
154

Lab Diagnosis of GBS
 blood culture, CSF culture
 Lancefield grouping
 hydrolyze sodium hippurate
 bacitracin-resistant, trimethoprim-sulfomethoxazole-
resistant
 CAMP test (Christie, Atkins, Munch-Peterson) –positive
based on synergistic effect of hemolysin of s.aureus and heat
stale CAMP factor produced by GBS
155
A. GBS
B. GAS
C. S.aureus

Treatment
 Penicillin G
 Combination of penicillin (ampicillin) for neonatal
infections and an aminoglycosides
Prevention
 Intrapartum prophylaxis of penicillin protective is for
neonates
 If third trimester vaginal culture positive give prophylaxis
 Vaccine is under development from capsule
156

Viridans Streptococci
 Heterogeneous collection of α and γ hemolytic streptococci
 Under Lancefield grouping, it is non groupable.
 S mutans, S salivarius, S mitis,S bovis etc.
 Formation of dental caries and other oral cavity (gingival /
gum) infections and bacterial endocarditis
 Treatment: combination of penicillin with aminoglycoside
157

Streptococcus pneumoniae (Pneumococcus)
 Encapsulated, Gram positive coccus, Lancet shaped, arranged in pairs
 Virulent strains are covered with polysaccharide capsule
 Fastidious in requirement.
 All colonies undergo autolysis due to amidase present in cell wall
 α hemolytic
 Colonies are bile soluble and optochin sensitive
 Based on capsular polysaccharide antigen this bacterium is serologically classified
into 90 serotypes
158

Virulence Factors
 Capsule : major determinant of virulence both antiphagosctic
and antigenic
 Secretory IgA protease – disrupts secretory IgA-mediated
clearance
 Pneumolysin: binds to cholesterol in host cell membrane and
creates pores. Destroys ciliated epithelial cells as well as
phagocytic cells
 Tissue destruction: Teichoic acid , Peptidoglycan, pneumolysin,
hydrogen peroxide
 Phagocytic survival: Capsule, pneumolysin 159

Clinical diseases
 Pneumonia – One of the most common causes of
community acquired disease
 Meningitis – Most common causes of bacterial
meningitis in most age groups
 Common cause of sinusitis, otitis media
 Can cause a variety of systemic infections like
bacteremia and endocarditis
160

Lab Diagnosis of S.pneumoniae
• specimen: Sputum,blood
• Microscopy : gram positive diplococcic
• Culture blood agar alpha hemolytic
• bile solubility test positive
• optochin sensitivity test
• capsule swelling test
161

Treatment
163
• Penicillin are drug of choice
• Penicillin resistant strains may be treated with
erythromycin, vancomycin,
• third-generation cephalosporins (meningitis)
Prevention
 23-valent polysaccharide vaccine is available

Enterococcus
• Enterococci (“enteric cocci”) are group D, -haemolytic
• Formerly group D Streptococcus, now separate genus.
• Enterococcus faecalis and Enterococcus faecium
• E. faecalis (formerly classified Streptococcus. faecalis) is the main
pathogen in the genus Enterococcus, causing about 95% of
enterococcal infections
• A minority of infections are caused by E. faecium.
• G+ cocci arranged in pairs and short chains
• Can tolerate exposure to harsh environmental conditions
• E.g., grow in the presence of 6.5% NaCl and 40% bile salts
• Enterococci are
• Inherently resistant to many commonly used antibiotics, e.g.
oxacillin, cephalosporins or have
• Acquired resistance genes, e.g. to aminoglycosides, vancomycin
(VRE)

Enterococcus…
• It is a normal commensal of the vagina and
GIT.
• Most infections from patient's bacterial flora
• Some caused by patient-to-patient spread
• Diseases:
– Urinary tract: urinary tract infection (UTI)
– Cardiovascular: infective endocarditis
– Skin: infection of ulcers (e.g. bed sores), wound
infections and intravenous (IV) line infection

Lab Diagnosis
• Morphology
– Enteroacoccus species are G+ cocci, occurring in pairs
or short chains.
– They are non-capsulate and the majority are non-
motile.
• Culture
– Enterococci are aerobic organisms capable of growing
over a wide temperature range, 10–45 ºC.
– Blood agar:
– Enterococci are mainly non-haemolytic but some
strains show alpha or beta-haemolysis.

Lab Diagnosis…
• MacConkey and CLED agar:
– E. faecalis ferments lactose, producing small
dark-red magenta (light purple) colonies on
MacConkey agar and small yellow colonies on CLED
agar.
– Enterococcus species are also able to grow in the
presence of 6.5% sodium chloride and 40% bile.
– When grown on media containing aesculin,
enterococci hydrolyze the aesculin, producing
black colonies.
– Growth on bile esculin agar, Black precipitate

Lab Diagnosis…
• Biochemical tests
• Enterococcus species:
– Ferment lactose (also mannitol and other sugars).
– Hydrolyze aesculin
– Reduce litmus milk
– Like streptococci, enterococci are catalase
negative.

