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 These are confirmed by the haemophilic character of the genus that reflects a requirement for either or

both of the two factors called X and V.

Culture and identification for Haemophilus

 Direct Examination

 Perform Gram stain

 Gram staining shows: Gram negative pleomorphic thin rods or coccobacilli.

Culture

o Inoculate samples onto chocolate agar media

o Incubate at 37°C in aerobic atmosphere containing 3-5 % CO2 (i.e. candle jar) for 24-48 hours.

Colony morphology

On chocolate agar: large flat, colorless to gray or opaque colonies.

Colonies are 0.5 – 1mm circular, low convex, smooth, and pale grey and transparent.

With a characteristic “mouse nest” odor. No haemolysis or discoloration is seen.

Encapsulated strains appear more mucoid (watery) and non capsulated strains appear as compact

grayish colonies.

Note: Growth is enhanced on chocolate agar and satellitism around Staphylococcus aureus is seen on blood

agar.

Biochemical reactions for differentiation:

Confirmatory tests for X and V factor requirements as follow:

1. Inoculate a single suspected colony from chocolate agar onto Mueller Hinton agar plates.

2. Place commercially available X, V, and XV factor discs/strips on streaked plates.

3. Incubate plates at 37°C in 3-5 % CO2 atmosphere for 18-24 hours.

4. Observe growth around the discs and H. influenzae will only grow around the combined XV disc.

Note that if only X and V factor discs (without XV) are applied, place them at least 2 cm apart and H.

influenzae will grow between the two discs.

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Serological identification (serotyping) of Haemophilus influenzae

Slide agglutination test.

1) Agglutinating antisera for serotypes “a” to “f” are available commercially. Such sera contain

antibodies directed towards somatic antigens present in patient’s sera which result in agglutination.

2) Apply one drop of normal saline on a slide and make a homogenous suspension with a single

suspected colony of H. influenzae.

3) Add one drop of specific antiserum and mix thoroughly.

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Lecture Eight

Sexually transmitted diseases (STD)

Genus Neisseria

The family Neisseriaceae includes the many genera, important one is genus Neisseria. The Neisseriae are gramnegative cocci that usually occur in pairs. Neisseria gonorrhea (gonococci) and Neisseria meningitidis

(meningococci) are pathogenic for humans and typically are found associated with or inside polymorphonuclear

cells.

Other neisseriae are normal inhabitants of the human respiratory tract, rarely if ever cause disease, and occur

extracellularly.

 N. catarrhalis (Moraxella or Branhamella catarrhalis)

 N. sicca

 N. subflava

 N. lactamica

 N. mucosa

Common or general characteristics of Neisseria are:

Gram negative diplococci, kidney shape, with flat or concave opposing edge, oxidase positive, pathogenic to

human only, Ferment carbohydrates and producing acid only, pyrogenic (produce pus), Non - hemolytic, nonmotile, non-spore forming, intracellular while non-pathogenic are extracellular, needs 48h of culturing time,

they are rapidly killed by drying, sunlight and many disinfectants, the non-pathogenic species can grow on

simple media while the pathogenic species need enriched media e.g. blood and chocolate agar, Gonococci and

meningococci are closely related with 70% DNA homology and are differentiated by few laboratory tests and

specific characteristics.

Neisseria gonorrhea (gonococcus)

Reservoir: human genital tract

Transmission: Sexual contact, birth

Sensitive to drying and cold

Gonorrhea; which differs in male than in female as in male it causes urethritis associated with yellowish

mucopurulent discharge from anterior urethra and dysuria, the infection can extend to the epididymis leading to

epididymitis and if not treated it will be complicated by fibrosis and urethral stricture.

In female the first site of infection is the cervix causing cervicitis, the infection is going to spread from cervix to

the vagina causing vaginitis and presented by vaginal discharge which is mucopurulent in nature, if not treated

it will spread upward to involve fallopian tube resulting in salpingitis plus PID (pelvic inflammatory disease)

which leads to fibrosis with tube stricture leading to infertility due to tubal damage.

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Rectal involvement will lead to proctitis; the patient presenting symptoms is purulent discharge from anus with

tensmus

Eye infection (infection of the conjunctival sac); occur in new born babies during their passage through

infected birth canal causing a condition called (ophthalmia neonatorum) which is a very serious condition and if

untreated it will lead to blindness. This case could be prevented through using antimicrobial agent as silver

nitrate and erythromycin

Throat infection (Gonococcal pharyngitis)

Sometimes Gonococcal can cause systemic infection as it disseminated by the blood stream to the distant

organs (skin, bone, joints) to gain arthritis or meningitis or endocarditis or vasculitis

Laboratory diagnosis

 Specimens: Pus and secretion are taken from the urethra, cervix, rectum, conjunctiva, throat or

synovial fluid for culture and smear.

