Lecture V, Microb

Download as pptx, pdf, or txt
Download as pptx, pdf, or txt
You are on page 1of 143

Medical Bacteriology: Specimen processing;

Medically important bacteria; Mycobacteria.


Susceptibility testing (pp. 1114-1153; 1187-1198)
GRAM STAIN

• Few would disagree that direct examination of a specimen


with Gram stain is one of the most valuable procedures
performed by the microbiology laboratory.
• The Gram stain result rapidly provides information that is
used by the clinician for selecting appropriate antimicrobial
therapy; it also helps the laboratory technologist assess the
quality of the specimen and the extent to which certain
organisms recovered in culture will be worked up.
• To prepare a smear for staining, an aliquot of the most purulent or
bloody portion of the specimen is placed on a clean microscopic slide
in a manner that provides both thick and thin areas.
• For sterile body fluids, a centrifuge may be used to concentrate the
specimen by 10 to 100 times. (We have to use the sediment to see
the microbes)
• The material on the slide is allowed to air-dry, is fixed with methanol
or gentle heat, and then is stained with Gram stain reagents (crystal
violet, Gram iodine, alcohol, and safranin).
• Organisms that have a gram-positive cell wall will resist
decolorization with methanol and will retain the purple color of the
crystal violet; organisms that have a gram-negative cell wall will be
decolorized and will stain red with safranin counterstain.
Media

• Demonstration of many bacteria on Gram stain that do not grow out


in culture may indicate unusual organisms that require more
specialized media or the inability of laboratory personnel to
recognize certain colonial types in culture, or it could suggest a false-
positive Gram stain result caused by contamination of reagents or
collection materials, such as swabs, or incorrect interpretation of
Gram stain results.
• Gram stain results could indicate the need to inoculate
additional media for a specific specimen.
• For example, finding many gram-negative coccobacilli in a
respiratory specimen could indicate the need for a chocolate
plate to recover Haemophilus spp. which would not be
recovered on a blood agar plate.
CULTURE TECHNIQUES
• Media for culture are selected to provide the optimal
conditions for growth of pathogens commonly encountered at
a particular site or in a particular type of specimen.
• Consideration is given to special growth requirements of
bacteria associated with a given type of infection or to the
necessity of selecting certain pathogenic bacteria from a mixed
population of indigenous (local) flora.
• Therefore, the media chosen may include selective and
differential media, in addition to standard enrichment agar.
• Blood-supplemented agar is a good general growth medium and
can be used to demonstrate the hemolytic action of colonies on
the red blood cells.
• Antibiotics or chemicals can be added to create a selective
medium such as colistin–nalidixic acid (CNA) agar or phenylethyl
alcohol agar, both of which are used to inhibit the growth of
gram-negative bacilli, while permitting gram-positive bacteria to
grow.
• Heating the blood to make chocolate agar and adding vitamin
supplements creates an enriched medium with available hemin (X
factor) and nicotinamide adenine dinucleotide (V factor) for the
isolation of Haemophilus spp. and other fastidious bacteria.
• Gram-negative bacilli may be separated from gram-positive bacilli
by using bile salts and dye in a medium such as MacConkey’s agar,
which additionally divides the colonies into lactose-positive and
lactose negative colonies, thus making it both selective and
differential.
• Bacterial cultures are generally incubated at 37 - 35°C and are
examined initially after 18 to 24 hours of incubation.
• For recovery of anaerobes, inoculated media should be placed into an
anaerobic environment as quickly as possible. Several types of
anaerobic culture systems are available.
• There is an anaerobic jar, in which water is added to a CO2 and
hydrogen (H2) generator package, and oxygen (O2) is catalytically
converted to water with palladium-coated alumina pellets contained
in a lid chamber.
• The blood agar- is an enriched medium in sheep whole
blood supplements the basic nutrients.
• The chocolate agar-is enriched with lysed sheep red blood cells,
which turn brown and give the medium the color for which it is
named.
• selective media are used to eliminate the large number of
inappropriate bacteria in these specimens.
1. Sodium azide-select- for gram Positive bacteria over gram Negative
bacteria
2. Bile salts- select- for gram Negative enteric bacteria, and inhibit
gram Negative mucosal and most gram Positive bacteria
3. Colistan- inhibit the growth of many gram Negative bacteria
• Examples of selective media is MacConkey agar ( contains
bile), that selects for the Enterobacteriaceae
CNA agar ( contains colistin), that select for Staphylococci and
Streptococci.
• CNA agar Base is prepared with the same formula as
Columbia Agar Base with the addition of 10 mg/L colistin and
15 mg/L nalidixic acid to inhibit the growth of gram-negative
bacteria and to support the growth of staphylococci, hemolytic
streptococci and enterococci
MEDICALLY IMPORTANT BACTERIA
GRAM-POSITIVE COCCI
Staphylococcus
Characteristics
• Staphylococci are catalase-positive and catalase negative
spherical cocci that often appear in grape-like clusters in
stained smears.
• They grow well on any peptone-containing nutrient medium
under aerobic and anaerobic conditions and may produce
hemolysis of various species of animal blood cells and yellow
or orange pigment on certain types of agar.
• Growth of staphylococci is readily detected on blood agar
plates or in various types of nutrient broth.
• A selective medium for the isolation of S. aureus is one
containing 7.5% to 10% sodium chloride (NaCl) with mannitol.
• S. aureus is differentiated from other species of staphylococci
principally by its production of coagulases, which are capable of
clotting plasma.