Treatment, prevention, and control
• Most enterococci are susceptible to ampicillin
and resistant to cephalosporins.
– Resistance is shown against penicillin.
– Vancomycin and ampicillin resistance appear to be
emerging, associated particularly with hospital
infections.
– Antibiotic resistance is becoming increasingly
common, and infections with many isolates
(particularly E. faecium) are not treatable with any
antibiotics
• Newer agents include linezolid, quinupristin/
dalfopristin, and selected fluoroquinolones

Reading assignment
 Micrococci
 Enterococci

 Gram-negative cocci
 Diplococcic (bean shaped)
 None motile, Intracellular
 Distinguished from many other Gram-negative pathogens
by being oxidase positive
 aerobic bacteria with carbon dioxide and enriched
medium for optimal growth
 2 species are pathogenic to humans
 The meningococcus, N. meningitidis
 The gonococcus, N. gonorrhoeae (more fastidus)

Morphology and Identification
 Both meningococci and gonococci are encapsulated
 Grow best under aerobic conditions but if alternative electron
acceptors (e.g., nitrites) are available they also can grow
anaerobically
 For growth both prefer medium containing complex organic
substances (e.g. blood, hemin, and animal proteins) and humid
atmosphere containing 5% CO2
 Meningococci and gonococci are rapidly killed by drying,
sunlight, moist heat and many disinfectants
 Poor survival at cooler temperature

 The natural habitat of Neisseria spp. is the mucous
membranes of the respiratory and urogenital tracts.
 N. gonorrhoeae is not considered to be part of the normal
biota and is always pathogenic.
 N. meningitidis may be found as a commensal inhabitant
of the upper respiratory tract of carriers, but it can also
become an invasive pathogen.
 Both N. gonorrhoeae and N. Meningitidis require iron for
growth.
 They compete with their human host by binding human
transferrin to specific surface receptor

Virulence factor and Pathogenicity
 Pathogenic Neisseria spp. have several characteristics that
contribute to their virulence, including the following:
Receptors for human transferrin
Capsule (N. meningitidis)
Pili (fimbriae)
Cell membrane proteins
Lipooligosaccharide (LOS) or endotoxin.
Immunoglobulin A (IgA) protease

Neisseria meningitidis
 Humans are the only natural carrier of N. meningitidis
 Can be a member of the normal microbiota of the upper respiratory
tract
 Causes life-threatening disease when the bacteria invade the blood or
cerebrospinal fluid
 Most common cause of meningitis in individuals under 20 years age
 Respiratory droplets transmit the bacteria among people living in
close contact, especially students living in dormitories, soldiers,
prisoners & household contacts

Neisseria meningitidis …..cont’d
 More than 13 known antigenic types based on
polysaccharide capsule
 Types A, B, C, X, Y & W135 are more commonly
associated with human disease
 Polysaccharide capsules resist lytic enzymes inside
phagocytes and antibody mediated immunity
 A, C and W-135 are commonly encountered
 Serogroup B polysaccharide capsule is a weak / non
immunogenic
 Ethiopia comes under African Meningitis Belt that
includes many African countries starting from Senegal

N.meningitidis
Areas with frequent
epidemics of
meningococcal disease.
This is known as the
Meningitis Belt of Africa,
and visitors to these
locales may benefit from
meningitis vaccine.

Meningococcal infections
•Meningococcal meningitis
•Meningococcal bacteremia
•Meningococcemia
•Respiratory tract infection
•Pneumonia
•Epiglottitis
•Chronic meningococcemia
•Purulent pericarditis
•Otitis media
•Focal infection
•Conjunctivitis
•Septic arthritis
•Urethritis

 Infection with N. meningitidis has two presentations:
1. Meningococcemia:- characterized by skin lesions, and
acute bacterial meningitis
 Infection is by inhalation of the bacteria, which attach to
epithelial cells of the nasopharyngeal and oropharyngeal
mucosa, cross the mucosal barrier, and enter the
bloodstream
Clinical diseases

 It leads to bacteremia
 Show symptoms of respiratory tract infection
 May lead to severe septicemia / meningococcemia with high
fever, hemorrhagic rash, circulatory collapse (Waterhouse-
Friderichsen syndrome), disseminated intravascular coagulation
DIC, thrombosis, myocarditis, arthritis the formation of minute
hemorrhagic lesions
Petechial skin rash
Hemorrhage (dark red areas) in the
adrenal glands in Waterhouse-Friderichsen

2. Meningitis
 Entry of meningococcus through nasal cavity – Enters
nasopharynx – then enters blood stream – it leads to
bacteremia
 Show symptoms of respiratory tract infection –
meningitis with sudden headache, vomiting, stiff neck,
photophobia, leading to coma within few hours
 Cases of meningococcemia with meningitis are also
present

Laboratory diagnosis
 Specimen: Cerebrospinal fluid, blood
 Smear: Gram-negative diplococcic (bean shaped)
 Oxidase positive
 Culture: Transparent or gray, shiny, mucoid colonies in chocolate agar
after incubation at 35-37Oc in a CO2 enriched atmosphere.
 intracellular diplococci
 Serology: Antibodies to meningococcal polysaccharides can be
measured using: latex agglutination or Haemagglutination tests

Species Glucose Lactose Maltose Sucrose
Neisseria gonorrhoeae Positive Negative Negative Negative
Neisseria meningitidis Positive Negative Positive Negative
Biochemical reactions
 Drug of choice: Penicillin, Chloramphenicol, Cefotaxime
ceftriaxone, ciprofloxacin, Rifampin for chemoprophylaxis
 Vaccination is available for serogroups A, C, Y and W135
 No effective vaccine for sero group B as it is poorly
immunogenic in humans is present

