Lab 1 Structure of bacterial cells. Microscopic observation of bacteria

Lab 1
Structure of bacterial cells. Microscopic observation of bacteria (light microscope and dark
field techniques).
Bacterial staining techniques. The simple staining technique (crystal violet or methylene blue)
Gram-staining technique.
1. Comparison the size of bacteria with: erythrocytes, epithelial cells and leukocytes.
2. Observation of typical shape and arrangement of some species of bacteria in microscope;
microscopic morphology – coccus (sphere); rod or bacillus, curved or spiral, (single, pairs,
clusters, chains, Chinese letters). Smears stained with positive and negative technique:
Staphylococcus spp. - arranged in irregular grapelike clusters
Streptococcus spp. - arranged in chain
Micrococcus spp. - arranged in tetrads
Escherichia coli - Gram-negative rods
Corynebacterium spp. - arranged in palisades or in V -or L shaped formation, resembling
Chinese letters.
3. Observation of nonessential component of bacterial cell in the light microscope
capsule - Streptococcus pneumoniae and Clostridium perfringens
spores - Clostridium spp.
flagella - Spirillum spp.
4. Observation of living bacteria:
hanging-drop technique
dark-field microscopy
5. The simple stain – with crystal violet, safranin, carbol fuchsin or methylene blue.
Stain is applied to the dried and fixed smear on the slide, next rinsed and dried with bibulous paper
or paper towel. Simple staining procedures require only that a fixed slide be bathed with a single
stain. All organisms will be one color. The type of procedures permits the observation mainly of
shape, size and arrangements or grouping of cells.
6. The complex stain – with more then one dye.
The Gram-staining technique
This method is most familiar and useful of staining protocols, will demonstrate the strategy of
differential staining (depending of cell wall structure).
This staining process has four steps:
a. flood the smear with crystal violet for 3 minutes
b. rinse with water and cover the slide with Gram’s iodine solution for 2 min
c. rinse with water and decolorize with alcohol or acetone for twenty seconds, than
d. counterstaine with carbol fuchsin (or safranin) for 30 second.
Gram-positive organisms appear dark blue-violet, while Gram-negatives are red-pink
Acid-fast stain – Ziehl-Neelsen method
Mycobacteria are Acid-fast rods. They stain with carbolfuchsin (basic dye, red in color), and
resist discoloration with acid alcohol (3% HCl in ethanol)
The procedures for staining bacteria to observe capsules, spores, flagella, fat globules,
granules, and nuclear material are described in most microbiology laboratory manuals.
Lab 2
Physiology of bacteria. Nutritional requirements and nutritional types. Cellular metabolism,
culture media. Specimen processing in laboratory.
Specimen collection and transport to the laboratory. Culture and growth of bacteria.
Almost all medically important bacteria are culturable outside of the host in artificial culture
media, (exceptions Treponema pallidium and obligate intracellular parasites).
The introduction of bacteria into liquid sterile media or on the surface of solidified media is
called inoculation. A population of bacterial cells is referred to as a culture.
In order to inoculate bacteria or clinical specimen into the media inoculating loop or pipette
may be used.
Nutritional types of bacteria:
Heterotrophs – organisms, that obtain carbon from organic materials
Autothrophs – organisms, that use inorganic carbon compounds, usually carbon dioxide as
their basic carbon source
Most microorganisms, including the pathogens are heterotrophic organisms.
Nutritional requirements
Each organism needs a source of energy and nutrient chemicals for growth and multiplication.
Bacteriologic media are essentially soup-like recipies prepared from digests of animal or
vegetable protein supplemented with substances such as glucose, yeast extract, serum or blood.
Types of culture media:
1. liquid media (broth)
2. solid media
A gelling agent (agar) added to a broth medium allows its preparation in solid form as plates in
Petri dishes. Agar is a polysaccharide extracted from certain types of seaweeds. In temperature less
than 42° C is solid. Bacteria grow as colonies on solid media. The cells in a colony are usually
descended from a single original cell. Colonies vary greatly in size, shape, texture, color and other
Differences in colony morphology are very useful for separating bacteria in mixtures and as clues
to their identity.
In liquid media bacteria may grow as: a turbidity, sediment or as a film on the surface of culture.
Both liquid and solid media are simple and enriched (e.g. with serum, blood or other
Nutrient media non-selective- allow the growth the widest range of bacteria.
Selective media - allow the growth of only a particular organism or group of organisms;
these media are used when specific pathogenic organisms are sought in sites with an extensive normal
flora, or when they are present in much smaller numbers, or when they are slow-growing.
Differential media - contain substances, designed to demonstrate biochemical or other feature
characteristics for specific pathogen. The addition to this medium one carbohydrate and a pH indicator
allows to observe fermentation (or oxidation) of the carbohydrate by the organisms (by a color
changing). In practice, nutrient, selective and indicator properties are often combined in the one
Demonstration of:
1. Sheep blood agar: nutrient medium allows to observe different types of hemolysis. (Sheep
blood however, inhibits the growth of Haemophilus spp.)
2. Chocolate agar: a medium containing heated blood with or without supplements
3. Mannitol - salt agar: selective and differential for Staphylococci
4. MacConkey agar: selective and differential for Gram-negative bacteria
5. Thioglycolate broth: for culture of most pathogenic anaerobes
6. Löffler agar: special selective medium for Corynebacterium diphtheriae
Demonstration: broth culture of Staphylococcus spp. (turbidity), Streptococcus spp. (sediment),
Bacillus subtilis (a film)
Conditions for incubation
Temperature. Bacteria can grow in wide ranges of temperature
1. psychophiles - that grow in 0-30º C (optimal 10-20º C)
2. mesophiles - that grow in 10-45º C (optimal 25-40º C)
3. thermophiles- those that grow in 40-70º C (optimal 50-55º C)
O2 requirements vary:
1. aerobes (strict aerobes) - require oxygen to grow
2. anaerobes (strict anaerobes) – can not grow in the presence of oxygen (lack superoxide
dismutase or catalase)
3. facultative anaerobes – (utilize O2 and use fermentation in the absence of O2)
4. capnophilic organisms - require for growth increased concentration of carbon dioxide (3-10%)
5. microaerophilic bacteria - grow better at low O2 concentration and increased CO2
Demonstration: growth in liquid medium Tg - Thioglycolate broth and solid medium Columbia blood
agar (culture in Gas Pack system)
Generation time varies (E. coli 20-30 min in vitro, 12 hrs in large intestine)
Generation time related to the time of onset of infectious illness – “incubation time”
18-24 h: for most human bacterial pathogens
2-5 days or longer: for obligate anaerobes
2-3 weeks or more: for Mycobacteria, some pathogenic fungi (Blastomyces, Histoplasma and
Coccidioides) and some others.
I. Blood cultures
1. Indications for obtaining blood cultures are:
a sudden relative increase in the pulse rate and temperature of patient
a change in sensorium
the onset of chils, prostration, and hypotension
a prolonged, mild and intermittent fever in association with a heart murmur
endocarditis, sepsis, FUO, (also in pneumonia, meningitis, osteomyelitis)
Blood for culture must be collected aseptically: first by cleaning the skin with the 70 %
alcohol to remove oils and fatty substances, 2) next by applying 2 % iodine - for at least 30
seconds, and 3) application of 70 % alcohol to remove the residual iodine from the skin. The
intended venipuncture site should not then be touched.
Specimen number, timing and volume
Bacteremia with constant fever and leukocytosis – at any time only once per day.
Intermittent bacteremia – blood should be collected just before, during, or immediately after a
fever’s peak.
In acute endocarditis – collection of three blood cultures from three different venipuncture sites
over a period of 1 to 2 hours is recommended
In subacute endocarditis – three blood cultures, obtained on day 1, preferably 15 minutes or more
apart are recommended. If these three cultures are negative after 24 hours incubation, three
additional cultures should be obtained from this patient.
Specimens from adults should contain min 10 ml of blood (20-30 ml), the desirable blood-tobroth ratio is approximately 1:10. Less blood is required for children. Most blood culture systems
specify the volume of blood, necessary for optimal recovery.
Blood culture methods
Conventional broth blood culture systems that use nutritionally enriched liquid media recover
most bacteria, including anaerobes. Traditionally 2 bottles are inoculated; one is vented to ensure
the recovery of strict aerobes, second-anaerobes. Bottles are examined daily for evidence of
growth, indicated by turbidity, hemolysis, gas production or presence of colonies (when biphasic
agar-broth culture is used). Subculture of the contents of the bottles is necessary. These systems
are inexpensive with regard to reagents, but they are relatively labor – intensive.
The fully automated continuosly monitoring blood culture systems are the newest type of
systems developed for the detection of bacteria and fungi in blood: Bactec (Becton Dickinson
Microbiology System), Bact/Alert (Organon Teknika Corp.) and others.
BACTEC System measures CO2 released during microbial metabolism. CO2 is detected by
infrared spectroscopy. A positive culture is based on CO2 exceeding a threshold level or a change
between two consecutive readings. A variety of media are available for use with the BACTEC
System: for isolation of bacteria and fungi including a small volume draw (pediatric) bottle.
The Bact/Alert System (Organon Teknika) is based on detection of CO2 by a colorimetric sensor
in the bottom of each bottle. The sensor pad is saturated with a pH-sensitive water solution. As the
hydrogen ions increase, the pH decreases and causes the sensor to change from dark green to
yellow. The instrument scans each bottle every 10 minutes.
Three types of media are available: aerobic, anaerobic and pediatric (possibility to detect of
fastidious organisms, mycobacteria and to inactivate antibiotics).
Lab 3
Assay of surrounding microflora. Antimicrobial susceptibility testing of different bacterial
Laboratory methods for detection of susceptibility to antibiotics.
Guidelines and recommendations for antimicrobial susceptibility testing are published by the CLS
(Clinical Laboratory Standard Institute) former National Committee for Clinical Laboratory Standards
(NCCLS), and every year are actualized.
The components of antimicrobial susceptibility testing that are standardized and controlled include e.g.
- bacterial inoculum size
growth medium used (e.g. Mueller – Hinton base)
incubation atmosphere, incubation temperature, incubation duration
pH, cation concentration, blood supplements
antimicrobial concentration tested
Three testing methods
1. Methods that directly measure the activity of one or more antibacterial agents against a
bacterial isolate
2. Methods that directly detect the presence of a specific resistance mechanism in a bacterial
isolate e.g. MRSA, MLSB
3. Method that measure complex reactions.
Conventional testing methods:
A. Dilution method: based on two – fold serial dilutions of antibiotic in liquid (broth dilution) or in
solid (agar dilution) bacteriologic medium. The media are inoculated with test bacteria and
incubated. The result is commonly reported as the minimal inhibitory concentration (MIC) or
minimal bactericidal concentration (MBC): amount of antimicrobial substance required to inhibit
the growth (MIC) or to kill the test bacteria (MBC). This method is time-consuming, and usage is
limited to special circumstances.
B. Diffusion method: A filter paper disk impregnated with antimicrobial substances is placed on
solid medium that has been heavily seeded with the test organisms. After incubation the diameter
of the clear zone of inhibition surrounding the disk is taken as a measure of the inhibitory power
of the drug against tested organism. If in the clear zone individual bacterial colonies are present, it
may be mixed culture, or resistant mutants of the test isolate.
Disc diffusion susceptibility testing conditions for same bacterial strains
S. aureus
0,5 McFarland
35º C, air
16-18 hrs
0,5 McFarland
35º C, air
24 h
0,5 McFarland
35º C, 5 % CO2
20-24 hrs
0,5 McFarland
35º C, 5-7 % CO2
16-18 hrs
Test medium
MRSA (oxacillin 1
µg or cefoxitin 30
S. pneumoniae
H. influenzae
Mueller-Hinton +
5% sheep blood
Haemophilus Test
Medium (HTM)
Interpretive categories
1. Susceptible (S)
2. Intermediate (I)
3. Resistant (R)
Diffusion in agar derivations. The E-test (combination of disk diffusion with the ability to generate
MIC date) uses plastic strip contains the antibiotic concentration gradient; the numeric scale indicates
the drug concentration. Mueller – Hinton plates (or others depending of studied the bacteria) are
inoculated as for disk diffusion and the strips are placed on the inoculum lawn. After incubation (24 or
48 h), the plate is examined and the number present at the point where the border of growth inhibition
intersects the E-strip is taken as the MIC value.