 Smears: Gram stained smears of urethral or endocervical exudates reveal many diplococci within pus

cells. In male the finding of numerous neutrophils containing gram negative diplococci in a smear of

urethral exudates permits a provisional diagnosis of gonococcal infection and indicates that the

individual should be treated. In contrast, a positive culture is needed to diagnose gonococcal infection

in female

 Culture: Immediately after collection of pus or mucous is streaked on enriched selective media such as

Thayer – Martin which is a chocolate agar + Enrichment element +Antibiotics like colistin (against G

– ve rods); vancomycin (against G + ve), Nystatin (against fungi)

 Modified Thayer - Martin: same as the above culture medium but plus trimethoprim (inhibit proteus)

It needs 5 – 7% CO2 (candle jar, CO2 generating kit); Humid atmosphere; 35 – 37 C◦, 72 incubation hours

 Biochemical test:

I. Oxidase test (N, N, N, N, tetramethyl-P-phenylenediamine) positive for all Neisseria species,

when get contact with oxidase enzyme they will gain purple color i.e. positive oxidase test.

II. Sugar fermentation test: (important in differentiation between Neisseria spp.)

Sucrose + Maltose + Glucose is (negative + positive + positive) regarding N. meningitidis, (negative + negative

+ positive) regarding N. gonorrhea and regarding M. catarrhalis all are negative

III- Nitrate reduction into nitrite it is positive, while in Moraxella catarrhalis it is negative

 Serology: It is not of great value in the diagnosis to detect antibodies against this bacterium; because

of the antigen diversity of Gonococci and there is delay in the development of these antibodies, so it

is more important to detect gonococcal antigens using elisa (EIA) or using radioimmunoassay (RIA).

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 PCR (molecular method): Detection of gonococcal nucleic acid using DNA probe which can detect

nucleic acid of the microorganism, this is the most sensitive and specific method.

Antigenic and virulence structures

Gonococci are antigenically heterogeneous both in vivo and in vitro. These antigenic changes help the

microorganism to escape from the immune system and it includes:

a- Pilli which is responsible for attachment to mucosal surfaces, also inhibit phagocytic uptake, and

antigenic (immunogenic) variation as more than 1 million variants are found

b- Porin (por) protein (protein I) that extends through the gonococcal cell membrane, each strain of

gonococcus expresses only one type of por.

c- Opacity (Opa) proteins (protein II) responsible for attachment to mucosal surfaces

d- RMP (protein III) It is associated with (Opa) protein in the formation of pores

e- Lipooligosaccharide (LOS) it has endotoxin effect and it is responsible for toxicity of gonococci

f- IgA protease it split IgA making it in non-functioning form, so IgA protease is a virulent factor that

enhances colonization of bacteria.

Treatment and prevention

More than 20% of current isolates of N. gonorrhoea are resistant to penicillin, A single intramuscular dose of

ceftriaxone is the recommended therapy for uncomplicated gonococcal infections. Intramuscular

spectinomycin is indicated in patients who are allergic to cephalosporines. Many patients with gonorrhea have

coexisting Chlamydia infections; therefore Doxycycline is effective against Chlamydia.

Prevention of gonorrhea involves evaluation and management of sexual contacts, plus antibiotic

prophylactically in an exposed individual even in the absence of symptoms. The use of barrier methods is also

a preventive measure against gonorrhea as is the case for all sexually transmitted infections. No vaccine is

available for gonorrhea.

Neisseria meningitidis (meningococcus)

N. meningitidis is one of the most frequent causes of meningitis. Infection with N. meningitidis can also take the

form of a fulminant meningococcemia, with intravascular coagulation, circulatory collapse, and potentially fatal

shock, but without meningitis.

In each case, symptoms can occur with extremely rapid onset and great intensity. Outbreaks of meningitis, most

common in winter and early spring, are favored by close contact between individuals, such as occurs in schools,

institutions, and military barracks. N. meningitidis tends to strike young, previously well individuals and can

progress over a matter of hours to death.