185
N. gonorrhoeae N. meningitidis

 Causes gonorrhea, a sexually transmitted disease
 Gonococci adhere to epithelial cells of the mucous membranes lining
the genital, urinary, and digestive tracts of humans spreading to
deeper tissue as they multiply
 As few as 100 pairs of cells are enough to cause disease
Neisseria gonorrhoeae

Clinical infections
 Lower tract infections
 Cervicitis
 Urethritis (male and female)
 Abscess formation in glands adjacent to the vagina, such as Skene
duct or Bartholin glands
 Upper tract infections
 Endometritis (uterine infection)
 Epididymitis
 Pelvic inflammatory disease (PID; infection of the fallopian tube
[salpingitis], the ovary, or adnexal tissues)

….cont’d
 Other (non-reproductive tract) localized sites
 Proctitis (rectal gonorrhea)
 Pharyngitis
 Ophthalmia neonatorum (bilateral conjunctivitis in infants born of
mothers infected with gonococci)
 Extension of the infection to areas contiguous with the pelvis
causing peritonitis or perihepatitis (Fitz-Hugh-Curtis syndrome)
 Disseminated gonococcal infection (DGI)
 Dermatitis–arthritis–tenosynovitis syndrome: fever, polyarthritis, and
tenosynodermatitis (vesicles or pustules on a hemorrhagic base) caused
either by immune complexes or by whole gonococci
 Rarely, endocarditis (infection involves heart valves) or meningitis
(infection of the central nervous system)

Common clinical diseases
 Gonorrhea in men
 Usually symptomatic producing inflammation that causes
painful urination and purulent discharge
 cause scarring and infertility if prostate and epididymis
are involved

…..cont’d
 Gonorrhea in women
 Often asymptomatic (50%)
 Can infect the cervix and other parts of the uterus, including the
Fallopian tubes
 Can result in pelvic inflammatory disease (PID)
 Can result in ectopic pregnancy or sterility
 Gonococcal infection of children can occur during childbirth
producing inflammation of the cornea (ophthalmmia neonatorum)
and sometimes blindness
 Infection of the respiratory tracts can also occur

Ophthalmmia
neonatorum
Discharges

Specimen: Urethral swab, cervical swab, eye swab.
Smear: Gram-negative intracellular and/or extra diplococci
Figure . ….. Left: N. gonorrhoeae Gram stain of pure culture
Right: N. gonorrhoeae Gram stain of a pustular exudates

Culture: Requires an enriched media like chocolate agar,
Modified Thayer-Martin agar and/or Modified New York
City medium
 Grows best in carbon dioxide enriched aerobic
atmosphere with optimal temperature of 35-370c

 On culture, Neisseriae species form convex, elevated and
mucoid colonies
 On Thayer - Martin and Chocolate agar media, colonies are
transparent or opaque and non - pigmented
Growth characteristics
 Most Neisseria grow best under aerobic conditions but some
grow under anaerobic situations
 Ferment carbohydrate and produce acid without gas

 Serology: Antibodies to gonococcal pili & outer
membrane proteins using RIA and/or ELISA.
 Genetic probes : For detection of nucleic acids

Treatment
 Drug of choice: Ceftriaxone, doxycycline, ciprofloxacin, or oflaxacin.
Penicillin resistance due to beta- lactamase enzyme producing N.
gonorrhoeae have been identified
 For ophthalmia neonatorum - 1% silver nitrate , 1% tetracycline or
0.5% erythromycin eye ointments
Prevention and control
 Early detection and treatment of cases (no effective vaccine)
 Using condom
 Health education

Moraxella catarrhalis
 Formerly called Neisseria catarrhalis or Micrococcus
catarrhalis
 Gram-negative, aerobic diplococcus
 a commensal of the upper respiratory tract
 belonging to the so-called non-gonococcal, non-
meningococcal neisseriae
 carriage rate in children is high (up to 75%) but1–3% in
adults because the age-dependent development of
secretory immunoglobulin A (IgA)
 upper respiratory tract infection healthy children and
elderly people

Pathogenesis virulence
 binding to and colonization of mucous surfaces;
 entry into host tissues;
 multiplication in the invivo environment;
 interference with host defense mechanisms
 damaging the host
Virulence
 Endotoxin
 Capsule
 Pili

Clinical disease
 important pathogen in respiratory tract infections, both in
children and in adults with underlying chronic
obstructive pulmonary disease
 occasionally causing systemic disease
 Cause respiratory infection in healthy children and
immunocompromised adults

1. Sinusitis: common in children with allergy
2. Otitis media: most serious and frequent
infection caused by M. catarrhalis in children
3. Lower Respiratory Tract Infections: mostly
occur in children under the age of 1 year
4. tracheitis and eye infection
Infection in children

Infection of adults
1. Laryngitis
2. Bronchitis and pneumonia
3. Nasocomical infection

 Direct smear from an otitis media sample showing
intracellular gram-negative diplococci
 Colonies appear smooth with a grayish- white color
 When colonies pushed with loop, they “scoot” across
media
 oxidase production DNase production; failure to produce
acid from glucose, maltose, sucrose,

Treatment
 β-lactamase-mediated resistance to penicillins and
inherent resistance to trimethoprim
 M. catarrhalis remains sensitive to most other
antibiotics used for treating respiratory infections
 No vaccine