• Staphylococci produce coagulase in two forms. One is bound to
the cell wall, the other is as free coagulase.
• Bound coagulase acts directly on the fibrinogen which causes
clumping of the staphylococcal cells, while free coagulase acts
on the prothrombin.
• many laboratories are now utilizing polymerase chain reaction
(PCR) assays for detection of S. aureus in nasal swabs and directly
from positive blood culture bottles. Many of these assays enable
detection of methicillin-resistant S. aureus (MRSA).
Clinical Manifestations and Pathogenesis
• S. aureus may be present among the indigenous flora of the skin,
eye, upper respiratory tract, gastrointestinal tract, urethra, vagina.
• Therefore, infection may arise from an endogenous or an exogenous
source.
• Factors of importance in the development of infection due to S.
aureus include breaks in the integrity of mucosal and cutaneous
surfaces, the presence of foreign bodies or implants, prior viral
diseases, antecedent antimicrobial therapy, and underlying diseases
with defects in cellular or humoral immunity.
Infections caused by S. aureus may affect multiple organ systems.
Among the most common are those involving the skin , such as
impetigo, folliculitis, mastitis, and infection of surgical wounds. S.
aureus is among the leading causes of bacteremia in hospitalized
patients, and it may cause endocarditis, particularly in persons with
heart disease and in intravenous drug users.
S. aureus is the most common cause of spinal epidural abscess and
intracranial phlebitis, and brain abscesses, typically following trauma.
Meningitis caused by S. aureus is uncommon and generally follows
head trauma or a neurosurgical procedure.
• S. aureus is responsible for many cases of osteomyelitis, is the
most common cause of septic arthritis in prepubertal children,
and is occasionally responsible for septic arthritis in adults.
• S. aureus is an infrequent cause of community-acquired
pneumonia but a common cause of nosocomial pneumonia,
which usually follows aspiration of endogenous nasopharyngeal
organisms.
• Predisposing factors include infection with measles or influenza A
virus, cystic fibrosis, and immune deficiency. Urinary tract
infections caused by S. aureus are rare, but cases of
pyelonephritis and intrarenal and perirenal abscesses can be
found.
Several factors play a role in the virulence of S. aureus. The
polysaccharide capsule, if present, has antiphagocytic
properties.
Cell wall peptidoglycans have endotoxin-like activity,
stimulating the release of cytokines by macrophages, activation
of complement.
Protein A, an immunologically active substance in the cell wall,
has antiphagocytic properties that are based on its ability to
immunoglobulin IgG.
Other surface proteins may play an important role in the ability
of staphylococci to colonize host tissues.
The significant involvement of superantigens of S. aureus in
sepsis, endocarditis, and acute kidney injury (abscess) has been
elucidated.
S. aureus produces numerous toxins.
The exotoxin TSST-1 is responsible for toxic shock syndrome,
and enterotoxins A to E are responsible for staphylococcal food
poisoning.
The exfoliative toxins—epidermolytic toxins A and B—cause
skin erythema and separation, as seen in scalded skin
syndrome.
Various enzymes are also produced, including protease, lipase,
and hyaluronidase, all of which destroy tissue.
Scalded skin syndrome occurs in infants infected with a
strain of S. aureus producing exfoliative toxin.
The illness begins abruptly with erythema, followed in 2 to 3
days by the formation of flaccid bullae, which slough, leaving
denuded areas that eventually resolve completely.
Staphylococcal food poisoning, characterized by nausea,
vomiting, abdominal cramps, and diarrhea, occurs 1 to 6
hours after ingestion of foods contaminated with preformed
staphylococcal enterotoxin, which is usually introduced into
the food product by food handlers who prepare and/or
serve the food.
Toxic shock syndrome (TSS) is primarily the result of a superantigen-
mediated cytokine storm and M protein-mediated neutrophil
activation, resulting in the release of mediators leading to respiratory
failure, vascular leakage, and shock.
The illness is most common in women 15 to 25 years of age who use
tampons during menstruation, but it also may occur in
nonmenstruating individuals, including women in the postpartum
period, male or female patients with a surgical wound or other focal
infection, and individuals who have had a surgical procedure in the
nose or sinuses.
Toxic shock syndrome begins abruptly with fever, myalgias, vomiting,
and diarrhea, followed by hypotension, hypovolemic shock, and an
erythematous rash that frequently involves the palms and soles and
flakes in 1 to 2 weeks.
The diagnosis is clinical; isolation of S. aureus from any site is not
required.
Over the past 10 to 15 years, cases of community-acquired
infection with S. aureus that are oxacillin resistant (CA-
MRSA) have become more common.
In these isolates, a toxin referred to as leukocidin toxin,
which has rarely been associated with hospital-acquired
strains of S. aureus, has been found.
leukocidin has been shown to be responsible for
necrotizing skin and soft tissue infections and has been
infrequently demonstrated to cause a necrotizing and
occasionally fatal pneumonia.
HA-MRSA strains are usually resistant to all antibiotics
except the glycopeptides, such as vancomycin.
S. aureus bacteremia can be caused by community and
nosocomial strains of S. aureus. A recent study compared
the risk factors, morbidity, and mortality due to bacteremia
related to health care–associated MRSA.
Patients with MRSA bacteremia were likely to have diabetes
mellitus, chronic liver disease, and HIV infection.
Coagulase-negative species
• Infections caused by coagulase-negative species (CoNS) of
Staphylococcus usually occur in association with foreign bodies,
especially implanted prosthetic valves, joints, and shunts.
• Isolates of CoNS are usually considered less pathogenic than S.
aureus, although that varies among the species and strains.