It is gram-negative, kidney coffee bean-shaped diplococci with large antigenic capsule, grows on chocolate (not

blood) agar in 5% C02 atmosphere and ferments maltose in contrast to gonococci

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It reservoir is human nasopharynx (5-10% carriers) and its transmission is through respiratory droplets; as it is

an oropharyngeal colonizer, so it spreads to the meninges via the bloodstream and disease occurs in only small

percentage of colonized individuals.

Antigenic and virulence structures

a- Capsule: At least 13 serogroups of meningococci have been identified by immunologic specificity and

capsular polysaccharides the most important serogroups associated with disease in human are

A,B,C,Y, W135 ,they are associated with fulminant sepsis with or without meningitis, which is used

for the preparation of vaccines

b- Outer membrane protein (OMP): about 20 antigenic types, used for serotyping

c- Pilli and outer membrane proteins important in ability to colonize and invasion

d- IgA protease it cleaves IgA and allows oropharynx colonization

e- Opa protein for attachment

f- Lipooligopolysaccharide (Endotoxin) responsible for fever, septic shock in meningococcemia.

Epidemiology

Transmission occurs through inhalation of respiratory droplets from a carrier or a patient in the early stages of

the disease. In addition to contact with a carrier

Risk factors for disease include:

1- Recent viral or Mycoplasma upper respiratory tract infection

2- Active or passive smoking

3- Complement deficiency (C5-C8)

In susceptible persons, pathogenic strains may invade the bloodstream and cause systemic illness after an

incubation period of 2 to 10 days. An incidence peak among adolescents and young adults led the Centers for

Disease Control (CDC) to recently recommend vaccination of this at risk group. Humans are the only natural

host

Diseases and pathogenicity

The source of the infection is either the patient or the carriers, the route of entry is through the nasopharynx by

droplet, and meningococci will attach to the epithelial cell by pili.

From the nasopharynx the meningococci will spread through the blood stream to the target organ causing

bacteremia, so the squeal is either meningitis or suffers from fulminating meningococcemia.

Meningitis: mainly in adult ages 11 to 55 years, it is usually begun suddenly with headache, vomiting and stiff

neck, this will progress to coma within few hours. Fulminating meningococcemia is more sever with high fever

and hemorrhagic rash; there may be disseminated intravascular coagulation and circulatory collapse (waterhouse- friderichsen syndrome).

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Laboratory diagnosis

a- Specimens: Blood, CSF, Nasopharyngeal swab are taken for carrier surveys

b- Smears as gram stained smear of CSF show typical Neisseria within polymorphonuclear leukocytes

c- Culture onto chocolate agar and Thayer - Martin media, incubated at 37 ºC

d- Serology as rapid detection test to measure antigens or antibodies to meningococcal polysaccharides

through latex agglutination test, to identify N. meningitidis capsular antigens in CSF, or by

Immunoelectrophoresis

e- PCR which is the most sensitive and specific method

Treatment

Bacterial meningitis is a medical emergency. Accordingly, antibiotic treatment cannot await a definitive

bacteriologic diagnosis.

Penicillin G is the drug of choice for treating meningococcal disease. In patient allergic to penicillin

chloramphenicol and cefotaxime (or ceftriaxone) can be used.

Prevention

a- Irradiation of the carrier states (major source)

b- Isolation of the patient

c- Chemoprophylaxis for contact people

d- Vaccination with a conjugate meningococcal vaccine which was approved in the United States in 2005

for use in adolescents and adults ages 11 to 55 years, and has replaced the Unconjugated

polysaccharide vaccine; this is a tetravalent vaccine that contains capsular polysaccharides from

serogroups A, C, W-135, and Y conjugated to diphtheria toxoid

Moraxella catarrhalis

Previously they are called Neisseria catarrhalis, the name changed to Branhamella and now they are a

separated genus “Moraxella”. They are normally found in upper respiratory tract especially among school

children as (50% of school children carry this microorganism), it may cause pneumonia, otitis media, sinusitis

and other infections.

It is nonmotile, gram-negative coccobacilli that are generally found in pairs. Moraxella are aerobic, oxidase

positive, non-fastidious organisms, can grow on nutrient agar, and does not ferment carbohydrates.

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Lecture Nine

Legionella

Legionella was first recovered from the blood of a soldier more than 50 years ago, but its importance as a

human pathogen was not recognized until 1976, when a mysterious epidemic of pneumonia struck members of

the Pennsylvania American Legion.