Genus Mycobacterium

Learning Objective
 Describe the virulence mechanisms used by pathogenic microbes
of each genus
 Describe the diseases caused by the genus Mycobacterium
 Explain how we diagnose individuals infected with Mycobacterium
 List the different treatment alternatives
 List the individuals that are at risk for infection

Genus Mycobacterium
 Classification of Mycobacterium
 M. tuberculosis complex
 Mycobacterium tuberculosis
 Mycobacterium bovis
 Mycobacterium africanum
 M. avium complex
 Mycobacterium avium
 Mycobacterium ulcerans
 M. Leprae

Genus Mycobacterium
General characteristics:
 Non motile rod-shaped, obligate aerobe and acid-fast bacteria
 Over 60% of the mycobacterial cell wall is a complex lipid rich:
 Impermeability to stains and dyes
 Resistance to many antibiotics
 Resistance to killing by acidic and alkaline compounds
 Killed by pasteurization (heating to 60°C for 30 minutes)
 Resistance to osmotic lysis via complement deposition
 Resistance to lethal oxidations and survival inside of macrophages
 Over 100 species, but few cause most human infection
 M. tuberculosis, M. bovis, M. leprae, M. avium complex, M. kansasii, M. fortuitum, M.
chelonae, and M. abscessus

 Mycobacterium tuberculosis:
 causative agent of tuberculosis in humans.
 Humans are the only reservoir for the bacterium.
 Mycobacterium bovis:
 cause TB in cows and rarely in humans.
 Humans infected by consumption of unpasteurized milk.
 can lead to the development of extrapulmonary TB
 Mycobacterium avium: causes a TB-like disease prevalent in
AIDS patients
 Mycobacterium ulcerans: skin diseases
 Mycobacterium leprae: cause leprosy

Mycobacterium tuberculosis
Structure:
 Can not be stained by simple stains due to their high lipid content
including mycolic acids
 Stained by Ziehl-Neelsen stain
 Acid fast: Once stained, resist decolourization with 3% acid
(H2SO4) & alcohol
 Appear as thin pink rods arranged singly or in small groups
 Slow proliferation (culturing 3–8 weeks)
 Facultative intracellular
 Obligate aerobe

Virulence factors:
 M. tuberculosis enters the respiratory airways and minute infectious
particles penetrate to the alveoli
 Bind to mannose receptors on macrophages and engulfed
 Can stay for life with out being killed
 Infection with M. tuberculosis confers on the patients two conditions
(resistance and hypersensitivity)
 Rather than specific virulence factors of the microbe
 Mechanisms of existence:
 Prevents fusion of the phagosome with lysosomes
 Evade killing by catalytically catabolizing oxidants
 Blocks acidification of phagosome
 Cord factor. It is known to be toxic to mammalian cells and to be an
inhibitor of PMN migration

Pathogenesis
 The mode of transmission is by direct inhalation of aerosolized
bacilli contained in droplet
 Air in a room occupied by a person with pulmonary TB may
remain infectious even after the person has left the room
 Infection also occurs infrequently by ingestion of infected milk.
 The initial infection with M. tuberculosis is referred to as a
primary infection.
 Subsequent disease in a previously sensitized person, either
from an exogenous source or by reactivation of a primary
infection, is known as secondary or reinfection tuberculosis
 Which have different pathological features.

……….. cont’d
 The site of the initial infection is usually the lung, following the
inhalation of bacilli.
 These bacilli engulfed by alveolar macrophages multiply and give
rise to a subpleural focus of tuberculous pneumonia, to form the
initial lesion or Ghon focus.
 Some bacilli are carried in phagocytic cells to the hilar lymph nodes
where additional foci of infection develop
 The Ghon focus, together with the enlarged hilar lymph nodes, form
the primary complex
 This is associated with the development of tuberculin
hypersensitivity

………….cont’d
 After infection, M. tuberculosis cells are phagocytized by alveolar
macrophages and are capable of intracellular multiplication.
 The pathologic features of TB are the result of a hypersensitivity
reaction to mycobacterial antigen.
 If there is a little antigen and a great deal of hypersensitivity
reaction, a hard tubercle or granuloma may be formed
 Granuloma, consists of three zones
 A central area of large, multinucleated giant cells containing
tubercle bacilli.
 A mid zone of pale epithelioid cells, often arranged radially.
 A peripheral zone of fibroblasts, lymphocytes, and monocytes.

Ggranulomas: They have rounded outlines. The center contains several Langhans giant
cells. Granulomas are composed of transformed macrophages called epithelioid cells
along with lymphocytes, plasma cells, and fibroblasts. The localized, small appearance of
these granulomas suggests that the immune response is fairly good
Collar of lymphocytes,
plasma cells
Central caseated necrosis
Giant multinucleated cells
(langhans type
Agrregation of epithliod
cells

……..cont’d
 The granuloma is an organization of lymphocytes, macrophages,
giant cells, fibroblasts, and capillaries
 Later, peripheral fibrous tissue develops, and the central area
under goes caseation necrosis. Such a lesion is called a tubercle.
Secondary Tuberculosis
 Secondary or adult type of tuberculosis is due to reactivation of
latent infection or exogenous reinfection
 Progression from infection to active disease varies with age and
the intensity and duration of exposure.
 Reactivation TB occurs when there is an alteration or a
suppression of the cellular immune system
 This favors replication of the bacilli and progression to disease.