Automated antimicrobial susceptibility test system
eg Vitek System (Vitek and Vitek 2 bioMerieux) and Walk-Away System (Dade international,
Sacramento) detect MIC value of investigative strains, but no mechanisms of resistance.
Supplemental methods for detection of resistance mechanism.
oxacillin agar screen (detection of staphylococcal resistance to beta – lactam antibiotics MRSA,
vancomycin agar screen (detection of enterococcal resistance to vancomycin - VRE)
gentamicin screen (detection of acquired enterococcal high-level resistance to aminoglicosides HLAR)
oxacillin 1µg disc screen (detection of Streptococcus pneumoniae resistant to penicillin)
Lab 4
Gram-positive cocci (Staphylococcus, Microccocus, Streptococcus and Enterococcus): methods
for isolation, identification and antibiotic susceptibility testing.
1. Demonstration of growth (blood agar and mannitol-salt agar)
a. Staphylococcus aureus
b. Staphylococcus epidermidis
c. Micrococcus sp.
2. Tests for identification of Staphylococci
a. Catalase detection - H2O2 degrading enzyme catalyzes conversion of H2O2 to H2 and O2.
Bubles indicate positive reaction.
b. Clumping-factor test (slide method)
c. Coagulase test (coagulase, tube method)
d. Novobiocin susceptibility – test for presumptive identification of coagulase- negative
staphylococci. Staphylococcus saprophyticus is novobiocin-resistant, S. epidermidis is
e. Biochemical identification of Staphylococci by API Staph
Antibiotic sensitivity testing
a. demonstration of disk - diffusion method
b. demonstration of methicillin - resistant MRSA and methicillin – sensitive MSSA strains
among Staphylococcus aureus isolates
c. detection of β-lactamase production (Nitrocefin test)
The discs are impregnated with Nitrocefin - a chromogenic cephalosporin. This compound
exhibits a rapid distinctive color change from yellow to red as the amide bond in the β-lactam ring is
hydrolyzed by a β-lactamase. Nitrocefin hydrolysis has been found to be highly efficient in detecting
of β-lactamase producing isolates of Neisseria gonorrhoeae, Haemophilus influenzae, Staphylococcus
spp., Moraxella catarrhalis, Enterococcus spp. and Bacteroides sp.
4. Staphylococci commonly associated with human disease
Characteristics of medically important Staphylococci
CF (clumping factor)
Mannitol (Chapman)
Protein A
Sensitivity to
β-hemolysis - complete lysis of red blood cells (clear zones around colonies).
Coagulase-positive strains produce a thrombin-like substance that catalyses clot formation when cells
and plasma are mixed together in a test tube.
Clumping factor: a component of cell wall of Staphylococcus aureus that binds to fibrinogen in
plasma, causing agglutination of bacteria
Protein A: presents in a cell wall of Staphylococcus aureus.
5. Types of hemolysis on blood agar plates (with 5 % sheep blood)
a. α-hemolytic Streptococci – a greenish discoloration of blood agar around colonies (due to a
reductant of hemoglobin). Red blood cells remain intact
b. β- hemolytic Streptococci (complete lysis of red blood cells)
c. γ-hemolysis – “nonhemolytic” Streptococci (non action on blood agar)
6. The classification of Streptococci
- β-hemolytic
a. susceptibility to bacitracin (Streptococcus group A - are sensitive, other β- hemolytic streptococci
are usually resistant)
b. CAMP test for presumptive identification of Streptococcus group B
c. Lancefield serogrouping for rapid grouping β-hemolytic streptococci e.g. Slidex Strepto-Kit,
Streptex or Pastourex. In this test is used latex coated with antibodies highly specific to each
group. Extraction enzyme removes specific antigen (polysaccharide C) from the cell wall of
Streptococci. Than, the group – specific antigens liberated are identified by latex particles
sensitized with immunoglobulins. This test facilitates the routine grouping of groups A, B, C, D, F
and G β-hemolytic Streptococci.
Non β-hemolytic
Streptococcus “viridans” (α- or γ-hemolysis)
Opportunistic pathogen viridans streptococci can cause disease when they are protected from host
defenses. Transient bacteremia may lead to endocarditis on damaged heart valve – subacute
endocarditis. Severe neutropenia may be accompanied with infection with opportunistic bacteria
e.g. viridans streptococci.
Biochemical identification e.g. API Strept.
Streptococcus pneumoniae
a. direct smear of sputum – Gram-positive diplococci with capsule
b. growth on blood agar – round, glistening colonies surrounded by a zone of α-hemolysis
c. identification – susceptibility to optochin (ethylhydrocupreine – HCl), inhibits S. pneumoniae,
bile solubility
d. detection of pneumococcal capsular antigens in body fluids (CSF, serum, urine) by slide latex
agglutination, or by the classical “quellung” reaction
Enterococcus spp.
Enterococci on blood agar plates (α-hemolytic or γ-hemolytic, sometimes β-hemolytic strains)
a. morphology – short chain, mainly diplococci
b. catalase-negative
c. PYR – a rapid and simple test for determination of PYRase activity. The discs are
impregnated with substrate for the detection of PYRase activity. Enzymatic hydrolysis of this
substrate by enterococci or Group A streptococci produces a red colour.
d. Slidex Strepto-Kit – group D
e. Biochemical identification e.g. API Strept
Demonstration of antibiotic susceptibility tests of Enterococcus faecalis
a) strain sensitive to high level aminoglycosides
b) strain of high level aminoglycosides resistant (HLAR)
c) VRE – Vancomycin-resistant Enterococci
Lab 5
Gram-negative cocci (Neisseria, Moraxella) and Haemophilus spp. methods for isolation,
identification and antibiotic susceptibility testing.
Neisseria meningitidis
1. Smear of spinal fluid
2. Culture of CSF (cerebro-spinal fluid) on Columbia blood, chocolate agar (incubated at 35°C in
5% CO2 atmosphere)
identification methods:
a. Gram-staining: Gram-negative diplococci
b. Oxidase test. Smear some test colonies onto a piece of filter paper and add a few drops of
oxidase reagent (dimethyl-p-phenylenediaminemonohydrochloride for the chemically
inclined). If the culture is oxidase positive the dye will be oxidized and the colonies will turn
from pink/red to black fairly quickly.
c. Biochemical identification e.g. API NH
3. Rapid diagnosis of meningococcal meningitis: Meningo-Kit (latex agglutination test to detect
capsular polysaccharide)
Neisseria gonorrhoeae
1. Smear of urethral discharge (Gram-negative diplococci within PMNs)
2. Culture on Thayer - Martin medium (incubated at 35-37° C in 5 % CO2)
Identification methods:
a. Gram-staining: Gram-negative diplococci
b. Oxidase test (positive)
c. Biochemical identification e.g. API NH
d. Gonozyme test
Haemophilus influenzae
Haemophilus spp. are small, polymorphic (from coccobacilli to filamentous), Gram-negative rods.
They are facultative anaerobic, nonmotile and usually encapsulated. For in vitro growth require
accessory growth factors: X factor - hemin, and V factor - nicotinamide adenine dinucleotide (NAD).
Haemophilus influenzae requires both of these compounds, whereas some of the other species of the
genus require only one.
Haemophilus spp. represent approximately 10 % the constant bacterial flora of the healthy upper
respiratory tract. The predominant species is Haemophilus parainfluenzae.
Nonencapsulated Haemophilus influenzae strains (multiple biotypes) are present in the pharynx of
most healthy children. Haemophilus influenzae serotype b may be also isolated from pharyngeal
culture of healthy individuals.
Encapsulate Haemophilus influenzae are divided into six serotypes (designated a, b, c, d, e, f) based on
the capsular polysaccharide antigen. Type b capsule consists of polyribitol phosphate, these surface
polysaccharides are strongly associated with virulence.
Clinical significance
Haemophilus influenzae is one of the three leading causes of bacterial meningitis. Virtually all of these
cases are caused by organisms that possess a serotype b capsule. Meningitis is often preceded by sings
and symptoms of an upper respiratory infection, such as nosopharyngitis, sinusitis, or otitis media.
Haemophilus influenzae b is the major causative agent of acute epiglottitis.
Invasion of the bloodstream by Haemophilus influenzae b may result in suppurative arthritis,
osteomelitis, cellulitis and pericarditis.
Haemophilus influenzae belongs to group of pathogens causing exacervation of chronic
Diagnosis is based on isolation of Haemophilus influenzae from the site of infection (e.g. CSF) or
from the blood - particularly in systemic infections (a large proportion of these cases are bacteremic):
isolation of Haemophilus influenzae on heated-blood („chocolate”) agar enriched with factors X
and V in 5 % CO2
presumptive identification on the basis of the requiring of both growth factor
growth on Mueller-Hinton agar with disks containing factors X, V and XV
satelitism around colonies of Staphylococcus aureus (based on V factor)
definitive identification
biochemical tests (e.g. API NH)
latex agglutination test for detection of capsular polysaccharides
capsular swelling reaction
fluorescent antibody staining
antibiotic sensitivity test
demonstration of cefinaze test to detect β-lactamases (about 25 % of Haemophilus influenzae
strains produce β-lactamases)
demonstration of HTM agar (special medium only for Haemophilus sp.)
Prevention – Haemophilus influenzae b conjugate vaccine is used in children (protection against
Haemophilus influenzae b infections)
Other species of Haemophilus
Haemophilus aegyptius - is associated with an acute purulent and contagious form of conjunctivitis
Haemophilus ducreyi - is the cause of the veneral disease - soft chancre
Haemophilus parainfluenzae
Haemophilus haemolyticus
Haemophilus aphrophilus
Haemophilus paraphrophilus
like several other oral bacteria, these species are occasionally implicated in endocarditis and abscesses
of internal organs
Moraxella catarrhalis
1. Smear of colony - (Gram-negative bacteria resembling Neisseria sp.)
Fermentation of glucose:
Moraxella catarrhalis is negative (non fermentative-blue)
Neisseria spp. are positive (fermentative-yellow)
Lab 6
Mycobacteria – laboratory diagnosis of tuberculosis. Nocardia.
Spore forming aerobic Gram-positive bacilli: Bacillus. Non spore forming Gram-positive rods
(Corynebacterium spp., Listeria sp., Erysipelotrix sp.).
Mycobaterium spp.
1. Direct smears of sputum - stained using Ziehl-Neelsen technique.
If present in sufficient numbers, acid-fast bacilli can be detected microscopically in direct smears.
These procedures are not specific only for Mycobacterium tuberculosis, because other
mycobacteria may have a similar morphology.
2. Demonstration of growth of Mycobacterium tuberculosis on the classic Loewenstein-Jensen
Colonies usually appear after 3 to 6 weeks of incubation. They become raised, warty, and adherent,
with a buff pigmentation, and are difficult to emulsify because of their high lipid content.
3. Classification
a. cultural features (nutritional and temperature requirements, growth rate, pigmentation of
colonies grown in light or darkness)
b. biochemical reactions - of particular importance: the ability of Mycobacterium tuberculosis to
produce large quantities of niacin (which is uncommon in other mycobacteria)
c. molecular techniques (PCR and non-PCR-based amplification techniques)
4. Susceptibility testing
by conventional procedures on the Loevenstein-Jensen medium with antibiotic
using radiometric detection of mycobacterial growth (more rapid results)
5. Treatment and prophylaxis of tuberculosis
combined therapy used to prevent resistance
chemoprophylaxis may use single drug (isoniazid alone)
In contrast to Actinomyces, they are strict aerobes.
Nocardia asteroides and Nocardia brasiliensis are weakly acid fast.
Nocardia spp. are commonly found in the environment, particularly in soil.
Clinical manifestations:
Pulmonary Nocardiosis (mainly Nocardia asteroides)
Skin and subcutaneous tissue infections (mainly Nocardia brasiliensis)
Clinical materials are: sputum and direct aspirates from skin or other purulent sites.
Direct smear: the microscopic morphology is similar to that of Actinomyces, but Nocardia tend to
fragment more reality and are found as shorter branched units.
Culture: growth is observed on ordinary culture media usually after 2 days. Sometimes Nocardia may
require longer time to grow e.g. 7 days. Colonies are dray, wrinkled and heaped up, (sometimes also
Identification can be made biochemically but it takes 2-3 weeks to complete.