• The presence of biofilms and antibiotic resistance of the species
appears to be associated with bacteremia, whereas specific
adhesion capabilities of the CNS were associated with prosthetic
joint infections.
• CoNS are one of the most commo organisms associated with
CSF infections; they are rarely involved in urinary tract
infections, pneumonia, or skin and soft tissue infections.
More than 20 species of CoNS are known, of which S.
epidermidis is the species most frequently involved in such
infections.
S. saprophyticus is an important cause of bacteriuria,
particularly among sexually active young women. S.
hemolyticus, reported to rank second in frequency to S.
epidermidis in clinical specimens, can be resistant to
vancomycin.
• More than 90% of staphylococci are resistant to penicillin due to
inducible plasmid-encoded β-lactamase.
• When β-lactamase is positive by either, isolates should be
reported as resistant to penicillin.
• Resistance to the penicillinase-resistant methicillin, oxacillin,
nafcillin- occurs in up to 80% of coagulase-negative staphylococci
and in more than 50% of isolates of hospital-acquired S. aureus.
• Clindamycin may be used to treat staphylococcal infection..
For coagulase negative staphylococci - using oxacillin.
• Several assays have been developed for rapid
detection of oxacillin resistance.
• These include nucleic acid amplification, nucleic acid
probe assays and latex agglutination assays.
Chromogenic media specific for the detection of MRSA require
overnight incubation but allow easy detection of specific-colored
colonies.
Those commercially available are MRSASelect (bioRad Laboratories,
Redmond, Wash.), CHROMagar MRSA (BD, Sparks, Md.), Brilliance
MRSA (Oxoid, Thermofisher Scientific, Lenexa, Kan.), and MRSA-ID
• Three molecular assays that can detect MRSA directly in
clinical specimens (nasal swabs, blood cultures, and other)
within 90 minutes (are GeneOhm Both Gene-OHM).
• MRSA in clinical specimens and from positive blood cultures.
• Although oxacillin-resistant staphylococci may appear to be
susceptible to cephalosporins, they should be considered
resistant to all β-lactam agents (penicillins, cephalosporins,
and carbapenems).
• Hospital-acquired strains usually are resistant to many non–β-
lactam antibiotics as well.
• Many of the CA-MRSA strains still remain susceptible to most
non–β-lactam antibiotics .
Vancomycin resistance, although rarely seen in S.
aureus, is a serious issue that laboratories need to be
aware of and should screen for.
Because the latter have not been uniformly detected in
automated systems for susceptibility testing, the Centers
for Disease Control and Prevention (CDC) recommends
that all S. aureus isolates tested on an automated
instrument should also be tested by a vancomycin
screen assay.
Streptococcus and Enterococcus
• Streptococci are catalase-negative, gram-positive,
shaped cocci, often seen in pairs or chains.
• They are facultatively anaerobic.
• Streptococci can be broadly classified according to
the hemolytic reaction on blood agar.
• Those strains that completely hemolyze the red cells
around their colonies are called β-hemolytic
Streptococci
• Important members of this group include Streptococcus pyogenes
(group A) and Streptococcus agalactiae (group B).
• Gram stain of S. pyogenes (group A streptococcus) in a specimen
from abscess material on the arm of a patient with cellulitis.
• Those gram-positive cocci in chains that produce partial hemolysis
(cause “greening” of the agar) are α-hemolytic.
• An important member of this group is S. pneumoniae. Streptococci
that do not hemolyze blood are γ-hemolytic.
• An important member of this group is Streptococcus bovis complex.
Some S. agalactiae may also be γ-hemolytic.
• Most of the remainder of the α- and γ-hemolytic streptococci are
collectively called viridans streptococci, including Streptococcus
mutans, Streptococcus sanguis, Streptococcus mitis, Streptococcus
salivarius, and Streptococcus anginosus.
Members of the genus Enterococcus
• They are facultatively anaerobic. Most enterococci are α- or γ-
hemolytic on blood agar, but some may exhibit β-hemolysis.
• The most common species are yellow motile strains of the
enterococci, usually nonpathogenic, include Enterococcus
casseliflavus and Enterococcus gallinarum.
• These latter two species are usually intrinsically vancomycin
resistant, and it is important to differentiate them.
Other genera of catalase-negative gram-positive cocci that may be
isolated from clinical specimens.
These organisms are considered to have low virulence potential
and generally are pathogenic only in the immunocompromised
host. However, some of these isolates may be confused with
viridans streptococci, in particular, and their differentiation is
important because of their lower virulence and their potential for
vancomycin resistance.
Further differentiation should be considered if a vancomycin-
resistant viridans streptococcus is thought to be clinically relevant.
One of the most common clinical manifestations of group A
streptococci is pharyngitis.
This may be accompanied by scarlet fever, a punctate exanthem
overlying diffuse erythema that usually first appears on the neck
or upper chest, becomes generalized, and then desquamates.
Skin infections caused by group A streptococcus include
cellulitis, erysipelas, and pyoderma.
Acute rheumatic fever, characterized by carditis, polyarthritis,
erythema marginatum, chorea, and subcutaneous nodules, may
occur 1 to 5 weeks after group A streptococcal pharyngitis. Acute
glomerulonephritis may develop 10 days to 3 weeks after group
A streptococcal pharyngitis or pyoderma.
Beginning in the late 1980s, serious group A streptococcal
clinical syndromes, including necrotizing fasciitis, myositis,
malignant scarlet fever, bacteremia, and toxic shock–like
syndrome, began to be seen with increasing frequency.