Clinical diseases
 Pulmonary tuberculosis (PTB)
 Tuberculosis may mimic other diseases, such as pneumonia, neoplasm, or
fungal infections.
 Primary tuberculosis typically is considered a disease of the respiratory
tract.
 Common presenting symptoms include productive cough, low-grade
fever, night sweats, fatigue, anorexia (loss of appetite), and weight loss
 Besides the lungs M. tuberculosis complex can affect other
organs called extrapulmonary tuberculosis (EPTB)
 Genitourinary tract
 Lymph nodes (cervical lymphadenitis)
 Central nervous system (meningitis)
 Bone and joint (arthritis and osteomyelitis)…

Mycobacterium tuberculosis multiplication in alveolar macrophages and
spread to lymph nodes and beyond

Outcomes of untreated
primary TB.

…..cont’d
 Coinfection with HIV
o Accelerates the progression of both diseases
o Requiring rapid diagnosis and treatment of both diseases
 Tuberculosis can produce atypical signs and symptoms in
infants, the elderly, and immunocompromised hosts and it can
progress rapidly in these patients

Epidemiology
 Worldwide; one third (1.7 billion) of the world's population is infected
 A total of 8.8 million new cases each year and 2 million deaths
 Disease most common in Southeast Asia, Sub-Saharan Africa, and
Eastern Europe
 Populations at greatest risk for disease are
 immunocompromised patients (children, elderly, therapy, chronic infections)
 Drug or alcohol abusers
 Homeless persons, and
 Individuals exposed to diseased patients
 Humans are the only natural reservoir
 Person-to-person spread by infectious aerosols

Laboratory Diagnosis
 The definitive diagnosis of tuberculosis is based on:
 Microscopy (Bright field microscopy, Fluorescent microscopy)
 Culture (LJ, BACTEC-MGIT)
 Experimental animals
 Demonstration of hypersensitivity to tuberculoprotein
 Tuberculin skin test, QuantiFERON-TB and interferon-γ release
tests are sensitive markers for exposure to organism
 Molecular diagnostic methods. (GeneXpert)
 X-ray
 Cytopathology/Histopathology
 EPTB

Microscopy
 Microscopy is considered sensitive and rapid procedure for
identification of Mycobacterium spp. in clinical specimens.
Acid-Fast (Ziehl-Neelsen) Stains
 The cell walls of mycobacteria contain high amount of mycolic acid
 Which contributes to the characteristic of acid-fastness that
distinguishes mycobacteria from other bacteria.
 Other species of Nocardia and Rhodococcus are also partially acid-fast

Fluorochrome Stain
 Fluorochrome staining is the screening procedure recommended for laboratories
that have a fluorescent (ultraviolet) microscope
 Fluorochrome stain is more sensitive than the conventional acid fast stains,
because the fluorescent bacilli stand out brightly against the background
 Procedures
1. Heat-fix slides at 80°C for at least 15 minutes or for 2 hrs at 65-70°C.
2. Add auramine-rhodamine reagent and allow to stain for 15 to 20 minutes at room
temperature.
3. Rinse with deionized water and tilt slide to drain.
4. Decolorizewith 0.5% acid-alcohol for 2-3 minutes.
5. Rinse with deionized water and tilt slide to drain.
6. Add 0.5% potassium permanganate for 2-4 minutes.
7. Rinse with deionized water and air dry.
8. Examine under low power (250×) for fluorescence.

Reporting

Culture
 It is a very sensitive diagnostic technique for tubercle bacilli,
detecting as few as 10 to 100 bacilli per ml
 The concentrated material is inoculated onto at least two bottles
of Löwenstein-Jensen (LJ) medium and incubate at 35 to 37°C.
 Growth of most strains of M. tuberculosis may appear in 2 to 8
weeks.

Tuberculin (Mantoux) Test
 The principle of this test is delayed hypersensitivity reaction.
 Purified protein derivative (PPD) is the skin test reagent that is
primarily used to detect hypersensitivity in these persons
 Uses of Tuberculin Test
1. To diagnose active infection in infants and young children.
2. To measure prevalence of infection in an area.
3. To select susceptible for BCG vaccination.
4. Indication of successful BCG vaccination.
5. Considered positive if area >=5mm
 Tuberculin testing of cattle has been of great value in the control of
bovine tuberculosis

……cont’d
 A positive tuberculin test result signifies cell-mediated
hypersensitivity to tubercular antigens. It does not
differentiate between infection and disease
 False-negative reactions may be produced by certain viral
infections, sarcoidosis, malnutrition, immunosuppression
 False-positive reactions may also result from infection by
atypical mycobacteria

Treatment
 The principles of antituberculous chemotherapy involve earlier,
combination, appropriate, regularly and durations.
 The critical issue in TB control is adopting the DOTS
(Directly Observed Treatment)
 Short-course therapy;
 Medicines used to treat tuberculosis are classified as
 First-line medicines include
 Isoniazid
 Rifampicin
 Pyraziniamide
 Streptomycine
 Ethambutol
 Second-line medicines
 Fluoroquinolone
 para-amino-salicylic acid
 kanamycin
 Amikacin
 Capreomycin, etc...