Clinical Significance of Atypical Mycobacteria
Environmental Sources
M. avium-intracellulare
Soil, water (including drinking water),
birds and other animals (especially
chicken, swine, and cattle), foods such as
meat, milk, and eggs
M. fortuitum-chelonae
M. genavense
M. haemophilum
M. kansasii
M. malmoense
M. marinum
M. scrofulaceum
M. simiae
M. szulgai
M. ulcerans
M. xenopi
Clinical Significance
Chronic pulmonary disease, local
lymphadenitis, bone and joint disease,
disseminated disease, skin and soft
tissue infections including abscesses
and corneal infections, rarely
genitourinary disease; disseminated
disease in patients with AIDS; also
responsible for the most important
mycobacterial diseases in animals
M. fortuitum is found almost everywhere Disseminated disease, cutaneous
in the environment including water, soil, lesions, pulmonary disease and a
and dust; the habitant of M. chelonae is
variety of miscellaneous infections:
not known for certain, although water
infection is often proceded by
may be a source
traumatic or surgical events
Natural resevoir unknown (? water
Disseminated disease in AIDS
Skin lesions, lymphadenitis, rarely
genitourinary disease
Natural resevoir unknown; has been
Chronic pulmonary disease, bone and
recovered from tap water and rarely from joint disease, disseminated disease,
tissues of cattle and swine; has not been cervical lymphadenitis, rarely
recovered from soil or dust
genitourinary disease
Chronic pulmonary disease,
Found in fresh and salt water as a result
Cutaneous disease
of contamination from infected fish and
other marine life; has been cultivated
from water of natural and constructed
swimming pools and aquariums; also
recovered from rough surface of
swimming pools
Soil, water (including tap water), raw
Cervical lymphadenitis in children,
milk, other dairy products, oysters
less commonly chronic pulmonary
disease in adults, occasionally
disseminated disease in children
Found in monkeys imported from India
Only rarely associated with chronic
(Macaeus rhesus) and isolated from tap
pulmonary disease, osteomyelitis, and
water in a hospital in Tucson, Arizona,
disseminated disease with renal
but these isolates were not associated
with disease
Although the distribution of this
Chronic pulmonary disease;
organisms appears widespread, little
extrapulmonary disease in uncommon
information is available in regard to its
but has included infections of elbow,
cervical lymphadenitis, and cutaneous
Environmental source seems likely, but
Bairnsdale ulcer, Buruli ulcer
has not been recovered outside the human
Hot and cold water taps, hot water
Chronic pulmonary disease
generators and storage thanks of
hospitals, birds
From: Barbara J. Howard “Clinical and Phatogenic Microbiology”
Bacillus spp. - aerobic, sporeforming, Gram-positive rods.
Bacillus anthracis - ow-virulence saprophytes widespread in air, soil, water, dust and animal
products. Bacillus anthracis causes the zoonosis anthrax, a disease of animals that is occasionally
transmitted to humans
a. direct smear of spleen of mice infected with Bacillus anthracis, stain with Löffler blue to
detect metachromically stained capsule
b. growth on liquid medium in sediment; in smear of sediment we observe very long chains of
rods with elliptic central spores (bamboo rods)
c. observation of colonies of Bacillus anthracis, colonies are characterized by a rough surface
with multiple curled extension at the edge, resembling a „Medusa head”
Bacillus anthracis virulence factors:
1. capsule - D-glutamic acid polypeptide - it has antiphagocytic properties, but is not immunogenic
2. exotoxin - with multiple activities (edema factor [EF], protective antigen [PA] and lethal factor
Other Bacillus species
Occasionally Bacillus cereus, Bacillus subtilis and some other species produce genuine infections,
including infections of the eye, soft tissues, and lung.
Bacillus cereus is most common opportunist, it can also cause food poisoning.
Corynebacterium (tab. 1)
In humans, corynebacteria inhabit the skin and or pharynx. Most Corynebacterium spp. (diphteroids)
are non pathogenic, but can cause opportunistic infection.
Pathogen: Corynebacterium diphtheriae causes diphtheria by producing an exotoxin.
Diphtheria – diagnosis
Primary diagnosis of diphtheria is clinical. Detection of Corynebacterium diphtheriae requires two
swabs taken from infected areas:
smear of the throat swab should be stained with both: Gram stain and methylene blue:
a. Gram-stain - to show cell morphology and typical arrangement in palisades or in V, X or L
shaped formations, resembling Chinese letter or palisade arrangements
b. Methylene blue-stain: to show volutin, a highly polymerised polyphosphate, which stains
metachromatically: the cell is yellow, granules of volutin are black
1. Culture on:
a. blood agar (suspicious colonies are greyish and may be surrounded by a small zone of hemolysis)
b. tellurrinum salts containing media (selective media e.g. Tinsdale or Clauberg medium containing
potassium tellurite) Corynebacterium diphteriae reduces the potassium tellurite to produce gray or
black colonies. The morphology differs with each of three types of organism: gravis, mitis and
c. Löffler medium - with horse serum (stimulats growth of Corynebacterium diphtheriae, useful for
selective growth of C. diphtheriae from throat swabs)
2. Biochemical differentiation and identification of the various species e.g. API Coryne (bioMerieux,
3. Toxigenicity testing
Toxigenicity testing should be done for all isolates of Corynebacterium diphteriae (also other
pathogenic strains - Corynebacterium ulcerans, and Corynebacterium pseudotuberculosis producing toxins). Diphtheria toxin can be detected in vivo, in the guinea pig lethality test, or in
vitro in the Elek agar immunodiffusion test.
a. In vitro Elek test. It is a double diffusion reaction in agar gel. On freshly prepared medium
enriched with serum a strip of sterile filter paper saturated with C.diphtheriae-ahtitoxin (750
u/ml) is placed. Then the cultured strains are inoculated perpendicullary to the paper strip. The
reference toxin-producing and nontoxigenic strains (positive and negative controls) are also
inoculated on this plate. If toxin is synthesized by the examined strain a characteristic
precipitation line arise like in positive control.
b. in vivo test – guinea pig inoculation
TABLE 1. Natural habitants and clinical significance of medically important corynebacteria and
related organisms
Species or groupa
C. ulcerans
C. kutscheri
C. renale group
Group JK,
C. mycetoides
Natural habitant
Proven or probable clinical significance in
Diphtheria, endocarditis
Cattle, horses
Sheep, goats, horses, etc.
Sore throat, diphtheria like illness
Granulomatous lymphadenitis, pneumonia
Human (nasopharynx,
skin, conjunctiva)
Human (nasopharynx,
skin), cattle
Human (pharynx, skin)
Endocarditis, pneumonia, arthritis, wound
infection, septicemia
Pleuropneumonia, lung abscess
Human (skin)
Human (skin),
Non known
Distilled and natural fresh
Chlorioamnionitis, septic arthritis
Abscesses (rectum, breast)
Endocarditis, urinary tract infection,
lymphadenitis, pneumonia, skin graft
Wound infections, urinary tract infection,
septicemia, endocarditis,
meningitis,peritonitis, lung infectionc
Erythrasma, bacteremia, endocarditis and
Tropical ulcers, septicemia
Endocarditis, meningitis, urinary tract
„C. bovis”b
C. matruchotii
Human (skin)
infection, peritonitis in continuous
ambulatory peritoneal dialysis, septicemia
Possibly urethritis
Human (skin)
Non known
Human (oral cavity),
Probably humans
Septicemia, eye infections, peritonitis
Eye infections
Rhodococcus spp.
Rhodococcus equi
Soil in association with
Soil in association with
Pharyngitis, sometimes diphthera- or
scarlet fever-like illness, skin ulcers,
abscesses, septicemia
Wound infections, meningitis, eye infection
Usually AIDS-related; tuberculosislike
manifestations, primarily of the lung,
septicemia, peritonitis, osteomyelitis,
abscesses, endophthalmitis
coryneform group
Non recognized as a species of the genus Corynebacterium by Bergey’s Manual of Systematic Bacteriology
CDC has received at least three group JK strains (one each from tibia, hip, and bone) isolated from patients with a diagnosis
of osteomyelitis.
From: A. Balows at all., Manual of Clinical Microbiology, Fifth Edition p278, Washington 1991
Listeria monocytogenes is Gram-positive rod morphologically resembling corynebacteria and in
culture on blood agar resembling streptococci, but is motile and catalase-positive.
- Gram–stain – short Gram-positive rods with rounded ends that may be in short chains, which may
be confused with streptococci in clinical samples. Organisms can “palisade” similar to
culture on blood agar - growth as small, smooth colony surrounded by a narrow rim of βhemolysis - the colonies resamble streptococci
observation of motility of Listeria monocytogenes by hanging drop-technique:
a. culture in 22° C - in this temperature it is actively motile
b. culture in 35° C - in this temperature it is non-motile
Listeriosis - epidemiology
The principal habitat of Listeria monocytogenes is the intestinal tract of mammals and birds.
Listeria monocytogenes may be also in human intestinal flora. It is continually shed in the soil, where
it may persist for months.
Foodborne transmission (food borne pathogens) is important in listeriosis, infections in human
are more frequently associated with exposure to milk products and contaminated vegetables than
contact with animals. The chicken is the animal most frequently found to harbor the organisms.
Listeria monocytogenes has ability to grow at refrigerator temperature.
cold culture (in refrigerator) of some clinical specimens
Erysipelothrix rhusiopathiae
micraerophilic, nonsporeforming, nonmotile, Gram-positive rod with a tendency to form filaments. It
resembles Corynebacterium diphteriae. Clinical materials - a skin biopsy specimen is placed into
thioglycolate, incubated overinight and transferred to blood agar. In very rarely cases when is caused
septicemia or endocarditis clinical material is blood. In blood bacteria are detected by fluorescence
Lab 7
Spore-forming Gram-positive anaerobic rods (Clostridium spp.) Non-spore-forming anaerobes.
Actinomycetes – laboratory diagnosis of infection caused by Actinomyces.
Clostridium - anaerobic, sporeforming, Gram-positive rods, are distributed widely in the environment,
and are found as normal flora in the human intestinal tract.
Clostridium botulinum
a. microscopic observation of Clostridium botulinum. Spores are subterminal.
b. virulence factors of Clostridium botulinum (toxins)
Clostridium botulinum is divided into 8 types based on the serological specificity of the toxin
produced. Types A, B, E - mainly, and F - rarely are associated with human infection. Type A is more
common in the western states, type B predominates in the central states and the eastern United States.
Botulism in Alaska is more often associated with type E.
All types of Clostridium botulinum produce toxin in foods even at low temperature (types A and B
produce toxin between 10 and 13° C; E and F, at 3-5° C).
All the toxins are heat labile and heating food at 100° C for 10 min. before eating will destroy the
toxin (method of prevention of botulism).
Laboratory diagnosis
Toxin can frequently be demonstrated in blood, intestinal contents, or remaining food by
inoculation into mice. Unprotected mice die, whereas those protected with specific antitoxin survive.
Trivalent (ABE) horse Clostridium botulinum antitoxin is given to neutralize any circulating
toxin. Antitoxin is the drug of choice.
Antimicrobic agents are necessary only in cases of wound botulism.
Clostridium perfringens
It is large, nonmotile, encapsulated rod with squarish ends. Spores are develop in the natural
habitat. Spores of Clostridium perfringens are resistant to all disinfectants and to boiling for brief
periods. The spores of some strains that cause food poisoning can withstand temperature of 100oC for
an hour or more, which account for their survival in cooked food.
Smear of culture of Clostridium perfringens. Spores are large, oval, central or subterminal and distend
the cell.
The growth on a blood agar plate in anaerobic conditions - round, smooth colonies, surrounded by a
double zone of hemolysis (hemolytic activity caused by alpha and theta toxins).
The growth in broth containing fermentable carbohydrates is accompanied by the production of large
amounts of hydrogen and carbon dioxide (much gas is also produced in vivo in necrotic tissues; hence
the tem gas gangrene).
Disease caused by Clostridium perfringens
1. Gas gangrene
2. Anaerobic cellulitis
3. Clostridial endometritis (septicemia and intravascular hemolysis associated with nonsterile
4. Clostridial food poisoning (after incubation period 8-24 hours, nausea, abdominal pain and
diarrhea without fever)
Diagnosis of Clostridium perfringens infections
Diagnosis is based ultimately on clinical observations. Isolation of Clostridium perfringens
from contaminated wound (pieces of necrotic muscle), or from the postpartum uterine cervix.
in direct smear of clinical material usually inflammatory cells are absent, and Gram-positive
encapsulated rods are present.