The disease was named Legionnaire's disease by the press. Within 6 months a bacterium, subsequently named

Legionella pneumophila, had been isolated and definitively established as the agent. A general term for disease

produced by Legionella species is legionellosis.

The most common presentation of Legionella pneumophila is acute pneumonia (legionellosis); potentially any

species of Legionella may cause the disease.

Extra pulmonary disease (e.g., pericarditis and endocarditis) is rare. Less often, disease presents as a

nonpneumonic epidemic, influenza like illness called Pontiac fever.

Structure, Classification, and Antigenic Types

Legionella species are Gram-negative bacilli. There are currently 39 species and 60 distinct antigenic types of

Legionella.

Legionella cells are thin, somewhat pleomorphic Gram-negative bacilli that measure 2 to 20 μm. Long,

filamentous forms may develop, particularly after growth on the surface of agar. Ultra structurally, Legionella

has the inner and outer membranes typical of Gram-negative bacteria. It possesses pili (fimbriae), and most

species are motile by means of a single polar flagellum.

Pathogenesis

Legionella bacilli reside in surface and drinking water and are usually transmitted to humans in aerosols. The

bacteria multiply intracellularly in alveolar macrophages.

Recruited neutrophils and monocytes, as well as bacterial enzymes, produce destructive alveolar inflammation.

Direct inoculation of surgical wounds by contaminated tap water has been described.

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Figure shows smear from the lung of a patient fatally infected with L pneumophila serogroup1,

demonstrating many thin, Gram-negative bacilli (arrows). These bacteria stain less intensely with safranin

than do enteric bacilli

Host Defenses

Nonspecific physical and inflammatory pulmonary defenses are important, but cell-mediated immunity is

critical. Immunologically activated monocytes and macrophages restrict intracellular bacterial growth. The role

of humoral immunity is unclear.

Epidemiology

Legionella species are widespread in nature. Interactions with other environmental organisms may facilitate

growth. Disease may be sporadic or epidemic and may occur in the community or in hospitals. People with

compromised host defenses are at increased risk.

The clinical manifestations of Legionella infections are primarily respiratory. The most common presentation

is acute pneumonia, which varies in severity from mild illness that does not require hospitalization (walking

pneumonia) to fatal multilobar pneumonia.

Typically, patients have high, unremitting fever and cough but do not produce much sputum. Extra

pulmonary symptoms, such as headache, confusion, muscle aches, and gastrointestinal disturbances, are

common. Most patients respond promptly to appropriate antimicrobial therapy, but convalescence is often

prolonged (lasting many weeks or even months).

Laboratory Diagnosis

There are no reliable distinguishing clinical features of Legionella pneumonia, so the diagnosis must come from

the laboratory.

Some clinical features suggest legionnaire's disease; however, and should prompt the selection of appropriate

laboratory tests. The diagnosis is confirmed in the laboratory by culture, demonstration of bacterial antigen in

body fluids, or a serologic response.

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The preferred diagnostic method is culturing, because it is both sensitive and specific; however, appropriate

specimens are not always available.

The laboratory must be alerted to the possibility of legionellosis, because specially designed media must be

used. The medium of choice is buffered charcoal-yeast extract - α-ketoglutarate medium.

This medium contains yeast extract, iron, L-cysteine, and α-ketoglutarate for bacterial growth; activated

charcoal to inactivate toxic peroxides that develop in the media; and buffer with a pH 6.9, the optimum for

growth of Legionella organisms.

Addition of albumin to the media may further facilitate growth of species other than L. pneumophila. For

contaminated specimens such as sputum, antibiotics should be added. Morphologically distinctive bacterial

colonies can usually be detected within 3 to 5 days and identified presumptively as Legionella species if the

isolated bacteria depend on cysteine for growth. The identification can be confirmed by specific immunologic

typing of the isolated bacteria or, in problematic cases, by molecular analysis.

Direct detection of bacterial antigen in clinical specimens is potentially much faster than culturing.

Unfortunately, direct immunofluorescence detection (DFA) of Legionella antigen in respiratory specimens is

neither sensitive nor specific enough to warrant general use. A commercially available radioimmunoassay for

bacterial antigen in urine is satisfactory, but is available only for serogroup 1 of L. pneumophila.

Serologic diagnosis is moderately sensitive and reasonably specific. It should be considered as an adjunct to

diagnosis by culture. Indirect immunofluorescence has been used most frequently.

It is important to use an assay that detects IgM and IgG. The advantages of serologic diagnosis are that it is

performed on easily obtained blood specimens and can detect mild or even asymptomatic infection.