Prevention and Control
 Prophylaxis for exposure to tuberculosis (latent TB)
 The best way to prevent tuberculosis is
 to diagnose infectious cases rapidly and
 administer appropriate treatment until cure
 BCG (bacille Calmette-Guerin) vaccination-an attenuated strain
of M. bovis first administered to humans in 1921 for new
infants
 Control of disease through active surveillance, prophylactic and
therapeutic intervention, and careful case monitoring

Drug Resistance and Tuberculosis
 M. tuberculosis: naturally resistant to
certain antibiotics due to presence of:
 Drug-modifying enzymes
 Drug-efflux systems
 Hydrophobic cell wall
 Development of increasingly resistant
strains mainly due to:
 Patient non-compliance
 Monotherapy (single drug treatment)
 Decreased treatment period

MDR and XDR Tuberculosis
MDR: Multidrug-resistant strains:
 Strains of tuberculosis resistant at least to rifampicin and
isoniazid.
 Mortality rate: 40-60%
 Estimated that 50 million people are infected with MDR-TB.
 MDR-TB is approximately 125 times more expensive to treat than
drug susceptible TB.
XDR: Extensively-drug resistant strains:
 Strains of tuberculosis resistant to rifampicin, isoniazid, in
addition to any Fluoroquinolone, and to at least one of the three
following injectable drugs used in anti-TB treatment:
Capreomycin, kanamycin and Amikacin.

 Read Mono-resistant, Poly-resistant, and TDR
What is it?

Mycobacterium leprae
Acid fast bacilli
Strict human pathogens
Cannot be cultivated in-vitro
Transmission - ? Air borne
- prolonged contact required
Low infectivity

Mycobacterium leprae
 Structure:
 Obligate intracellular bacillus
 Produces no known toxins and is well adapted to penetrate and
reside within macrophages
 Not been cultivated on artificial medium or tissue culture
 M. leprae grows best in cooler tissues
 The skin, peripheral nerves, anterior chamber of the eye, upper
respiratory tract, and testes
 Scant in warmer areas of the skin (the axilla, groin, scalp, and
midline of the back)
 Virulence factors:

Hansen’s disease (Leprosy) caused by M. leprae
 Hansen’s disease is a chronic, slowly progressive
 granulomatous disease involving ectodermally derived
 The disease is usually limited to the cooler parts of the body such
as the skin, nose and upper respiratory tract.
 It rarely affects internal organs such as the brain, liver, spleen,
kidneys, and bones.
 It has a specific predilection for peripheral nerves.
 Generally, the disease may be classified into two types; these
are:
1. Lepromatous.
2. Tuberculoid.

Tuberculoid Leprosy Lepromatous Leprosy
Skin lesions: Few erythematous or
hypopigmented plaques with flat centers
and raised, demarcated borders
 peripheral nerve damage with
complete sensory loss
 visible enlargement of nerves
Skin lesions: Many erythematous
macules, papules, or nodules
 Extensive tissue
 Diffuse nerve involvement with
patchy sensory loss
 Lack of nerve enlargement
Infectivity: Low Infectivity: High
Immune response (IR): Reactivity to
lepromin
IR: Nonreactivity to lepromin
Immunoglobulin: Normal Immunoglobulin: Hyper
Erythema nodosum: Absent Erythema nodosum: Usually present

Lepromatous form
Tuberculoid form

Epidemiology
 Fewer than 300,000 new cases were reported in 2005, with
most cases in India, Nepal, and Brazil
 Approximately 100 new cases reported in United States
annually
 Lepromatous form of disease, but not the tuberculoid form, is
highly infectious

Transmission
 Route of transmission of leprosy remains uncertain and may be
multiple
 Nasal droplet infection: the nose is a major portal of exit
 Contact with infected soil, fomites like clothes
 Direct dermal inoculation (e.g., during tattooing) may transmit M.
leprae
 Prolonged contact with infected patients
 Leprosy has a long incubation period, an average of about three to
five years
 Person-to-person spread by direct contact or inhalation of
infectious aerosols

Diagnosis
 Microscopy is sensitive for the lepromatous form but not the
tuberculoid form
 Skin testing is required to confirm tuberculoid leprosy
 Culture is not useful
 Experimental animal (Armadillo, Mouse, Chimpanzees, Monkeys..)
Treatment, Prevention, and Control
 Tuberculoid form is treated with rifampicin and dapsone for 6
months;
 clofazimine is added to this regimen for treatment of the
lepromatous form, and therapy is extended to a minimum of 12
months

Armadillo

243
FAMILY ENTEROBACTERIACEAE

244
Learning objectives
At the end of this chapter, the student will be able to:
1.List the coliform bacteria
2.List medically important lactose fermenting and non-lactose
fermenting Enterobacteriaceae.
3.Discuss the E. coli strains associated with diarrhoeal diseases.
4.Differentiate Salmonella from Shigella species.
5.Discuss the general characteristics of the genus Yersinia.

245
 Enterobacteriaceae are Gram negative rods, found primarily in
Intestine tract of humans and animals.
 Many are part of the normal flora.
 All are facultative anaerobes.
 They have simple nutritional requirements and ferment glucose.
 They are catalase positive, oxidase negative and reduce nitrates to
nitrites.