These have been associated with high morbidity rates and
mortality rates of up to 30% or more.
The reason for this increase is not completely understood but
appears to be related to changes in the prevalence of
organisms having an enhanced virulence potential.
S. pyogenes produces numerous virulence factors.
One of the most important is the antiphagocytic cell wall M
protein. Antibodies against the specific M protein confer lifelong
type-specific immunity; however, because more than 60 M
protein types exist, infection with a group A streptococcus
possessing a different M protein may occur.
Another important cell wall component is lipoteichoic acid,
which permits bacterial adherence to the respiratory
epithelium.
S. pyogenes also elaborates about 20 extracellular products,
including enzymes (streptolysins, hyaluronidase, streptokinase,
Streptolysin O, an antigenic, oxygen-labile enzyme, produces
subsurface hemolysis on blood agar plates; streptolysin S, a
nonantigenic, oxygen-stable enzyme, produces surface
hemolysis.
Neither streptolysin has a proven role in the pathogenesis of
human disease.
Streptokinase promotes fibrinolytic activity by converting
plasminogen to plasmin, and hyaluronidase may enhance the
spread of the organism through connective tissue.
The pathogenesis of acute rheumatic fever is not fully understood.
Certain M protein types of S. pyogenes may be rheumatogenic.
• The presence of complexes of immunoglobulin and the C3
component of complement along the sheaths of cardiac myofibers
from individuals with rheumatic carditis suggests that myocarditis
results from the production of antibodies directed against a
streptococcal cell wall M protein that cross-reacts with myocardial
tissue.
Renal damage in acute glomerulonephritis is caused by deposits of
circulating streptococcal–antistreptococcal immune complexes in the
• glomeruli and subsequent activation of complement.
• Cell-mediated reactions to an altered glomerular basement
membrane or activation of the alternate complement pathway also
may be involved.
• Isolates of S. pyogenes have been linked to toxic shock syndrome,
with a clinical picture very similar to S. aureus; streptococcal
superantigens (Sags) are thought to be responsible for the toxic
shock–like syndrome caused by strains of S. pyogenes .
The most common infections caused by Group B
streptococci (GBS) are neonatal sepsis, pneumonia, and
meningitis.
Colonization of the maternal genital tract is associated
with colonization of infants and risk of neonatal
disease, with early-onset infections occurring within
the first few days after delivery and late-onset
infections appearing after 1 week of age.
• To reduce the incidence of neonatal disease, the CDC
published specific guidelines to facilitate early
identification and treatment of women colonized with
GBS and identification and treatment of neonates at
risk for developing disease.
• All pregnant women at 35 to 37 weeks of gestation should have
vaginal/rectal specimens collected and processed for detection of
GBS.
• If urine is positive, screening of vaginal/rectal cultures may not be
necessary. Molecular tests that can rapidly identify the presence of
GBS are available and are used in some laboratories at the time of
labor and delivery.
• Group B streptococcal infections in adults include postpartum
endometritis, urinary tract infection, bacteremia, skin and soft tissue
infections, pneumonia, endocarditis, meningitis, arthritis, and
osteomyelitis.
Infections caused by S. pneumoniae include pneumonia,
meningitis (especially in infants and the elderly),
spontaneous bacteremia (in persons who do not have a
spleen), otitis, sinusitis, and spontaneous peritonitis.
S. pneumoniae is seen in the normal flora of the upper
respiratory tract of 25% to 50% of preschool children, 36%
of primary school-age children, and nearly 20% of adults,
termed carriers. Its spread is enhanced by upper
respiratory tract infections and crowding.
• Pneumonia may develop when the host immune
defenses are impaired.
• Most cases are endogenous, following aspiration
of oral secretions containing normal flora that
includes S. pneumoniae. Person-to-person
transmission during epidemics occurs by droplet
aerosols.
Vaccines designed to protect against infection by pneumococci of
many of the predominant capsular polysaccharide types are available.
The CDC recommends that infants receive the 13-valent conjugated
vaccine starting at age 2 months.
It also is recommended that adults 65 years of age and older receive
both the 13-valent conjugated vaccine and the 23-valent
polysaccharide vaccine.
• Immunosuppressed patients of any age should receive both vaccines
.
Bacterial endocarditis is the most common infection caused by viridans
streptococci; others include abscesses in the brain or liver, bacteremia, and dental
caries.
The milleri streptococci complex (S. constellatus, S. intermedius, and S.
anginosus) consists of the most common viridans streptococci responsible for
liver, spleen, and brain abscesses; they often are more susceptible to antibiotics
than other strains of viridans streptococci, although resistance to penicillin is
increasingly being recognized.
Streptococcus bovis
S. bovis bacteremia has been associated withmalignancies of the gastrointestinal
tract. S. bovis is now recognized as a complex of seven strains and/or subspecies.
Biochemical differentiation remains difficult within the species.
• Enterococci are not highly pathogenic; however, they are
a common cause of urinary tract infection in hospitalized
persons.
• They may also cause endocarditis, bacteremia, and
wound infection. Vancomycin-resistant enterococci offer
a greater potential for infection, especially in
immunocompromised patients and patients with
implanted foreign devices.
• Urinary tract infections are most common, but rare cases
of more serious infection, including bacteremia, are seen.
Laboratory Diagnosis
• Streptococci grow well on blood or chocolate agar. Blood agar is
preferred because the hemolytic properties of the organism can be
assessed.