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Lecture Ten

Bordetella

Bordetella pertussis and Bordetella parapertussis is the human pathogens of this genus. The former causes the disease

pertussis (also known as whooping cough), and the latter causes a mild pertussis-like illness. Whooping cough is a highly

contagious disease and a significant cause of morbidity and mortality worldwide (51 million cases and 600,000 deaths

each year). Members of the genus Bordetella are aerobic. They are small, encapsulated coccobacilli that grow singly or

in pairs. They can be serotyped on the basis of cell-surface molecules including adhesins and fimbriae.

Epidemiology

The major mode of transmission of Bordetella is via droplets spread by coughing, but the organism survives only briefly

outside the human respiratory tract. The incidence of whooping cough among different age groups can vary substantially,

depending on whether active immunization of young children is widespread in the community. In the absence of an

immunization program, disease is most common among young children (ages 1 to 5 years). Adolescent and adult

household members, whose pertussis immunity has disappeared, are an important reservoir of pertussis for young

children.

Pathogenesis

B. pertussis binds to ciliated epithelium in the upper respiratory tract. There, the bacteria produce a variety of toxins and

other virulence factors that interfere with ciliary activity, eventually causing death of these cells.

Clinical significance

The incubation period for pertussis generally ranges from 1 to 3 weeks. The disease can be divided into two phases:

catarrhal and paroxysmal.

1. Catarrhal phase: This phase begins with relatively nonspecific symptoms, such as rhinorrhea, mild conjunctival

infection (hyperemia, or bloodshot conjunctivae), malaise, and/or mild fever, and then progresses to include a dry,

nonproductive cough. Patients in this phase of disease are highly contagious.

2. Paroxysmal phase: With worsening of the cough, the paroxysmal phase begins. The term “whooping cough” derives

from the paroxysms of coughing followed by a “whoop” as the patient inspires rapidly. Large amounts of mucus may be

produced. Paroxysms may cause cyanosis and/or end with vomiting. Pertussis typically causes leukocytosis that can be

quite striking as the total white blood cell count sometimes exceeds 50,000 cells/μl (normal range = 4,500–11,000 white

blood cells/μl), with a striking predominance of lymphocytes. Following the paroxysmal phase, convalescence requires at

least an additional 3 to 4 weeks. During this period, secondary complications, such as infections (for example, otitis

media and pneumonia) and central nervous system (CNS) dysfunction (for example, encephalopathy or seizures), may

occur. Disease is generally most severe in infants.

Laboratory identification

Presumptive diagnosis may be made on clinical grounds once the paroxysmal phase of classic pertussis begins. Pertussis

may be suspected in an individual who has onset of catarrhal symptoms within 1 to 3 weeks of exposure to a diagnosed

case of pertussis.

Culture of B. pertussis on Bordet-Gengou or Regan-Lowe media (selective and enrichment media) from the nasopharynx

of a symptomatic patient supports the diagnosis. The organism produces pin- point colonies in 3 to 6 days on selective

agar medium (for example, one that contains blood and charcoal), which serves to absorb and/or neutralize inhibitory

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substances and is supplemented with antibiotics to inhibit growth of normal flora. More rapid diagnosis may be

accomplished using a direct fluorescent antibody test to detect B. pertussis in smears of nasopharyngeal specimens.

Serologic tests for antibodies to B. pertussis are primarily useful for epidemiologic surveys.

Treatment

Erythromycin is the drug of choice for infections with B. pertussis, both as chemotherapy (where it reduces both the

duration and severity of disease) and as chemoprophylaxis for household contacts. For erythromycin treatment failures,

trimethoprim-sulfamethoxazole is an alternative choice. Patients are most contagious during the catarrhal stage and

during the first 2 weeks after onset of coughing. Treatment of the infected individuals during this period limits the spread

of infection among household contacts.

Prevention

Pertussis vaccine is available and has had a significant effect on lowering the incidence of whooping cough. It contains

proteins purified from B. pertussis and is formulated in combination with diphtheria and tetanus toxoids. To protect

infants who are at greatest risk of life-threatening B. pertussis disease, immunization is generally initiated when the

infant is 2 months old. Until the middle of the first decade of the 21st century. However, because neither disease- nor

vaccine-induced immunity is durable, there has been resurgence, with reported cases in 2010 the highest since the

1950’s. A new vaccine, licensed for adolescents and adults, and vaccination of women even during the last trimester of

pregnancy.