In clostridial food poisoning, isolation of more than 105 Clostridium perfringens per gram of the
ingested food in the absence of any other cause is usually sufficient to confirm the etiology of
Clostridium tetani
It is slime, strict anaerobic and motile rods. It forms spores readily in nature and in culture.
Typical terminal spores give the organism the characteristic apparence of “tennis racket”. The spores
are resistant to most disinfectants and withstand boiling for several minutes. They are killed by
autoclaving at 121° C for 15 minute. C.tetani produces a powerful exotoxin-neurotoxin (tetanospasmin
and tetanolysin). Toxin becomes anatoxin (toxoid) under the formaldehyde treatment.
1. Wound infection. Tetanus may result from small wounds or splinters. The contamination of the
wound with soil, manure, dust containing spores of C.tetani leads to the infection.
2. In many less developed countries, the majority of tetanus cases occur in delivered babies (when
the umbilical cord is severed or bandaged in an uncleanly manner), and their mothers.
Large doses of human tetanus immune globulin (TIG), which is derived from the blood of
volunteers hyperimmunized with toxoid.
Routine active immunization with tetanus toxoid, combined with diphtheria toxoid, and
pertussis vaccine (DPT) for primary immunization in childhood, can completely prevent the disease.
Five doses of DTP are recommended, to be given at the ages of 2, 4, 6, and 18 month, and once again
between the ages of 4 and 6 years. There after a booster of adult - type tetanus diphtheria toxoid
should be given every 10 years.
Unimmunized subjects (or persons without fixed data about vaccination) with tetanus suspected wound should be given passive – active immunization with tetanus toxoid and tetanus
immune globulin (TIG) as soon as possible.
Those who had a full primary series of immunization, and appropriate boosters, within last 8
years need only antitoxin injection.
Clostridium difficile
1. Pseudomembranous colitis (PMC)
2. Antibiotic - associated diarrhea (AAD)
Endoscopic examination of the colon
1. Detection of toxin A and B in stool
a. toxin B by tissue culture
b. both toxin A and B by enzyme immunoassays
2. Culture of stool samples on selective CCCA medium.
Treatment - with oral antibiotics such as vancomycin or metronidazole.
Alternative treatment – Saccharomyces boulardii, Lactobacillus spp., (enema with feces of healthy
Though Louis Pasteur is credited with the first cultivation of strictly anaerobic bacteria, it was
Veillon and Zuber (1897, 1898) who demonstrated clearly the involvement of Gram-negative nonsporeforming strictly anaerobic bacteria in infections such as brain and lung abscesses, peritonitis,
bartholinitis and otitis media. They named the most abundant and frequently observed bacterium, in
appendicitis and peritonitis Bacillus (now Bacteroides fragilis)
Decision about culturing anaerobes must be based on the estimation of clinical symptoms.
All specimens collected for microbiological studies may be divided into 2 groups:
4. not contaminated with the physiologic bacterial flora (are suitable for culturing anaerobes)
5. contaminated with the physiologic bacterial flora (are suitable for culturing anaerobes only by
using selective media for isolation of suspected, etiological agent)
Non-sporulating anaerobes are important as etiological agents of:
1. endogenous infections
2. mixed infections
3. infections of each tissue and each part of body
They can cause diseases with considerably differentiated clinical symptoms
Family: Bacteroidaceae
Genus: Bacteroides
Group: Bacteroides fragilis
Bacteroides fragilis group (small Gram-negative pleomorphic rods-shaped) members:
Most important species in human pathology belonging to this group are B.fragilis and
B.thetaiotaomicron. These bacteria are causative agents of intraabdominal, vaginal, perianal and brain
abscesses and soft-tissue infections. B.fragilis is also the most common cause of anaerobic septicaemia
with a potential mortality of to 19%. The risk of B.fragilis infection after surgical operations of the
lower intestinal tract makes necessary prophylactic antibiotic cover.
Haemin and vitamin K are stimulatory for the growth of many Bacteroides spp., they are also biletolerant, eskulin hydrolysed and saccharolytic. Major metabolic end products are acetate and
succinate. Fimbrial and extracellular polysaccharides are two surface structures very important for
virulence factors. Encapsulated B.fragilis strains are resistant to complement-mediated killing.
Capsule is responsible also in abscess formation (studies in an animal model). Recently (1984)
enterotoxigenic B.fragilis strains were described as a cause of watery diarrhoeal disease in newborns
lambs, calves and also humans. The enterotoxin is zinc-dependent metalloprotease of ≈ 20 kDa. The
enterotoxin can be detected in culture supernatant by alteration of the morphology of the colonic
epithelial cell line HT 29/C1. Majority of B.fragilis strains produces ß-lactamases and are resistant to
Family: Bacteroidaceae
Genus: Fusobacterium
Gram-negative, anaerobic, unencapsulated, non-spore-forming rods. Major metabolic end products is
butyric acid. The most important species in human pathology are F.necropharum, F.nucleatum,
F.varium (especially isolated from mixed infections).
The other medically important Gram-negative anaerobic bacteria are Prevotella, Porphyromonas and
Bilophila (Table 6)
Table 6
Some virulence factors of anaerobic non-sporing Gram-negative bacteria (after Lorber, 1995)
Virulence factor
Capsular polysaccharide
Bacteroides fragilis
Cell adherence
Prevotella melaninogenica Abscess formation
Inhibition of phagocytosis
Porphyromonas gingivalis
Bacteroides spp.
Lacks Lipid A – low endotoxicity
Potent endotoxic action
Inhibition of phagocytosis and intracellular
Many species
Bacteroides spp.
Bacteroides spp.
Spread in tissues
Prevotella maleninogenica Tissue damage
Cell membrane damage
Succinic acid
Phospholipase A
Among the Gram-positive non-sporeforming anaerobes, there are cocci, primarily Peptostreptococcus
and bacilli of the genera Actinomyces, Eubacterium and Propionibacterium.
Family: Actinomycetaceae
Genus: Actinomyces
Species Actinomyces israelii
Morphology: Gram-positive club shaped rods with branching filaments. In liquid media, at the bottom
of tube, they develop small granules. On solid media they grow like small, smooth “spider-like” or
rough “molar tooth surface” resembling colonies.
Different types clinically manifested Actinomycosis can be distinguished:
1. cervico-facial-hard infiltration and necrosis of tissue, containing yellow “sulfur-granules”
(Actinomyces colonies)
2. pleuro-pulmonary
3. abdominal
4. pelvic infection (usually after using intra-uterine anticonception)
Microscopic smears are prepared for:
a. unstained, under cover-slip observation
b. Gram staining
c. Ziehl-Nielsen staining (for distinguish from acid-fast Nocardia)
In unstained smeares typical picture with radially arranged rood-shaped cells at the colonies-edge is
observed. Actinomyces spp. are metronidazole resistant, but sensitive to penicillin, tetracycline and
Family: Peptostreptococcaceae
Genus: Peptostreptococcus
Gram-positive anaerobic cocci, frequently isolated from clinical specimens.
Laboratory diagnosis is based on the presence of Gram-positive cocci and growth under anaerobic
conditions. Volatile acid analyses and gas chromatography are useful for identification.
Majority of Peptostreptococci are metronidazole-resistant.
Material for direct smear and culture should include as much pus as possible to increase the
chance of collecting the diagnostic sulfur granules.
Direct smear
Sulfur granules are crushed between two slides and staining by Gram (Gram-positive bacteria
with individual braching rods at periphery are observed).
Granules should also be selected for culture because material taken from a draining sinus gives
usually growth only superficial contaminants. Actinomyces spp. is usually very slow growing obligate
anaerobes. There are no selective media for the isolation of Actinomyces.
Identification - biochemical e.g. API for anaerobic bacteria.
Identification of non-spore-forming anaerobes
Clinical samples
(pus, tissue, swabs, punctuates)
Inoculation into selective
IF (direct immunofluorescence
blood-enriched medium
(2-7 days incubation
Inoculation into
blood-agar plates
(aerobic control)
in anaerobic condition)
Gram stain
India ink stain
tests (API 20A)
susceptibility testing
Lab 8
Spirochetes Treponema pallidum, Leptospira and Borrelia. Morphology and antigenic structure.
Clinical manifestations and laboratory diagnosis of syphilis and gonorrheae.
I Treponema
The pathogens - which are termed nonculturable treponemas:
Treponema pallidum subspecies pallidum (syphilis)
Treponema pallidum subspecies pertenue (yaws)
Treponema pallidum subspecies endemicum (endemic syphilis, bejel)
Treponema careteum (pinta)
The diseases caused by these four human pathogens are collectively termed treponematoses.
The „nonpathogens” – which are termed culturable treponemas
Treponema phagedenis
Treponema refringens
genital flora
Treponema minutum
Treponema denticola
Treponema vincentii
Treponema socranskii
oral flora
Treponema scoliodentum
„Nonpathogens are part of normal flora of oral cavity, genital tract and intestinal tract. Some of them
may be associated with the gingivitis and periodontal disease.
Treponemas in AIDS patients with diarrhea: Treponema hyodysenteriae, Treponema innocens and
Treponema large treponeme.
Morphology - Treponema pallidum is a slime spirochete 5 to 15 µm long with r e g u l a r spirals of a
wavelength of 1 µm. It cannot be visualized with the usual bacteriologic stains (silver impregnation
techniques are used to demonstrate it in histologic preparations).
Antigenic structure
proteins of the outer membrane (weakly antigenic)
somatic proteins
cardiolipins – an important component of antigen which stimulate nontreponemal antibodies (reagin) it may be a product of the spirochete itself or modified component of host cells.
capable of staining Treponema pallidum by indirect IF – fluorescent treponemal antibody
immobilizing and killing live motile Treponema pallidum and fixing complement in the presence of a
suspension of Treponema pallidum or related spirochetes
reagin – is a mixture of IgM and IgA antibodies directed against some antigens widely distributed in
normal tissues
Syphilis - clinical manifestations
The primary syphilitic lesion is an ulcer (hard chancre) with regional lymphadenophaty. The median
incubation period from contact until the appearance of the primary syphilitic chancre is about 21 days.
They always heal spontaneously.
Secondary syphilis - may develop 2 to 10 weeks after the primary lesion has healed. It is characterized
by a symmetric red maculopapullar rash anywhere on the body and generalized non tender lymph
node enlargement.
Primary and secondary lesions are rich in spirochetes and highly infectious.
Latent syphilis – no lesion appear, but serologic tests are positive.
Tertiary syphilis - one third of untreated cases progress to tertiary stages. The manifestations may
appear 5 years after infection, but characteristically occur after 15 to 20 years:
granulomatous lesions (gummas) in the skin, bones and liver
degenerative changes in the central nervous system (c. n. s.)
cardiovascular lesions
In tertiary stages treponemas can occasionally be found in the eye or c. n. s.
Congenital syphilis – Treponema pallidum can transmit to the fetus through the placenta,
beginning in the 10th to 15th weeks of gestation.
Laboratory diagnosis of syphilis
a). dark-field microscopy
b). indirect immunfluorescence test
ad. a) Dark-field microscopy requires considerable skill and experience (other spirochetes than
Treponema pallidum may be present in oral and rectal lesions). This technique is practically used in
primary syphilis, to detect bacteria in syphilitic chancre.
ad. b) IF – tissue fluid or exudate is spread on a glass slide, air dried and sent to the laboratory. It is
fixed, stained with a fluorescein labeled antitreponema serum, and examined by means
immunofluorescence microscopy.
Serologic tests:
nonspecific serologic tests (nontreponemal tests to detection nonspecific antibodies - reagin) use
cardiolipin antigen – RPR, VDRL and others. This tests are used for screening and as tests of response
to treatment. False positive test results may be in other diseases.
Reagins can be detected in patients serum after 2-3 weeks of untreated syphilis and in cerebrospinal
fluid after 4-8 weeks of infection. Positive VDRL or RPR tests revert to negative in 6-18 months after
effective treatment of syphilis.
“Biologic” false positive results – other infections caused by treponemas and other infections e.g.
malaria, leprosy, measles, infectious mononucleosis. Vaccinations, collagen-vascular disease e.g.
systemic lupus erythematosus, polyarteritis nodosa, etc.
Specific serologic tests (treponemal tests to detect specific antibodies)
1. FTA-ABS is an indirect immunofluorescent test (antibodies can be detected in early syphilis
and are usually present many years after effective treatment of early syphilis. The test cannot
be used to judge the efficacy of treatment.)