• When culturing vaginal/rectal swabs from pregnant women for
group B streptococci, specimens should first be inoculated to a
selective broth, such as Lim (Lysine, Indole, Motility Medium)
(Formula :Dextrose, Peptone, L-Tryptophan.)
• or carrot broth, or on to selective agar, such as Granada agar, to
enrich for this organism.
Tests that may be used in the clinical microbiology laboratory to presumptively
name the β-hemolytic species of Streptococcus.
More than 99% of isolates of group A streptococcus are susceptible to
bacitracin.
All isolates of group A streptococcus and more than 99% of isolates of
Enterococcus are PYRase positive.
Pyrrolidonyl arylamidase (PYR), also known as pyrrolidonyl aminopeptidase, is
a bacterial enzyme.
Testing by rapid PYR methods.
• PYR (Pyrrolidonyl Aminopeptidase) Test is used for the
detection of pyrolidonyl arylamidase (also called pyrrolidonyl
aminopeptidase) activity in Streptococcus pyogenes (group A
strep), Enterococcus spp., some coagulase-negative
staphylococci, and some Enterobacteriaceae.
• It is also known as PYR (L-pyrrolidonyl-β-naphthylamide)
which serve as a substrate for the detection of pyrrolidonyl
peptidase.
• A nucleic acid probe (Gen-Probe, San Diego) is also available for
direct detection of group A streptococcus on throat swabs.
• Group A streptococcal antigen may be detected directly in throat
swab specimens by using commercial kits designed to generate a
rapid result.
• These tests are highly specific but, given their low sensitivity, which
varies among studies from 31% to 95% , a negative antigen test in
children should be followed by culture or probe.
• More recently, clinical guidelines recommended that negative
antigen assays in adults need not be followed up with culture.
• Serologic tests to detect streptolysin O and DNase B antibodies in
acute and convalescent serum samples are used primarily to
diagnose acute rheumatic fever and acute glomerulonephritis
following infection with group A streptococcus.
• Latex agglutination assays are available for direct detection of group
B streptococcus (as well as S. pneumoniae, some serotypes of N.
meningitidis, E. coli, and H. influenzae type b) in CSF, serum, and
urine. These assays have been shown to have sensitivities
equivalent to or lower than a Gram stain and may give false-positive
results; hence, they do not in general provide additional useful
information above that provided by the CSF Gram stain. The rapid
bacterial antigen tests are much more expensive and labor intensive
than Gram stain; most laboratories no longer offer these tests or
strictly limit their use.
There are no vancomycin-resistant streptococci, but occasionally a
• “viridans”-like isolate is reported as vancomycin resistant.
• Usually this is an enterococcus, but it could also be a member of
some more uncommon genera such as Leuconostoc or Pediococcus.
If vancomycin resistance has been demonstrated, it would be
important to differentiate the intrinsically vancomycin-resistant
Leuconostoc and Pediococcus from enterococci that have acquired
vancomycin resistance.
Bacillus
Characteristics
Members of this genus are strictly aerobic or facultatively anaerobic,
rodshaped, spore-forming, gram-positive, and catalase-positive organisms.
With the notable exception of Bacillus anthracis, they are usually motile by
means of lateral or peritrichous flagella.
Some strains stain gram-negatively and, because of their variable oxidase
reactions, can be confused with gramnegative bacilli.
The most reliable diagnostic characteristic of the genus is spore formation,
which occurs optimally and on a variety of media under aerobic conditions
at 25° to 30°C. In Gram-stained smears, endospores are detectable by the
presence of unstained defects or holes within the cell.
The spores themselves can be stained by any of several methods.
• Of the numerous species of Bacillus, B. anthracis is the only one that
is uniformly and highly pathogenic. Great care must be exercised
when handling material suspected of harboring this species. Work
should be in biological safety cabinets by gloved, gowned, masked,
and personnel; work surfaces must be disinfected with 5%
hypochlorite or 5% phenol; and all supplies, materials, and
equipment must be decontaminated.
• Because B. anthracis spores have been used as a means of
bioterrorism, cultures containing suspect B. anthracis should be
handled only by reference or public health laboratories.
• Bacillus anthracis is the only bacterium with a capsules composed of
protein.
• This capsule prevents Phagosytosis
• Humans are exposed to the spores of bacillus anthracis during direct contact
with infected animals or soil
• Human-to-human transmission never been reported.
is most common in Africa and central and southern Asia.
• The pathogenicity of B anthracis depends on two virulence factors: a poly-y-
D-glutamic acid polypeptide capsule, which protects it from phagocytosis.
• The toxin consists of three proteins: protective antigen (PA) , lethal factor
(LF) , and edema factor (EF) .
• Three forms of anthrax are recognized: cutaneous, inhalation, and intestinal.
• Inhalation anthrax
• Inhaled anthrax is the most dangerous form of this infection, but also the
rarest.
• It causes symptoms that start out like the flu. These symptoms include fever,
chest discomfort, malaise (weakness), tiredness, and dry cough.
• The signs of illness appear as early as 48 hours after the spores of the bacteria
have been inhaled.
• If the symptoms are not treated quickly, the infection can rapidly turn into a
severe infection similar to pneumonia, and Meningitis.
• Fortunately, early treatment decreases the risk of death.
Intestinal anthrax

• When a person breathe in anthrax spores, they can develop


inhalation anthrax.
• People who work in places such as wool mills, butchery, and
tanneries may breathe spores when working with infected animals
or contaminated animal products
• Inhalation anthrax starts primarily in the lymph nodes of the chest
before spreading throughout the body, ultimately causing severe
breathing problems and shock.