246
Antigens
Possess a wide variety of antigens which are used in serotyping,
particularly salmonellae, shigellae, and E.coli
Cross-reactions however, can occur due to a sharing of antigens
1. O antigens: found in the bacterial cell wall and are heat stable
2. K antigens: capsular polysaccharide antigens, surround the cell wall.
3. H antigens: flagellar protein antigens possessed by motile
Enterobacteriaceae. They are heat labile (destroyed at 60-100 ºC).

Virulence factors (common and species specific)
248
1. Endotoxin
 Released during cell lysis (is lipid A).
 Responsible for systemic manifestations
- Activation of complement system
- Release of cytokines
- leukocytosis
- fever, DIC
- decreased blood circulation shock  death
2. Capsule
 Interferes binding of antibody to the bacteria
 poor immunogen/activator of complement system

3.Type III section system
 Tube like structure by which virulence factors are delivered/injected
to the host (Salmonella, Yersinia, Shigella, E.coli, Pseudomonas)
4. Sequestration of Growth factors
 Iron is important for growth of bacteria and for host cell
 Bacteria compute iron with the host and have iron chelating or iron
binding proteins like enterobactin, aerobactin etc.,
5.Resistant for serum killing
 Systemic infection producing bacteria have capsule, long O
polysaccharide etc.
6. Antimicrobial resistance
 producing enzymes, efflux mechanisms, protective mechanisms to
counteract drugs actions.
 Some possess plasmids that may mediate resistance to antibiotics
249

Members of Enterobacteriaceae
Lactose fermenting Non-lactose fermenting
 Escherichia 1
 Enterobacter1,2
 Klebsiella1
 Citrobacter1,2
 Salmonella
 Providencia2
 Shigella
 Serratia2
 Proteus
 Yersinia
 Morganella 2
 Edwardsiella
 Hafnia2
250
1Often referred to as coliforms.
2 Less common human pathogen, often opportunistic or hospital-acquired.

251
Genus Escherichia
 Gram negative bacilli consisting six species
• E. adecarboxylata
• E. ballatae
• E. fergusonii
• E. hermanii
• E. vulneris
• Escherichia coli is the most common and clinically important.
 It is the most abundantly found bacteria in colon and faeces.
 Found in soil, water and vegetation.
 Most are motile; some are capsulated.

252
Virulence factor
1. Antiphagocytic surface properties: capsules, K antigens and LPS.
2. Adhesins: fimbriae / pili, Intimin (non-fimbrial adhesin)
3. Invasins: hemolysisn, siderophores and siderophore uptake systems
and Shigella-like "invasins" for intracellular invasion and spread.
4. Toxins: Heat labile(LT) toxin, heat stable (ST) toxin, Shiga -like
toxin, cytotoxin and endotoxin (LPS).

253
Pathogenesis and clinical manifestations
Non Diarrhoeal disease:
E. coli causes:
 Urinary tract infections. E. coli is the commonest pathogen
isolated from patients with cystitis. Recurring infections are
common in women.
limited to bladder (cystitis) or can spread to kidneys
(pyelonephritis) or prostate (prostatitis).
 Infections of wounds, peritonitis, sepsis and endotoxin
induced shock.
 Meningitis and bacteraemia in neonates. E. coli capsular
type K1 is associated with neonatal meningitis.
 Diarrhea: At least six different pathogenic groups cause
gastroenteritis (ETEC,EPEC, EIEC, EHEC, EAEC)

254
Diarrhoeal disease: infantile gastroenteritis, traveller’s diarrhoea,
dysentery, and haemorrhagic diarrhoea which may progress to
haemolytic uremic syndrome.
E. coli strains associated with diarrhoeal disease
1. Enterotoxigenic E. coli (ETEC): Causes watery (secretory)
diarrhoea due to the production of plasmid mediated toxins (LT,
ST) in infants and adults.
ETEC are the most important cause of traveler’s diarrhea in visitors
to developing countries
Pathogenic serogroups includes O6, O8, O15, O25, O27, O63,
O119, O125 - O128 and O142.

255
 Transmission is by consumption of food and water contaminated
by infected human or convalescent carriers.
 Uncooked foods such as salads or marinated meats and vegetables
are associated with the greatest risk.
 Direct person-to-person transmission is unusual, because the
infecting dose is high.

256
2. Enteropathogenic E. coli (EPEC): Causes vomiting, fever, and
prolonged diarrhoea mainly in infants (less than 2 year).
Nursery outbreaks and endemic diarrheas occur in developing world
Due to bacteria adhering to epithelial cells, multiplying and causing
lesions.
Pathogenic serogroups includes O26, O55, O86, O111, O114, O125-
O128 and O142.

257
3. Entero-invasive E. Coli (EIEC): Causes dysentery (similar to
shigellosis), fever and colitis, with blood, mucus, and many pus
cells in faecal specimens.
Due to bacteria invading and multiplying in epithelial cells.
Produces shiga like toxin which is responsible for dysentery by
 destructing cells and
 inhibiting protein synthesis by ribosomal modification
Pathogenic serogroups includes O78,O115, O148,O153, O159 and
O167.

258
4. Enterohemorrhagic E. coli (EHEC): Causes life-threatening
haemorrhagic diarrhoea (colitis) in all ages, without pus cells, and
often without fever.
 It can progress to haemolytic uremic syndrome with renal failure.
EHEC is due to cytotoxin damaging vascular endothelial cells, and is
mainly associated with the serogroup O157:H7.
It is sometimes referred to as VTEC (verocytotoxin-producing E. coli,
because it is toxic to vero monkey cells in culture).
Infection occurs by ingesting contaminated meat products,
unpasteurized milk and dairy products.