2. TPHA is a hemagglutination test – the test is positive later than FTA
3. TPI - Treponema pallidum immobilization test - this test is recommended in extreme cases,
since it requires a viable source of freshly harvested treponemas and the maintenance of a
large colony of rabbits.
The most recent recommendation by the World Health Organization is to screen sera with a VDRL,
RPR, or automated reagin test and confirm positive sera with the FTA-ABS test. A Treponema
pallidum immobilization test will definitively determine equivocal reactions.
Cerebrospinal fluid should be tested to diagnose symptomatic neurosyphilis and latency, also in AIDS
Diagnosis of congenital syphilis - maternal IgG passes the placenta. At birth, the baby will be
seropositive for at least 3 to 6 months as a result of residual maternal antibodies. Some attempts have
been made to detect IgM against Treponema pallidum; in congenital infection, the child makes IgM
antitreponemal antibody. The presence of IgM ELISA (Captia - syphilis M.) in the blood of newborns
is good evidence of in utero infection.
Leptospirosis is a worldwide disease of a variety of animal species. It can be transsmitted to humans,
usually through water contamined with animal urine.
The pathogenic member of the genus Leptospira is Leptospira interrogans, it has many serogroups
(18), and serotypes (more than 170).
Leptospira interrogans
It is a slime spirochete 5 to 15 µm long, with a single axial filament, fine closely wound spirals and
hooked ends. Bacteria are not visualized with the usual bacteriological staining technique. Detection is
easy in dark-field or immunofluorescences microscopy.
Bacteria are aerobic, and can grow in special medium containing rabbit serum (e.g. Fletcher or Stuart)
and those enriched with bovine serum albumin – Tween 80. Semisolid 0,2 % agar media are
recommended. Seroidentification is accomplished by microscopic agglutination and absorption test by
agglutination tests using highly specific absorbed antisera against the various antigenic components.
The zoonotic reservoirs of the leptospirosis constitute particularly rats, cattle and dogs.
Infection occurs through small skin lesions or the conjunctiva or through upper alimentary tract
The subject to exposure are sewer workers, miners, farm workers, veterinians, and also children
playing in irrigation ditches or other bodies of water receiving farmland drainage (in North America).
Incubation period is 7 to 13 days, after these we observe influenza - like illness with fever, chills,
headache, conjunctival suffuzjon, and muscle pain. This disease is associated with bacteriemia,
bacteria may be present also in cerebrospinal fluid, but without clinical or cytologic evidence of
meningitis. First phase lasts about 7 days.
The second phase of the disease usually lasts 3 or more weeks, and many present as aseptic
meningitis resembling viral meningitis, or may be generalized; clinical manifestation depends partly
on the serogroup involved.
The most sever form of leptospirosis is Weil’s disease, caused by the Leptospira icterohaemorrhagie.
Weil’s disease with hemorrhage, hepatitis, and renal involvement has significant mortality.
Laboratory diagnosis
specimens - blood or cerebrospinal fluid during the first week and urine after
microscopic examination – dark-field and IF
culture – all cultures are incubated in the dark at 30º C for up to six weeks. Tubes are examined
weekly for macroscopic growth in the form of a ring 1-3 cm below the surface and by dark-field
animal inoculation – intraperitoneal inoculation of young hamsters or guinea pigs with fresh plasma
or urine. Within a few days, Leptospires become demonstrable in the peritoneal cavity (dark-field)
serology – antibodies begin to appear within the first week of clinical disease. Slide agglutination tests
using serotypes common in particular regions as antigen are preformed. A titer of 1 : 100 or greater is
suggestive of infection. A fourfold increase in titer is diagnostic.
Treatment - penicillin and tetracycline, but treatment is effective if is start very quickly, within the first
4 days of bacteremic phase. Later treatment is ineffective.
Borrelia recurrentis
It is large spirochete with spirals, 10-30 µm long, containing 15-20 axial flagella, Borrelia are Gramnegative bacteria, may be stained with aniline dyes, smears from blood are stained with Giemza or
Wright staining.
Borrelia recurrentis infect a range of small rodents and other mammals and is pathogenic to humans.
Relapsing fever
endemic infection - tick-borne (ticks of the genus Ornithodorus)
The epidemics are associated with overcrowding, poverty and war; it does not occur in the USA.
Laboratory diagnosis
specimen – blood taking during fever
microscopic examination - direct blood smears stained with Giemsa or Wright technique.
Borrelia burgdorferi
Large spirochete with irregular spirals, containing 7 to 11 flagella. Like others borrelias, Borrelia
burgdorferi is Gram-negative, but is most easily visualized with Giemsa, Wright or acridine orange
stains. It is micraerophilic and can grown in culture, but culture is very difficult, bacteria are fastidious
in nature and have prolong doubling time (8-24h).
Vectors: Ixodes dammini
Ixodes pacificus
Lyme Borreliosis
Infection is most common in summer months. The clinical findings have been divided into 3 stages:
The primary lesions - occurs at the site of thick bite 3 to 30 days after the feed. The lesion, known as
erythema chronicum migrans (in 85 %).
Next stage begins week to months after the resolution of the primary lesion. Some untreated patients
develop fluctuating meningitis or cardiac manifestations.
The third stage develops week to years after the onset of infection, fluctuating arthritis is observed
(large joints, particularly the knee).
Laboratory diagnosis
Specimens - blood, joint fluid, cerebrospinal fluid and skin lesions
Culture and direct microscopic observation are possible, but not usually available, (a low diagnostic
Is possible to demonstrate of Borrelia burgdorferi by amplifying Borrelia burgdorferi specific DNA
sequences in body fluids and tissue. It is rapid, very sensitive and specific.
Detection of IgG and IgM antibody by enzyme immunoassay test. The most widely used tests are the
indirect fluorescent antibody (IFA) and enzyme immunoassays (EIA or ELISA). But methods are not
well standarised. Western immunbloting procedures is sometimes performed to confirm results
obtained by other tests.
Patients with Lyme disease do not give positive VDRL and other cardiolipin antigen tests for syphilis.
Treatment - Tetracycline and Penicillin - are effective in the treatment of early Lyme disease, arthritis
often responds to large doses of penicillin. In refractory cases, ceftriaxone has been effective. Longstanding Lyme arthritis can be treated with doxicycline or amoxicillin plus probenecid for 30 days or
Prevention – attachment of the tick for 24 hours or more is necessary before there is transmission of
Borrelia burgdorferi. Protective clothing and insect repellents, careful search for nymphs after
potential exposure, and removal of the tick by its head with tweezers is only prophylaxis. No vaccine
is available.
Early diagnosis and treatment prevent the more serious complications.
Lab 9
Enterobacteriaceae I: Escherichia coli – characteristics, antigenic structure, methods of
identification Klebsiella spp., Proteus spp., Yersinia spp. and others. Inflammatory diarrhea
(determination of pathogens in fecal specimens). Urinary tract infections (UTI): pathogenesis,
microbial factors and general diagnostic approaches.
Enterobacteriaceae II: Salmonella spp. and Shigella spp. - characteristics, clinical manifestations,
and laboratory diagnosis of diseases. Treatment and prevention. Vibrio, Campylobacter and
Helicobacter - characteristics, clinical manifestations and diagnosis of infection.
The Enterobacteriaceae are large Gram-negative rods, members are free living bacteria in nature and
part of the indigenous flora of humans and animals. They are facultative anaerobes, and are
metabolically active: all ferment glucose, are oxidase-negative, and reduce nitrates to nitrites. Some of
them are motile and some encapsulated.
Growth - Enterobacteriaceae grow readily on simple media under aerobic or anaerobic conditions.
Antigenic structure:
O antigens (cell wall LPS)
K antigens (some are polysaccharides, others are proteins)
H antigens (flagellar proteins)
Serotyping system: based on O, K and H antigens. Enterobacteriaceae possess more than 150
different heat-stable somatic antigens O (lipopolysaccharide), more than 100 heat-labile K (capsular)
antigens, and more than 50 H (flagella).
Habitat - most Enterobacteriaceae are primarily inhabitants of lower gastrointestinal tract of human
and animals. They colonize also female genital tract and as transients, colonize skin. They are scant in
the upper respiratory tract of healthy persons (their number may increase in hospitalized patients).
SALMONELLA, SHIGELLA and YERSINIA species are intestinal pathogens and not constitute part
of the normal flora.
Escherichia coli
Escherichia coli is most common member of the intestinal flora and can cause opportunistic
infections. Most strains ferment lactose rapidly and produce indole.
Escherichia coli-associated diarrheal diseases
1. enterotoxigenic Escherichia coli (ETEC) – traveler’s diarrhea
2. enteropathogenic Escherichia coli (EPEC) – infant’s diarrhea
3. enteroinvasive Escherichia coli (EIEC) – disease very similar to shigellosis
4. enterohemorrhagic Escherichia coli (EHEC) – bloody diarrhea, hemolytic uremic syndrome
5. enteroaggregative Escherichia coli (EAEC) – acute and chronic diarrhea in developing countries
These Escherichia coli are classified by specific characteristic of their virulence properties, and
each group causes disease by using different mechanism.
Nearly all of these enteropathogens share the property of plasmid-mediated adherence to the
epithelium of the small and/or large bowel as first step of infection.
O, K and H antigens of Gram-negative rods may be identify by slide agglutination. In practice
this test is required for identification of diarrheal Escherichia coli. Demonstration: slide agglutination
used to identify of EPEC strains, isolated from stool samples taken from infants.
Slide agglutination test (identification of EPEC strain, isolated from diarrhoeal babies stool
Latex agglutination test (detection of E.coli K1 antigen in CFS)
Latex agglutination is used also for detection Escherichia coli O157 cell surface antigen and
Escherichia coli H7 flagella antigens characteristic for verocytotoxigenic strains (EHEC).
Urinary tract infections (UTI)
Escherichia coli strains account for more than 90 % of cystitis (bladder) and pyelonephritis (renal
pelvis and kidney) infections, known as urinary tract infection (UTI), that develop in otherwise healthy
persons. Nephropathogenic Escherichia coli typically produce α-hemolysin. Pyelonephritis is
associated with a specific type of pilus, P pilus (P fimbriae), which bind to the uroepithelial receptors
by recognizing the α-D-Gal-(1-4)β-D-Gal carbohydrate sequence of specific glycosphingolipids.
Demonstration: Orion Diagnostica PF test to detect P fimbriae in Escherichia coli isolated from UTI.
Meningitis (Escherichia coli K1)
Escherichia coli is one the most common cause of neonatal meningitis. Escherichia coli colonize the
infant via ruptured amniotic membranes or during childbirth. Fully 75 % of cases are caused by strains
possessing the K1 capsular polysaccharide.
Other opportunistic infections:
it may follow mechanical damage, such as a ruptured intestinal diverticulum or intestinal trauma, or
involve a generalized impairment of immune function. When normal host defenses are lowered
Escherichia coli may reach the bloodstream and cause sepsis. Sepsis may be a complication, occurs
secondary to urinary tract infection (urosepsis).
1. Klebsiella pneumoniae (a primary pathogen)
Klebsiella pneumoniae in sputum - important respiratory tract pathogen
urinary tract infection (UTI)
bacteremia with focal lesions in debilitated patients
nosocomial infections (also Klebsiella oxytoca)
2. Klebsiella ozenae – a fetid, progressive atrophy of mucous membranes
3. Klebsiella rhinoscleromatis - a destructive granuloma of the nose and pharynx.
Opportunistic pathogen’s
1. Enterobacter spp. – UTI, sepsis, nosocomial infections
2. Serratia marcescens and others spp. – pneumonia, bacteremia, UTI ,endocarditis, nosocomial
3. Proteus vulgaris and Proteus mirabilis – UTI, bacteremia, pneumonia, focal lesions, nosocomial
4. Morganella morgani – nosocomial pathogen
5. Providencia rettgeri (P. alcalifaciens, P. stuartii are members of the normal intestinal flora) - UTI
and bacteria occasionally - nosocomial infections.
6. Citrobacter – UTI, sepsis, nosocomial infections
Laboratory diagnosis
a. specimens – urine, blood, pus, sputum, CSF and others
b. culture – blood agar and differential media e.g. MacConkey agar
c. biochemical identification (e.g. API 20 E)
d. antibiotic sensitivity test. Demonstration of antibiogram of multiple antibiotics resistant strain of
Klebsiella pneumoniae:
disc diffusion technique
E test for ESBL detection (Extended Spectrum β-lactamases)
automatic methods e.g. Vitek Systems
Gram-negative rods (intracellular parasite) which have animals as their natural host, but they may
be etiological agent of serious disease in human.