• Very high mortality associated with pulmonary anthrax, it is felt to be an
ideal candidate for biological terrorism and warfare.
• This fear came to United Stases in October 2001 when 22 cases of anthrax
were identified.
• This initial cases were discovered in postal workers in New Jersey and
Washington DC. Three letters were found which contained anthrax spores.
• 11 affected individuals developed cutaneous anthrax and recovered with
appropriate therapy.
• Of 9 cases of intestinal anthrax 4 died.
• 2 cases of inhalation anthrax both patient died.
• Inhalation of anthrax spores can lead to acute bronchopneumonia,
mediastinitis, and septicemia (“woolsorter’s disease”). The mortality in
recognized cases of this form of disease is nearly 100%.
• Intestinal anthrax follows the ingestion of contaminated food and is
manifested by nausea, vomiting, and diarrhea. In some cases,
gastrointestinal bleeding is followed by prostration, shock, and death.
Septicemia can occur in all three of anthrax and may lead to a fatal,
purulent meningitis.
• More recently, a fourth type of anthrax, injectional anthrax, has been
recognized in heroin addicts, resulting in severe soft tissue infections with
an increased risk of shock and higher level of mortality than is associated
with cutaneous infections
Cutaneous anthrax

• Cutaneous anthrax is much less dangerous than the inhaled form


When infection occurs on the skin develops symptoms like spider
bite. Within a day or two, develop that usually turn black in the
center.
• Usually, people with cutaneous anthrax feel only mildly ill. Early
antibiotic treatment has good result.
• If cutaneous anthrax is not treated, the bacteria may get into the
bloodstream and cause more serious symptoms and disease
• In its cutaneous form, anthrax produces a small, red, macular lesion
that progresses to a vesicle and finally to necrosis with formation of
a characteristic black eschar. Regional lymphadenopathy and
septicemia may occur.
• Mortality in untreated cases with this form of disease is
approximately 20%.
Laboratory Diagnosis
• In cases of suspected cutaneous anthrax, vesicle fluid and material
under the edge of the eschar should be collected with a swab for
smear and culture.
• For suspected inhalation anthrax, sputum should be collected for
and culture.
• Cultures of stool should be considered in the intestinal form. Smears
and cultures consisting of CSF should be requested in suspected
meningitis. In the septicemic stage, blood cultures should be
performed.
• Finding large, boxcar-shaped gram-positive cells in smears
of any of these specimens should raise suspicion for the
diagnosis.
• Fluorescent, available in some state health laboratories and
at the CDC,provide a rapid presumptive diagnosis.
• Species of Bacillus grow well on sheep blood agar. Colonies
of B. anthracis are usually flat, with an irregular margin
(“Medusa head”), appear with a ground glass surface, and
are usually nonhemolytic.
Gram-negative bacteria
• Pseudomonas aierugnosa
• Pseudomonas aeruginosa is from bacterial family
Pseudomonadaceae. The family includes only members of the genus
Pseudomonas.
• Pseudomonas aierugnosa has become increasingly recognized as an
emerging opportunistic pathogen of clinics.
• Several different epidemiological studies - as a nosocomial pathogen
The ability of Pseudomonas aeruginosa to invade tissues depends
production of extracellular enzymes and toxins that break down
physical barriers and damage host cells, as well as resistance to
phagocytosis and the host immune defenses.
• Pseudomonas aeruginosa produces other soluble protein it is a
cytotoxin or leukocidin.
• Two extracellular proteases : elastase and alkaline protease.
• Elastase has several activities that relate to virulence.
The cytotoxin is a pore-forming protein. It was originally named
leukocidin because of its effect on leukocytes.
Of the two hemolysins, one is a phospholipase and the other is a
lecithinase.
• They appear to act to break down lipids and lecithin.
• The cytotoxin and hemolysins contribute to invasion through their
cytotoxic effects on neutrophils, lymphocytes and other eukaryotic
cell.
• Elastase disrupts the respiratory epithelium and interferes with ciliary
function.
• Alkaline protease interferes with fibrin formation and lyse it.
• Together, elastase and alkaline protease destroy the ground substance
of the cornea and other supporting structures composed of fibrin and
elastin.
• Elastase and alkaline protease together are also reported to cause the
inactivation of gamma interferon (IFN) and tumor necrosis factor (TNF).
• Pseudomonas pigment is probably a determinant of virulence for
the pathogen.
• The blue pigment, pyocyanin, impairs the normal function of human
nasal cilia, disrupts the respiratory epithelium, and cause a
proinflammatory effect on phagocytes.
• A pyocyanin is produced under low-iron conditions to isolate iron
from the environment for the growth of the pathogen.
• It can play a role in invasion if it extracts iron from the host to
permit bacterial growth in a relatively iron-limited environment.
• Serious Pseudomonas infections usually occur in people in the hospital and/or with
weakened immune systems.
• Infections of the blood, pneumonia, and infections following surgery can lead to
severe illness and death in these people.
• Pseudomonas aeruginosa causes bacteremia primarily in immunocompromised
patients, with AIDS, neutropenia, diabetes , and severe burns.
• Most Pseudomonas bacteremia is acquired in hospitals and nursing homes.
• Pseudomonas accounts for about 25 % of all hospital acquired Gram-negative
bacteremia.
• Pseudomonas aeruginosa causes meningitis and brain abscesses.
• Pseudomonas aeruginosa can produce disease in any part of the gastrointestinal
tract from the oropharynx to the rectum.