259
5. Enteroaggregative E. coli ( Eagg EC)
 Causes chronic watery diarrhoea and vomiting, mainly in children.
 There are more than 50 pathogenic serogroups responsible for the
infection.
 Adherence and biofilm cause diarrhea
Generally:
• Dysuria and frequency are features of UTIs by E.coli
 ETEC and EPEC diarrhea is watery
 EHEC diarrhea is bloody
 HUS begins as oliguria and may progress to renal failure

260
 Specimen: Urine, pus, blood, stool, body fluid
 Smear: Gram-negative rods
 Culture: Lactose-fermenting mucoid colonies on Macconckey agar
and some strains are hemolytic on blood agar .
 Biochemical reaction
• Lactose -------positive
• Indole--------- positive
• Lysine decarboxylase (LDC)…… positive
• Hydrogen sulphide …… negative

261
Treatment
 Trimethoprime- sulphamethoxazole, ampicillin, cephalosporines,
aminoglycosides and Cefotaxime.
 Reducing rise of nosocomial infections such as restricting use of
antibiotics and avoiding use of urinary catheters.
 Maintenance of hygienic standards to reduce gastroenteritis.
 Proper cooking of beef reduces risk of EHEC infections.

262
Genus Klebsiella
 Non-motile, lactose-fermenting, capsulated, gram-negative rods.
 Main species of medical importance:
- K. pneumoniae
- K. rhinoscleromatis
- K. ozenae

263
Virulence factors
 Pili ……. adherence to respiratory and urinary epithelium.
 Capsule …….. Prevent phagocytosis.
 About 80 capsular (K) antigens are presently recognized.
 Type K1, K2, K3 and K21 are of particular significance in human
disease.

264
Pathogenesis and clinical manifestations
Klebsiella pneumoniae causes chest infections and occasionally
severe bronchopneumonia with lung abscesses.
Infections are often opportunistic, occurring in those with existing
chest disease or diabetes mellitus, or in malnourished persons.
It also causes urinary tract infection, Septicaemia and meningitis
(especially in neonates) wound infection and peritonitis

265
Klebsiella rhinoscleromatis causes rhinoscleroma (granulomatous
disease) of the nose and pharynx.
Chronic inflammatory growths can lead to deformity of the nose
or distortion of the respiratory passages.
Klebsiella ozaenae. It causes ozena manifesting with foul smelling
nasal discharge leading to chronic atrophic rhinitis.

266
 Specimen: Sputum, urine, pus, CSF, body fluid
 Smear: Gram-negative rods
 Culture: Large, mucoid, lactose-fermenting colonies on Macconckey
agar.
 Serology: Capsular polysaccharide serotyping. More than 80
serotypes of K. pneumoniae recognized

267
Treatment
 Since isolates from hospital -acquired infection are frequently
resistant to multiple antibiotics, the choice of drugs depends on
results of sensitivity testing.
 However, gentamycin and cefotaxime can be used.
 Removing of urinary catheters when they are no longer needed.
 Taking proper care of respiratory therapy devices.

268
Genus Enterobacter
 It is gram-negative lactose fermenting motile rods, and found as a
commensal in the intestinal tract of humans and animals and moist
environments.
 Medical important species ………….. Enterobacter aerogens.
 It produces mucoid colony resembling Klebsiellae on MacConkey
agar.
 Enterobacter aerogens is associated with urinary tract infection,
wound infection and septicaemia in immunocompromised and
chronically debilitated patients.

269
Genus Citrobacter
 It is gram-negative lactose fermenting motile rods, and opportunistic
pathogen.
 Medical important species is Citrobacter freundii.
 Citrobacter freundii may cause infections of the urinary tract, gall
bladder, middle ear, meninges, wound infection and septicaemia in
immunocompromised and chronically debilitated patients.
 C. koseri occasionally causes neonatal meningitis

270
Genus Salmonellae
 Gram-negative, facultative rod-shaped bacteria
 Motile, non-spore forming
 Salmonellae live in the intestinal tracts of warm and cold
blooded animals.
 Some species are ubiquitous.
 Other species are specifically adapted to a particular host.

271
Classification of salmonella
1. Old classification: based on epidemiology, host range,
biochemical, serology-mainly classified into genus and species
e.g. S. typhi
2. Current classification: based on molecular characterization
classified into Genus–species-subspecies-serotype
 There are two species (salmonella bongori, salmonella
enterica)

Salmonella
Salmonella bongori salmonella enterica
enterica salamae arizonae diarizonae houtenae indica
99% human infection
Typhoid non-typhoidal
S.typhi S. typhimurium, entritidis, dublin etc.
S. paratyphi A,B,C
Kauffmann-White Scheme of Classification
272

273
Antigenic Structure
1.Somatic (O) or cell wall antigens
 Somatic antigens that are heat stable and alcohol resistant
 Are lipopolysaccharide layer on surface of bacterial cell wall.
 Used for serological tests
 Not always antigenically identical and over 60 different O
antigens are recognized

274
Figure . …. Antigenic structure of Salmonella typhi

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