1. Yersinia pestis – the cause of plague
Bubonic plague (fever and lymphodenitis) is transmitted to humans by infected rodent fleas.
Pneumonic plague – can be transmitted by the droplet rout during direct contact with infected
animals and humans.
2. Yersinia enterocolitica – mainly enterocolitis in infants and young children, but also:
abdominal pain suggesting appendicitis (acute mesenteric lymphadenitis). Ileitis terminalis
and in adults – erythema nodosum, reactive arthritis, septicemia and recurrent fever. Very
rarely also pneumonia and meningitis
3. Yersinia pseudotuberculosis – mainly abdominal pain suggesting appendicitis (acute
mesenteric lymphadenitis), rarely other diseases like Yersinia enterocolitica.
Diagnostic laboratory tests
1. Plague
specimens: blood and aspirate of enlarged lymph nods, sputum, CSF
smears – aspirate is staining with Giemza stain and IF
culture – blood agar and MacConkey agar
biochemical identification (e.g. API 20E)
All cultures are highly infectious and must be handled with extreme caution. Detailed
identification of Yersinia pestis is performed in a reference laboratory.
2. Yersinia enterocolitica and Yersinia pseudotuberculosis infections
specimens: stool, blood, lymph nods obtained at surgical exploration and other
culture: MacConkey agar, but they grow slowly and better grow in room temperature. If tisssue
specimen is cultured “cold enrichment” is recommended (material is placed in saline pH 7,6 and
incubated in 4º C for 2-4 weeks).
To culture Yersinia special Yersinia selective agar also is used. Growth in room temperature and
motility only in this temperature (not in 37º C) is used for identification of Yersinia spp.
serology: tube agglutination test to detection agglutinating antibodies in paired serum specimens,
taken 2 or more weeks apart.
UTI (urinary tract infection)
Community-acquired UTI
primarily in women
the typical pathogen is Escherichia coli but very often typical for woman are also coagulasenegative Staphylococci. Staphylococcus saprophiticus (the cause as much as 20 % of systemic
UTI in young, sexually active woman), and Staphylococcus epidermidis.
Nosocomial UTI
in both men and women
typical pathogen is also Escherichia coli, but other microorganisms like Klebsiella, Proteus,
Pseudomonas, Serratia, Acinetobacter, Enterococcus or fungi are also frequent.
UTI may be: - symptomatic or asymptomatic
acute or chronic
primary or recurrent
UTI: pyelonephritis, cystitis, urethritis or prostatitis.
Microbial factors:
Escherichia coli accounts for more than 90 % of acute infections in patients with structurally
normal urinary tract. Uropathogenic Escherichia coli all possess chromosomally mediated virulence
factors such as the P pilus, alpha-hemolysin, and/or siderophores such as aerobactin.
Hospitalised patients are particularly susceptible to cross-infection with nosocomial strains of
Staphylococci and Enterococci. Proteus strains appear to be particularly virulent.
In the cases of catheterized patients also yeast’s, particularly Candida spp. are important
causative agents.
Recent evidence indicates that Corynebacterium urealyticum (D2) can causes both acute and
chronic cystitis as well as pyelonephritis.
Other organisms: - Ureaplasma urealyticum (uretritis in males)
Mycoplasma pneumoniae (pyelonephritis)
Mycoplasma genitalium (may be uretritis in males)
Mycobacterium tuberculosis
Specimen collection
In patients with symptoms of UTI, urine is collected for culture at the onset of symptoms and may be
repeated 48 to 72 hours after institution of therapy.
1. Clean-voided midstream urine
2. Suprapubic aspiration (when an anaerobic infection is suspected)
3. Catheterization (when obtaining of midstream of urine is impossible)
Urine culture
1. Quantitative culture
a calibrated loop that holds 0.01 or 0.001 ml of urine can be used to streak the culture
serial 10-fold dilutions can be made and samples from the dilutions streaked
a screening procedure suitable for the physician’s involves an agar-covered „paddle” that is dipped
into the urine.
If bacteriuria is asymptomatic culturing of, two or three specimens may be necessary to confirm an
initial positive culture.
1. Significant bacteriuria has been ≥ 105 CFU/ml
2. 102 - negative
3. > 103 - 104 - result is doubtful
4. Isolation of three or more different bacteria from a clean - catch, midstream urine specimen is
mainly indicated as contamination.
Automated detection systems
Bac-T-Screen (bioMérieux Vitek)
UTI Screen System (Coral Biomedical)
VITEK System (bioMérieux Vitek)
Features of Automated Urine Screens
Colorimetric filtration:
bacteria and
leukocytes are trapped in
a filter and detected by a
residual pink color
remaining from safranin
O dye
Detection time
≤ 2 min
Reagent format
Filter card
bacterial adenosine
triphosphate (ATP) is
measured by an
bioluminescence reaction
of ATP with luciferin and
Yes, room temperature
10-45 min
Photometry: detection of
growth is based on
changes in light
VITEK system
Yes, 35° C in instrument
1-13 hr; also enumerates
and identifies common
urinary pathogens
12- × 50-mm polystyrene 57- × 91-mm sealed
tubes with 25 µL of
plastic triple-port card;
dehydrated somatic
releasing agent
25 µL
200 µL
Urine volume
1 mL
from: Howard J. B., Clinical and pathogenic Microbiology, 1994, p. 204-205
Salmonella spp.
1. Enteric fever:
typhoid fever - Salmonella typhi
typhoid-like disease - Salmonella paratyphi A, Salmonella paratyphi B (Salmonella shottmuelleri),
Salmonella paratyphi C (Salmonella hirschfeldii)
2. Enterocolitis or gastroenteritis (Salmonella typhimurium, Salmonella enteritidis, Salmonella
newport and other serotypes)
3. Bacteremia with focal lesions – commonly Salmonella choleraesuis, but may be caused by any
salmonella serotype
Laboratory diagnosis of typhoid fever
Bacteriologic methods
1. Clinical materials
a. blood - in first two weeks of the disease
b. stool (and in some cases urine) - after two weeks
Isolation of Salmonella typhi from the blood or feces confirm the diagnosis
c. bone narrow culture may be useful
d. duodenal drainage (in carriers)
2. Culture on special selective media e.g. sodium hydrogen selenite broth (SEL) and differentiating
component to presumptive identification e.g. Mac Conkey agar, Salmonella Shigella agar or
Hektoen agar
3. Biochemical identification (e.g. API 20E)
4. Serological identification – slide agglutination test
Serologic methods
5. Tube dilution agglutination test (Widal test) - most patients with typhoid fever develop
agglutinating antibodies to the O and H antigens of Salmonella typhi between the second and
fourth weeks of illness. The results are interpreted as follows:
a. high or rising titer of O ( ≥ 1 : 160) suggests that active infection is present (anti O IgM
b. high titer of H (IgG ≥ 1 ; 160) suggests past immunization or infection
c. high titer of antibodies to the Vi antigen occurs in some carriers
6. Laboratory diagnosis of gastroenterocolitis: clinical material - stool.
Laboratory diagnosis of sepsis: clinical material - blood.
On the basis of differences in O antigens and some biochemical reactions the genus is divided into
four species (or group).
1. Shigella dysenteriae (group A)
2. Shigella flexneri (group B)
3. Shigella boydii (groupC)
4. Shigella sonnei (group D)
Among these species more than 30 individual serotypes were recognized. Shigella dysenteriae type 1
known as the Shiga bacillus, is more severe and cause classic tropical bacillary dysentery.
In the USA most common are:
Shigella sonnei - in children (less than 10 years),
Shigella flexneri - in sexually active gay man.
Clinical manifestations - acute inflammatory colitis and bloody diarrhea, which is also known as most
characteristic dysentery-syndrome.
A clinical trial consisting of:
painful straining to pass stools (tenesmus)
small-volume blood, mucid discharge
(but in cases if Shigella sonnei diarrhea is watery, syndromes are often very similar to the other
bacterial or viral diarrhea).
Clinical diagnosis
1. Isolation of Shigella sp. from stool on differential and selective media
2. Biochemical identification (e.g. API 20E)
3. Serological identification of group and serotype by slide agglutination test.
Vibrio sp.
Vibrio spp. are curved, Gram-negative rods, motile (with a single polar flagellum), non spore forming,
oxidase-positive and can grow under aerobic and anaerobic conditions (better in aerobic), require
alkaline media.
Vibrio cholerae - water-loss diarrhea called cholera
Vibrio parahaemolyticus - infection from undercooked or raw seafood
Vibrio vulniticus - septicemia or wound infection (cellulitis), gastroentertis
Vibrio cholerae:
V. cholerae has O lipopolysaccharides that confer serologic specificity. There are at least 139 O
antigen groups. V. cholerae strains of O group 1 and O group 139 cause classic cholera.
serogroup O 1 contains 2 biotypes (biochemical variants): El Tor and cholerae (classical)
belong to (Ogawa, Inaba, Hikojima).
Serogroups other than O 1 and other than O 139 are causative agents of cholera-like-disease.
Epidemic and pandemic cholera is limited to group O 1 and O 139 strains including EI Tor biotype
(sometimes called Vibrio eltor). Epidemic cholera is spread primarily by contaminated water under
conditions of poor sanitation - via drinking, food preparation or bathing. In early 1993 in Bangldesh
was detected a novel vibrio serotype, that cause severe disease.
Vibrio cholerae has a low tolerance for acid, but grows under alkaline conditions (pH 8.0 to 9.5).
To produce disease, Vibrio cholerae must:
reach the small intestine in sufficient number (for colonization - approximately 1 billion of
bacteria must be ingested)
be able to penetrate the surface mucus covering of the intestinal mucosa (motility and chemotaxis)
be able to adhere to cells of the brush border of the gut by using Tcp pili - Toxin Co-regulated
after adhering, the organism multiplies and secretes an protein (choleragen) enterotoxin
Cholera is not an invasive infection, the rods do not reach the bloodstream but remain within the
intestinal tract.
Laboratory diagnosis:
isolation of Vibrio cholerae from the stool („rice-water” stools), culture on selective media e.g.
thiosulfate-citrate-bile-sucrose medium (TCBS).
Biochemical, serological (slide agglutination test with polyvalent O 1 or non O 1 antiserum)
Campylobacter sp.
Microaerophilic (require less oxygen, optimum level is 6 %), comma or S-shaped Gram-negative
motile rods with a single polar flagellum.
Campylobacter jejuni is a major causative agent of diarrhea in the USA.
Laboratory diagnosis:
The diarrheal stool is cultured on special selective blood agar supplemented with antibiotics. The
antibiotic supplements reduces the total flora without loss of Campylobacter. Media are incubated in
an atmosphere with reduced O2 (5 %) level and with increased CO2 (10 %) level. Incubation of
primary plate should be at 42-43° C at least 3 days.
Identification - Gram-stained smears show typical morphology: ability to grow at 42° C on selective
media in microaerophilic atmosphere and oxidase and catalase positivity results are usually all tests
necessary for identification of Campylobacter spp. Nitrate reduction, H2S production, hippurate tests
and antibiotic susceptibility testing required for further identification of species.
Helicobacter pylori
Microaerophilic, comma or S-shaped Gram-negative rods. Helicobacter pylori has many
characteristics in common with campylobacters. It has multiple flagella at one pole and is actively
motile. Bacteria colonize the gastric mucosa mainly region of the antrum. The consequence of
colonization may be: gastritis, duodenal (peptic) ulcer disease, and possibly gastric ulcers and
carcinoma. H. pylori is carcinogenic.
Laboratory diagnosis:
Specimens: gastric biopsy specimens for culture and blood for determination of serum antibodies.
Smears: observation of typical S-shaped Gram-negative rods in Gram-stained smears of biopsy
specimens. Smears may be stained also with Giemza stain or special silver stain.
Culture: the growth on e.g. blood agar and chocolate agar at pH of 6,0 - 7,0 in 5 % O2 and 10 % CO2
atmosphere at 37oC 4-6 days.
in microscope - morphology and motility
biochemical tests: urease, catalase and oxidase positivity,
Antibiotic sensivity test (e.g. E-test)
Serology: the serum antybodies persist even if the Helicobacter pylori infection is eradicated, and the
role of antibodies determination for diagnostic purposes of active infection is therefore limited.
PCR techniques are also used for detection of Helicobacter pylori in biopsy specimens.