MYCOBACTERIUM TUBERCULOSIS
COMPLEX
• The tubercle bacilli that make up the M. tuberculosis complex
(MTBC) include Mycobacterium tuberculosis, Mycobacterium bovis,
M. bovis Bacille Calmette-Guérin strain (BCG),
• Mycobacterium caprae, Mycobacterium pinnipedii, Mycobacterium
africanum, Mycobacterium microti, and Mycobacterium canettii.
The species that most commonly infects humans is M. tuberculosis. M. bovis,
which infects many warm-blooded animals (e.g., cattle, dogs, cats, pigs,
badgers, deer, elk, and some birds) as well as primates and humans, is
responsible for a small fraction of tuberculosis (TB) cases today.
M. bovis BCG is used for vaccine purposes in many countries outside the
United States.
It also is administered intravesically to induce immunostimulation against
bladder cancer, and in such situations rarely may cause disseminated disease.
M. africanum causes TB in humans in tropical Africa but has been reported
elsewhere, including the United States, primarily in persons who had lived in
Africa.
• According to the World Health Organization, the global burden of TB
has declined slowly over the past several years, but it remains high.
2012, there were an estimated 8.6 million (range, 8.3 million to 9.0
million) incident cases of worldwide. Of these, 0.5 million occurred in
children, 2.9 million in women, and 1.1 million in persons infected
with HIV.
An estimated 450,000 of these cases were multidrug resistant (MDR-
TB), defined as resistance to rifampin or any two of the other primary
antituberculous drugs (discussed later in the chapter).
58% of the total TB cases occurred in Southeast Asia and Western
Pacific regions, 28% in the African region, 8% in the Eastern
Mediterranean region, 4% in the European region, and 3% in the
region of the Americas. The five countries with the highest number of
cases were India, China, South Africa, Indonesia, and Pakistan. The
lowest rates were in the United States, Canada, Western Europe,
Japan, Australia, and New Zealand. An estimated 1.3 million people
died from TB in 2012, of which 320,000 were infected with HIV and
170,000 had MDR-TB.
Tubercle bacilli are transmitted primarily via inhalation of dried
residues of small infected droplets (1 to 10 µm in diameter).
Infection also may be acquired by direct inoculation of abraded skin—
an event most likely to occur when pathologists or other laboratory
personnel handle infected tissues.
In addition, M. bovis may be transmitted from cattle to humans by
drinking contaminated raw milk or by respiratory exposure to live
infected cattle or their carcasses, from humans to cattle via exposure
to urine from persons with urinary tract infections due to M. bovis,
and from cattle and wild reservoirs to cattle probably by respiratory
secretions.
The most important source of infection is an undiagnosed person with
cavitary (and sputum smear–positive) pulmonary disease.
The risk for active pulmonary disease is low after one exposure to the
organism but increases under conditions of stress or in a closed
environment where repeated exposures to the organism occur.
persons who become infected with M. tuberculosis do not develop
active disease.
The lifetime for active disease is 5% to 10% for immunocompetent
persons.
For persons infected with both MTBC and HIV, in contrast, the risk for
developing TB is 7% to 10% per year.
Increased risk
Persons at increased risk for MTBC infection are close contacts of
those with known or suspected active TB, foreign-born persons from
an area with a high incidence of active TB (e.g., Asia, Africa),
persons who visit a country with a high prevalence of active TB,
residents and employees of whose clients are at increased risk for
active TB (e.g., correctional facilities, homeless shelters), health care
workers who care for patients at increased risk for active TB, and
persons who abuse drugs or alcohol.
The usual host response to infection with MTBC is activation of the
cell-mediated immune system.
During primary (initial) infection, inhaled bacilli travel to the alveolar
spaces, where they are ingested by resident macrophages.
These macrophages are unable to kill the tubercle bacilli, which
multiply intracellularly during the first several days after infection.
Macrophages infected with mycobacteria migrate to regional
tracheobronchial lymph nodes and present sensitizing antigen(s) to
immunocompetent T cells, or they enter to blood and travel back to
the lungs and to organs such as lymph nodes, kidneys, epiphyseal
areas of the long bones, vertebral bodies, and meninges, where bacilli
continue to multiply until the cellular immune response is activated.
Immunocompetent T cells migrate from regional lymph nodes
to the site of infection in the lung.
There they release chemotactic, migration inhibitory, and
mitogenic cytokines, which stimulate recruitment of blood
derived monocytes and lymphocytes, macrophage and
lymphocyte division, and macrophage activation.
Activated macrophages produce cytokines such as interleukin-
1, interferon-γ, and tumor necrosis factor, which stimulate or
regulate other components of the immune system, properties
that help control infection.
• The cytokines and lytic enzymes released by the macrophages also
contribute to concomitant local tissue destruction.
• Over time, the activated T cell population declines and is replaced
by long-lived memory immune T cells, which protect against
reinfection with MTBC and provide some cross-protection against
infection with other mycobacteria.
The primary focus of pulmonary infection, called the Ghon lesion, is
usually subjacent to the pleura in the lower part of the upper lobes or
in the upper part of the lower lobes of one lung, corresponding to
areas of the lung that receive the greatest volume of air.
Lesions(tubercles) are well-circumscribed, 1- to 2-cm–diameter areas
of grayish consolidation with soft to necrotic centers. Similar-appearing
tubercles typically are found in the regional tracheobronchial lymph
nodes.
Over time, these lesions are replaced by hyalinized fibrous tissue and
eventually calcify.
• For optimal recovery of mycobacteria from clinical specimens,
inoculation of both a broth and a solid medium is recommended.