CLO-test – rapid test to detect urease activity used for presumptive identification of Helicobacter
pylori in specimens. Gastric biopsy material can be placed onto a urea-containing medium with a color
indicator. If Helicobacter pylori is present, the urease rapidly splits the urea and the resulting shift pH
yields a color change in the medium.
Another rapid test for urease activity testing is recommended in vivo.
C- or
C- labeled urea is
ingested by the patients. If Helicobacter pylori is present, the urease activity generates labeled CO2
that can be detected in the patient’s exhaled breath.
Lab 10
Determination of bacterial resistance mechanisms. Pseudomonas aeruginosa, Acinetobacter spp.
and other opportunistic Gram-negative "nonfermenters".
The laboratory must detect potential microbial pathogen, identify them to species and
perform susceptibility testing. The microbiology laboratory should also monitor multidrug-resistant
organisms. Significant findings should be immediately reported to the infection control practitioner.
The laboratory works with the Infection Control Committee by:
saving all isolates
culturing possible reservoirs (patients, personnel or the environment)
performing typing of strains to establish relatedness between isolates of the same species.
Biotyping – analyzing unique biologic or biochemical characteristics.
Antibiograms – analyzing antimicrobial susceptibility pattern (hospital strains of bacteria are
very often multidrug-resistant).
Serotyping – serologic typing of bacterial or viral antigens.
Bacteriocin typing – susceptibility to bactriocin. Bacteriocin – producing strains are resistant to their
own bacteriocin; thus, these substances can be used for typing of organisms.
Bactriophage typing – ability of bacteriophages to attack certain strains of bacteria.
Genotypic or molecular methods
1. Demonstration of materials frequently investigated in patients with nosocomial infections
urine (UTI) – Escherichia coli, resistant to antibiotics, strains producing ESBL
pus (from surgical wound) – mixed infection Staphylococcus aureus (MRSA) + Enterococcus
faecalis + Psedomonas aeruginosa
blood (sepsis) - Staphylococcus epidermidis (MRCNS)
2. Demonstration of antibiograms of strains isolated from hospitalized patients
3. Film – sources of infectious agents and their spreading.
Methods that directly detect specific resistance mechanisms e.g.:
β-lactamase detection with the use of a chromogenic cephalosporinase test. This simple test detect
Staphylococcal resistance to penicillin, Neisseria gonorrhoeae resistance to penicillin,
Haemophilus influenzae resistance to ampicillin, enterococcal resistance to penicillin and
ampicillin, anaerobic bacteria resistance to penicillin
genotypic methods
Accurate and relevant testing need using a mixture of: conventional, automated, screening
methods and also molecular techniques as well as predictor drug panels.
Important examples in which more than one method is required to obtain complete and accurate
susceptibility testing data are methicillin-resistant staphylococci, vancomycin-resistant enterococci
and extended-spectrum β-lactamase- producing Gram-negative bacilli.
The Pseudomonas group
1. Pseudomonas aeruginosa
An aerobic, (obligate aerobe) not ferment glucose and oxidase-positive, motile Gram-negative
rods. Psedomonas aeruginosa is widely distributed in nature and is commonly present in moist
environments in hospitals.
it does not require enriched media for growth
it can multiply over a wide temperature range (2-42° C)
it uses oxidative energy
it form nonfluorescent blue pigment pyocyanin (typical for only Pseudomonas aeruginosa),
and yellow, fluorescent pigment - pyoverdin. Some strains may produce a rust-colored
pigment – pyorubin, or black pigment pyomelanin.
The main pathogen among Pseudomonas is Pseudomonas aeruginosa. It possesses two virulence
factors: endotoxin and exotoxin A which blocks protein synthesis by a mechanisms of action identical
to diphtheria toxin. Strains isolated from cystic fibrosis possess glycocalix which allow adherence to
mucous surfaces and protect against antibody. P. aeruginosa forms mucoid colonies, as a result of
overproduction of alginate, an exopolysaccharide.
1. in the healthy people – can cause only superficial and self limited infections e.g. mild otitis
externa in swimmers
2. in patients with serious underlying disease (immunocompromised) and extensive burns can cause
hospital infections, sepsis and others
3. in cystic fibrosis patients – can cause different complications
Laboratory diagnosis:
1. Presumptive identification - on the basis of morphology of colonies (on MacConkey agar are
colorless, form metallic and fluorescent pigment and typical fruit smell – grape-like or corn-takolike odor). Characteristic green colonies on Pyocyanosel agar.
2. Confirmatory tests - biochemical identification e.g. API NE
3. Identification for epidemiologic purposes (hospital infection)
pyocins typing
bacteriophages typing
lipopolysaccharide immunotypes
4. Antibiotic sensitivity test - hospital strains are usually resistant to antibiotics, susceptibility test
should be done for to selection of antimicrobial therapy.
Prevention – vaccine from appropriate types administered to high-risk patients provides some
protection against pseudomonas sepsis (is possible in patients with leucemia, burns, cystic fibrosis and
Other Pseudomonas:
acidovorans, Pseudomonas alcaligenes - all are opportunistic.
2. Burkholderia pseudomallei - causes primary infection in otherwise healthy persons. The disease
is mieloidosis (usually an acute pneumonia).
3. Burkholderia mallei - causes glanders in horses, transmission to human is extremely rare, (local
suppurative or acute pulmonary infection)
4. Burkholderia cepatia
hospital infections
5. Stenotrophomonas maltophilia
hospital infections
Other opportunistic nonfermentive rods:
Actinobacillus actinomycetemcomitans (is often found in actinomycosis)
Cardiobacterium hominis
Chromobacterium violaceum
Eiknella corrodens
Chryseobacterium spp.
Kingella kingae
Lab 11
Bordetella: morphology, growth requirement and diseases (diagnosis, treatment and prevention).
Legionella – bacteriologic features, and diseases (legionnaires disease and Pontiac fever)
Chlamydia – laboratory diagnosis, treatment and prevention.
Mycoplasma and Ureaplasma – clinical manifestations, laboratory diagnosis and treatment.
Ricketssia, Ehrlihia and Coxiella – clinical diseases.
Basic mycology – laboratory diagnosis of systemic mycoses.
Bordetella sp.
Bordetella pertussis is a coccobacillus strict aerobic and nonmotile. The surface exhibit pili and a
rodlike protein called the filamentous hemagglutinin (Fha), which can bind to and agglutinate
Laboratory diagnosis of whooping cough (pertussis)
isolation of Bordatella pertussis from nosopharyngeal swabs on Bordet-Gengou medium (specimens
collected early in the course of disease - during the catarrhal or early paroxysmal stage - provide the
greatest chance of successful isolation)
The swab is collected by the pernasal route and plated directly onto a special medium. Cephalosporins
are usually added to this medium in low concentration to inhibit members of the normal flora. Small
and silvery colonies appear after 3 to 5 days of incubation in 35°C
differentiation of Bordatella pertussis based on certain biochemical tests (e.g. oxidase, urease) and
motility material from the swab may be also used for the fluorescent antibody testing: smear is stained
directly with fluorescein-conjugated antipertussis serum (results should be confirmed be other
Treatment - Erythromycine (is effective in early stage of disease, in catarrhal phase).
Vaccines are produced from:
inactivated whole cell suspension (it has side effects)
partially purified preparations derived from whole cells (acellular vaccines are less effective and are
used in booster later in childhood).
Legionellae are fastidious, aerobic, Gram-negative rods. Bacteria cannot grow on common enriched
media; for growth they require iron, cystein and low pH. They stain faintly with Gram method and are
not seen in direct smears of clinical specimens. Gram method is used usually for staining of Legionella
from suspected colonies on agar medium. Basic fuchsin should be used as the counterstain (not
Legionella pneumophila is the prototype bacterium of the 34 species of Legionella (among some
species existes multiple serotypes), and is the major cause of disease in human.
Pathogenesis. Legionellae are present in warm moist environments. The organism is known to survive
as long as a year in unchlorinated tap water. Infection of immunocompromised humans commonly
follows inhalation of the rods from aerosols generated from contaminated air – conditioning systems,
shower heads and similar sources. Person-to person spread does not occur. Legionella pneumophila is
one of the agent of atypical pneumonia, usually causing a lobar, segmental or patchy pulmonary
infiltration. The severe pneumonia is called Legionnaires disease.
Legionella is a facultative intracellular pathogen, may survive and multiply within cells of the
monocyte-macrophage series. Intracellular multiplication is the key of virulence.
The incidents of clinical significant disease is higher in man over age 55 years, with high risk factor:
smoking, alcoholics
chronic bronchitis and emphysema
steroid and immunosuppresive treatment
cancer chemiotherapy
diabetes mellitus
Laboratory diagnosis
specimens – bronchial waschings, transtracheal aspirate, pleural fluid, lung biopsy specimens or
blood. In sputum bacteria are found very rarely.
smears – direct fluorescent antibody test of specimens can be diagnostic (low sensitivity compared
with culture)
culture - on special media required for isolation, e.g. buffered charcoal yeast extract (BCYE), pH 6.9 growth after 2 to 5 days (usually 3 days). Colonies are identified by direct immunfluorescence
Specific tests – demonstration of Legionella antigens in the patients urine.
Serologic tests - detection of specific antibody titer in serum in convalescent phase by the indirect
immunofluorescence assay.
Treatment: The drug of choice is Erythromycin (or Erythromycin with Rifampin). The most strains
of Legionella produce β-lactamases. Tetracyclines and the newer quinolones are also used for
Prevention is nonspecific (avoide aerosol sources in cooling tower, and in water supplies of
buildings). Preventive measures involve cleaning chlorination and heating of water sources.
Other disease caused by Legionella pneumophila is: “Pontiac fever” – fever, chills, myaglia, malaise
and headache also dizziness, photopholia, neck stiffness and confusion, mild cough and sore throat.
Legionella pneumoniae is the major causative agent of diseases (in Legionella pmeumoniae 90%).
Other species with medical importance: L. micdadei, L. gormanii, L. dumoffii, L. bozemanii and
Chlamydia trachomatis - the etiologic agents of:
inclusion conjunctivitis
infantile pneumonia
NGU - nongonococcal urethritis and cervicitis
Lymphogranuloma venerum
Chlamydia trachomatis are solely human pathogens.
Chlamydia psittaci
Ornithosis (Psittacosis) - faecal excretions of birds contain many microorganisms; they are the major
source of infection for humans and birds. Humans usually acquire ornithosis by the inhalation of dried,
infected feces. After an incubation period of 1 to 3 weeks develops pneumonia.
Chlamydia pneumoniae (strain TWAR)
this organism causes an acute lower respiratory tract infection - pneumonia and bronchitis. The
bacteria seems to be spread from person to person.
mainly on the basis of the pathologic findings
conjunctival scrappings may be included into cell cultures or the yolk sacs of chick embryos
identification of the characteristic inclusion bodies with fluorescein - labeled antibody
Inclusion conjunctivitis:
isolation of Chlamydia trachomatis
demonstration of cellular inclusion with fluorescein – labeled antibody
Genital tract infections:
Growth on the cell culture
Serologic techniques are often used for identification of these organisms. Commercially available
kits that contain fluorescently labeled polyclonal antibodies that can bind to elementary bodies.
These labeled antibodies can be used to stain smears taken from patients. An enzyme–linked
immunoassay for detection chlamydial antigens in the scrapings or swab specimens from infected
sites. Anti-chlamydial antibodies are linking to horse-radish peroxidase
In situ DNA hybridization of cervical scrapings. These last two (b and c) techniques are very
sensitive, greater than 90 %.
Lymphogranuloma venerum:
on the basis of the clinical picture and history
complement-fixation test for antibodies
biopsy of infected nodes for isolation of the organisms
Frei skin test - the killed organisms are injecting into the skin and delayed - type skin reaction is
observe (not specific and will give positive results in person with psittacosis).
isolation of the etiologic agent from the blood or sputum samples taken in acute-stage of disease:
culture is possible only by using cell cultures or embryonated eggs, and identification-with
fluorescently label specific antibody.
Treatment - tetracycline and macrolides.