• because broth is more sensitive than solid media and provides more
rapid detection of mycobacterial growth.
• However, use of a broth system alone may be considered if data show
that adding a solid medium does not enhance recovery of
mycobacterial pathogens.
• Two types of solid media are used for mycobacterial culture: egg-based
and agar-based (Middlebrook 7H10, 7H11, and selective 7H11). These
media are available in screw-cap tubes and flasks; agar-based media
also are available in Petri dishes.
• Gold standard for sensitivity and specificity Use of culture increases
the number of TB cases found by 30–50% over smear alone
• – ~10 viable bacilli/ml of sputum needed for culture
• compared to at least 5000 bacilli/ml of sputum for microscopy
• • Culture used for species identification, drug susceptibility testing
(DST), and genotyping
• Culture also used to monitor patient response to treatment
• All cultures should be incubated at 37° C in an atmosphere of 5% to 10%
carbon dioxide (CO2).
• All cultures should be examined weekly for at least 6 weeks.
• The colony appearance and growth characteristics of commonly
encountered mycobacteria are outlined in.
• For laboratories with a low number of requests for mycobacterial culture,
the manually read MGIT tubes, which can be used to culture all specimen
types except blood and urine, may be the most cost-effective option.
These tubes contain modified 7H9 broth and have a fluorescent indicator
embedded in silicone in the bottom.
• Tubes are inoculated with bacteria and a mycobacterial growth
enrichment are added, and the tubes are capped and incubated at 37°C for
at least 6 weeks.
Automated Systems
• Continually monitor the media for detection of mycobacteria for 6
weeks
• Most widely used FDA-cleared automated systems rapid detection
of mycobacteria using liquid media
• – Biomerieux BacT/ALERT® 3D
• – Becton Dickinson BACTEC MGIT™
• – Thermo Scientific VersaTREK™
• • MGIT and VersaTrek are also FDA-cleared for
• susceptibility testing of MTB
MYCOBACTERIUM LEPRAE
• M. leprae is the etiologic agent of leprosy, also called Hansen’s disease.
• is unique among the mycobacteria by virtue of the fact that it has not been
cultivated.
• Useful animal models of infection are the mouse footpad model and the
nine-banded armadillo.
• M. leprae has been recognized in nearly every part of the world at some
time.
• The reported number of new cases worldwide registered each year remains
between 500,000 and 700,00.
• Persons born in Oceania were the population with the highest rate of
leprosy in the United States.
• Leprosy predominantly affects humans but is also a natural infection
of wild armadillos in Louisiana, Arkansas, and Texas, and
spontaneous cases have been described in monkeys.
• The mechanism of transmission of M. leprae is unknown, but
person-to person spread via aerosolization of organisms from the
nose of a person with active lepromatous disease , which then
contacts the nasal mucosa of another individual, is the privileged
theory.
Transmission also may occur through intact skin or via penetrating
wounds, such as thorns or the bite of an arthropod.
• Breast milk from lactating women with lepromatous
• disease contains bacilli that may be transmitted to infants, and
transplacental transmission of M. leprae is possible.
• Cases of human leprosy following contact with armadillos have been
reported.
• sporadic cases of leprosy occur in
• persons who have no known contact with human leprosy suggest that
nonhuman sources of M. leprae may exist
• Most persons effectively resist infection with M. leprae. Resistance
depends on an effective cell-mediated immune response to M.
leprae antigens, as occurs in tuberculoid leprosy.
• In persons who lack specific cell mediated immunity to these
antigens, bacilli multiply within macrophages, eventually resulting in
widely disseminated lepromatous leprosy.
• The lesions of leprosy develop after a 2- to 5-year incubation period,
varying in appearance depending on the host’s immune response.
Leprosy always involves peripheral nerves, almost always involves
the skin, and frequently involves mucous membranes.
The leprosy—lepromatous leprosy, the widespread anergic form of the
disease, and tuberculoid leprosy, the localized form— stable clinically.
• Lepromatous leprosy is characterized by cutaneous lesions ranging from
diffuse generalized skin involvement to widespread, symmetrically
distributed nodules (called lepromas) filled with organisms.
• Lesions generally involve the cooler parts of the body surface: the
anterior third of the eye, the nasal mucosa, and the superficial peripheral
nerve trunks. In advanced disease, lesions are accompanied by sensory
loss from involvement of dermal nerve fibers.
Diagnosis and Treatment
• Skin lesion biopsy. The skin is tested to look for skin conditions or
diseases. A skin biopsy can help health care provider diagnose or
rule out problems such as skin cancer or psoriasis.
• Treatment
• Several antibiotics are used to kill the bacteria that cause the
disease. These include rifampin, clofazimine, macrolides, and
minocycline. More than 1 antibiotic is often given together
• A mild, less severe form of leprosy. People with this type have only
one or a few patches of flat, pale-colored skin (paucibacillary
leprosy).
• The affected area of skin may feel numb because of nerve damage
underneath. Tuberculoid leprosy is less contagious than other forms.
Tuberculoid leprosy
A sample of the biopsy was sent for polymerase chain reaction (PCR)
for Mycobacterium leprae (M. leprae) with M. leprae-specific-
repetitive-element PCR positive, M. leprae Ag 18-kDa PCR positive
and GenoType Leprae-DR negative.
• exudate smear microscopy was negative.
• https://microbiologyinfo.com/pyr-test-principle-uses-procedure-and-r
esult-interpretation/

• https://www.sciencedirect.com/topics/medicine-and-dentistry/tuberc
uloid-leprosy
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6957169/

You might also like