Ricketssia, Orientia, Ehrlihia and Coxiella – aerobic, gram-negative bacilli mainly intracellular
They are small (0,3 x 1 to 2 µm)
Stain poorly with the Gram stain, are seen best with Giemsa or Gimenez stains
Grow only in the cytoplasm of eukaryotic cells (are strict intracellular parasites)
Are structurally similar to Gram-negative bacilli
Contain DNA and RNA
Multiply by binary fussion
Are inhibited by antibiotics
Possess an animal and arthropod reservoirs
Are transmitted by arthropod vectors (ticks, mites, lice, fleas) – except Coxiella burnetii
Ricketssia – are subdivided into two group:
1. Spotted fever group. 12 species have been associated with human disease. R.rickettsii
(Rocky Mountain spotted fever) is most common rickettsial pathogen in USA.
Culture: bacteria can be isolated in tissue culture or embryonated eggs, only in reference laboratory.
Diagnosis primarily by serology:
1). Weil-Felix test – it is insensitive and nonspecific, and it is not recommended now
2). Indirect fluorescent antibody (IFA) test. The IFA test is sensitive (94-100%) and specific (100%).
A positive result: an initial titer of 1:64 or more (1: ≥64), or the finding of a four-fold rise in the
antibody titers.
Treatment: The tetracyclines are drugs of choice; alternative antibiotics include chloramphenicol and
fluorochinolones (e.g. ciprofloxacin)
Typhus group
Ricketssia prowazekii – etiologic agent of
1). Epidemic typhus (louse – borne typhus). Humans are the primary reservoir of typhus;
person–to–person transmission is by louse vector.
2). Recrudescent disease (Brill-Zinsser disease) can occur years after the initial disease
3). Sporadic typhus (rural areas of the eastern states) – from squirrels to human by squirrel
Diagnosis – The IFA test
Treatment: The tetracyclines and chloramphenicol
Prevention: formaldehyde – inactivated typhus vaccine is available for high – risk populations.
Ricketssia typhi – etiologic agent of endemic or murine typhus
Scrub typhus group: Orientia tsutsugamushi (formerly – Rickettsia tsutsugamushi),
etiological agent for scrub typhus, mite-born disease, reservoir – mites (chiggers), wild
Treatment: Tetracyclines and chloramphenicol
Erlichia organisms
Ticks are the vectors of ehrlichiosis with exception Erlichia sennetsu (Sennetsu fever, disease
primarily restricted to Japan, associated with ingestion of raw fish)
Tick-borne ehrlichiosis (USA 1986):
1. Human monocytic ehrlichiosis (E. haffensis)
2. Human granulocytic ehrlichiosis (E. evingii, E.phagocytophila)
Diagnosis: Giemsa stain direct smears of peripheral blood – detection of intracellular organisms called
morulae is diagnostic. Culture not typically useful. Serology. Species-specific DNA probes
Treatment: Doxycycline
Coxiella burnetii – formerly Rickettsia, now recognized as more closely related to Legionella and
Francisella – etiological agent of Q fever.
Reservoirs: mammals, birds and ticks (cattle, sheep, goats, cats, dogs)
Coxiella is highly resistant to desiccation, may be live in the environment for months to years.
Route of infection – inhalation (in most cases), ingestion (consumption of contaminated unpasteurized
1. Acute disease – long incubation period average 20 days, the sudden onset of severe headache,
high fever, chills and myalgia. Diseases include influenza – like syndrome, atypical pneumonia
hepatitis, pericarditis, myocarditis, meningoencephalitis.
Chronic disease – the incubation period can be months to years. The most common presentation
of it is subacute endocarditis (on a prosthetic or previously damaged heart valve). Others –
hepatitis, pulmonary disease and infection of pregnant women.
Diagnosis: Culture – not commonly performed. Serology:
LPS with a complex carbohydrate is a phase I antigen (weakly antigenic)
Modified LPS, protein surface is a phase II antigen
Acute disease – IgM and IgG against phase II antigen.
Four-fold increase in antibody titers
An IgM titer of at least 1:50 or
An IgG titer of at least 1:200
The complement fixation titer is positive after 2 weeks (in nontreated patients) and than reverts to
negative after 12 weeks. The IgG titer is positive after 12 weeks, and persists for more than 1 year in
the most of patients (90%).
Chronic disease – antibodies are present against both Ags: to phase I antigen (the titers are typically
higher), and to phase II antigen
Nucleic acid amplification techniques
Treatment: Acute disease – tetracyclines
Chronic disease – e.g. rifampin + doxycycline or trimethoprim – sulfamethaxazole
Prevention: Phase I antigen vaccines are used in a single dose, both in animals and humans.
Basic mycology
A clinical classification of some fungal infection (table 29.4 from Clinical and phatogenic
II. Diagram of possible clinical courses in agents of systemic mycoses caused by dimorphic fungi
symptomatic pneumoniae
asymptomatic pneumoniae
progressive lung disease
chronic lung disease
endogenous reactivation
extrapulmonary dissemination
Atlas of clinical fungi ed. Hoog G. S., Guarro J. P.22
Opportunistic mycoses - predisposing factors for the development of e.g. candidiasis:
a. underlying diseases: malignancy, congenital diseases, viral infections, diabetes mellitus,
endocrinopathy, immune deficiency (e.g. in neonates), AIDS, neutropenia
b. physiological factors: pregnancy, oral contraconception, unbalanced, carbohydrate - rich diets,
c. mechanical trauma: skin damage (wounds, burns, maceration), deep trauma, surgery
d. chemical factors: indwelling catheters/prostheses, heroin addiction, iatrogenic factors
(corticosteroids, antibiotics, cytostatics, irradiation)
1. Direct microscopic examination - sputum, exudates and body fluids
a. Unstained wet preparations may be examined microscopically. In this procedure a drop of 10
% KOH is placed in the center of a clean glass slide. The material to be examined is added and
mixed carefully. A coverslip is placed over the preparation, and it is gently heated by passing
through a flame two or three times. After cooling, the slide is examined under low power.
b. If cryptococcosis is suspected, Indian Ink preparations should be made to demonstrate
capsules around yeast cells present in cerebra-spinal-fluids (CSF)
c. Immunofluorescence microscopy is used to detect Histoplasma capsulatum, Cryptococcus
neoformans, as well as several other fungi directly in tissue samples or fluids.
d. Exudates should be examined microscopically, for granules; when present, these should be
stained with Gram and PAS (periodic acid - Schiff staining technique).
2. Isolation
Primary isolation media - e.g. Sabouraud Dextrose Agar
Special media - e.g. caffeic acid agar is used to recognize Cryptococcus neoformans
3. Identification
observation of colonies morphology
microscopic examination of cell morphology
study of conidia and conidiofores
germ tubes with human serum test for Candida albicans
biochemical identification ATB fungus or tests on identification media
4. Antifungal susceptibility testing of yeast
Demonstration of:
a. ATB fungus
b. E-test - selection of MIC and Points of yeast such as Candida albicans other Candida spp. and
Cryptococcus neoformans
5. Immunology
detection of fungal antigens in culture filtrates
fluorescent antibody microscopy for fungal identification in tissue
test for antibodies
antigens delayed hypersensitivity (skin tests)
6. Molecular diagnostics
DNA probes for the recognition of the main agents of systemic mycosis. (Blastomyces dermatidis,
Coccidioides immitis, Cryptococcus neoformans and Histoplasma capsulatum are commercially
A Clinical Classification of Some Fungal Infections
Nature of
Body Sites
Skin (thick) or palms,
Keratinized tissue
(hair, nail, skin)
Black piedra
White piedra
Pityriasis versicolor
Tinea nigra
Representative Etiologic Agents
Piedraia hortae
Trichosporon beigelii
Malassezia furfur
Phaeoannellomyces werneckii*
Skin, nails
Epidermophyton floccosum,
Microsporum canis,
Trichopyton rubrum
Candida albicans
Aspergillus fumigatus, C. albicans,
Scopulariopsis brevicaulis
Scytalidium dimidiatum †
Aspergillus flavus, Bipolaris
spicifera, C. albicans,
Fusarium solani
Aspergillus niger, C. albicans
Sporothrix schenckii
Madurella mycetomatis,
Pseudallescheria boydii
Skin and lymph nodes
Skin and subcutaneous
tissue and bone (often
feet and hands)
Skin and subcutaneous
tissue (often legs)
Skin and subcutaneous
Mucosa of nose
Skin and subcutaneous
Skin and subcutaneous
Any organ system may Blastomycosis
be affected
All organs
Disseminated candidiasis
Nasal sinuses, lungs,
gastrointestinal tract
Any organ, deep tissue, Systemic fungal disease
Cladosporium carrionii, Fonecaea
pedrosoi, Phialophora verrucosa
Basidiobolus ranarum,
Conidiobolus coronatus
Rhinosporidium seeberi
Loboa loboi
Exophiala jeanselmei,
Wangiella dermatitidis
F. solani
Blastomyces dermatitidis
Coccidioides immitis
Cryptococcus neoformans
Histoplasma capsulatum
Paracoccidioides brasiliensis
C. albicans
A. fumigatus
Rhizopus arrhizus
Xylohypha bantiana ‡
Bipolaris hawaiientsis,
Penicillium marneffei,
Pseudallescheria boydii,
Torulopsis glabrata,
Trichosporon beigelii,
virtually any other fungus
* Previously classified as Exophiala werneckii.
† Previously classified as Scytalidium synanamorph of Hendersonula toruloidea.
‡ Previously classified as Cladosporium bantianum.
From: Barbara J. Howard “Clinical and Phatogenic Microbiology”
Lab 12
Laboratory diagnosis of viral and prion diseases.
Specimen selection and collection.
Specimen selection depends on:
the specific syndrome
viral etiologies suspected
time of year
Specimens for the detection of virus should be collected as early as possible following the onset of
disease. Virus may no longer be present as early as 2 days after the appearance of symptoms.
1. Throat or nasopharyngeal swab or aspirate
2. Bronchial and bronchoalveolar washing
3. Rectal swabs and stool specimens
4. Urine (10 ml of a clean voided, first-morning urine)
5. Skin and mucous membrane lesions
6. Sterile body fluids (cerebrospinal, pericardial, pleural)
7. Blood (5 to 10 ml of anticoagulated blood collected in a Vacutairer tube)
8. Bone marrow
9. Tissue specimens
10. Blood for viral serology
Specimens should be placed in ice and transported to the laboratory at once. If a delay is unavailable,
the specimen should be refrigerated. For storage up to 5 days, hold the specimen at 4° C. Storage for 6
or more days, should be at -20° C or preferably -70° C. Specimens for freezing should first be diluted
(1 : 2 to 1:5) or emulsified in viral transport medium.
Blood for viral culture, must be kept at 4° C until processing.
Blood for viral serology testing should be transported to the laboratory in the sterile tube in which it
was collected. Serum should be separated from the clot as soon as possible. Serum can be stored for
hours or days at 4° C or for weeks or months at -20° C or below before testing. Testing for virusspecific IgM should be done before freezing (IgM may form insoluble aggregats upon tchawing).
Requisition : patient identification
source of specimen
clinical history or viruses suspected
date and time of specimen collection
Processing viral specimens should occur in a biological safety cabinet. Latex gloves and a laboratory
coat should be worn during manipulation.
any specimen that might be contaminated with bacteria or fungi or any swab specimen should
be added to viral transport medium
viral transport medium or fluid specimens should be vortexed just before inoculation
blood viral culture requires special processing to isolate leukocytes, which are than inoculated
to cell culture tubes.
Detection of viruses in patient specimens –
1. cytologic or histologic examination for the presence of characteristic viral inclusions. Smears are
stained Papanicolau or Giemza technique e.g. A Tzank test to detect VZV or HSV inclusions in
cells of skin vesicle.
2. electron microscopy – esp. for the detection of viruses that do not grow readily in cell culture e.g.
viruses causing gastroenteritis and encephalitis
3. immunodiagnosis
-direct and indirect immunofluorescent antibody methods
-enzyme immunoassay (ELISA)
-radioimmunoassay (RIA)
-latex agglutination (LA)
-immunoperoxidase staining
4. molecular detection using nucleic acid probes and polymerase chain reaction assays
5. conventional cell culture
host cells grow into a monolayer on the sides of glass or plastic test tubes. Cells are in special cell
culture medium, prepared with e.g. Eagle’s minimal essentional medium or Earle’s balanced salt
solution. To each medium are added antibiotics (vancomycin, gentamycin and amfotericin), and
bovine serum
Cell lines are classified as – primary cell lines
- low-passage cell lines
- continuos cell lines
Viruses are most often detected in cell culture by the recognition of cytopathic effect (CPE).
Some viruses (influenza, parainfluenza, mumps), which produce little or no CPE, can be detected by
Lab. 13
Course review.