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Lecture 11

(Part-one)

Anaerobic Bacteriology

Infections caused by anaerobic bacteria are common. The infections are often polymicrobial; that is, the anaerobic

bacteria are found in mixed infections with other anaerobes, facultative anaerobes

Anaerobic bacteria are found throughout the human body; on the skin, on mucosal surfaces, and in high concentrations

in the mouth and gastrointestinal tract, as part of the normal microbiota

Infection results when anaerobes and other bacteria of the normal microbiota contaminate normally sterile body sites.

Several important diseases are caused by anaerobic Clostridium species from the environment or from normal flora:

botulism, tetanus, gas gangrene, food poisoning, and

Pseudomembranous colitis

Aerobic bacteria: Bacteria that require oxygen as a terminal electron acceptor and will not grow under anaerobic

conditions (i.e., in the absence of O2). Some Bacillus species and Mycobacterium tuberculosis are obligate aerobes (i.e.,

they must have oxygen to survive).

Anaerobic bacteria: Bacteria that do not use oxygen for growth and metabolism but obtain their energy from

fermentation reactions. A functional definition of anaerobes is that they require reduced oxygen tension for growth and

fail to grow on the surface of

solid medium in 10% CO2 in ambient air. Bacteroides and Clostridium species are examples of anaerobes.

Facultative anaerobes: Bacteria that can grow either oxidative, using oxygen as a terminal electron acceptor, or

anaerobically, using fermentation reactions to obtain energy. Such bacteria are common pathogens. Streptococcus

species and the Enterobacteriaceae (e.g., Escherichia coli) are among the many facultative anaerobes that cause disease.

Often, bacteria that are facultative anaerobes are called “aerobes.”

Anaerobic bacteria do not grow in the presence of oxygen and are killed by oxygen or toxic oxygen radicals.

Aerobes and facultative anaerobes often have the metabolic systems listed below, but anaerobic bacteria

frequently do not.

1. Cytochrome systems for the metabolism of O2

2. Superoxide dismutase (SOD), which catalyzes the following reaction:

O2−

+O2−

+ 2H+ → H2O2 +O2

3. Catalase, which catalyzes the following reaction:

2H2O2 → 2H2O + O2 (gas bubbles)

Anaerobic bacteria do not have cytochrome systems for oxygen metabolism. Less fastidious anaerobes may

have low levels of SOD and may or may not have catalase. Most bacteria of the Bacteroides fragilis group have

small amounts of both catalase and SOD. There appear to be multiple mechanisms for oxygen toxicity.

Presumably, when anaerobes have SOD or catalase (or both), they are able to negate the toxic effects of oxygen

radicals and hydrogen peroxide and thus tolerate oxygen. Obligate anaerobes usually lack SOD and catalase

and are susceptible to the lethal effects of oxygen; such strict obligate anaerobes are infrequently isolated from

human infections, and most anaerobic infections of humans are caused by “moderately obligate anaerobes.”

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Facultative anaerobes grow as well or better under anaerobic conditions than they do under aerobic

conditions. Bacteria that are facultative anaerobes are often termed aerobes. When a facultative anaerobe such

as E coli is present at the site of an infection (e.g., abdominal abscess), it can rapidly consume all available

oxygen and change to anaerobic metabolism, producing an anaerobic environment, and thus allow the

anaerobic bacteria that are present to grow and produce disease.

Gram-Negative Anaerobes

A. Gram-Negative Bacilli

1. Bacteroides: The Bacteroides species are very important anaerobes that cause human infection. They are a

large group of bile-resistant, non–spore-forming, slender gram negative rods that may appear as coccobacilli.

Many species previously included in the genus Bacteroides have been reclassified into the genus Prevotella or

the genus Porphyromonas. Those species retained in the Bacteroides genus are members of the B fragilis

group. Bacteroides species are normal inhabitants of the bowel and other sites. Normal stools contain 1011 B

fragilis organisms per gram (compared with 108

/g for facultative anaerobes).

Other commonly isolated members of the B fragilis group include Bacteroides ovatus, Bacteroides distasonis,

Bacteroides vulgatus, and Bacteroides thetaiotaomicron. Bacteroides species are most often implicated in intraabdominal

infections, usually under circumstances of disruption of the intestinal wall as occurs in perforations related to

surgery or trauma, acute appendicitis, and diverticulitis.

These infections are often polymicrobial. Both B fragilis and B thetaiotaomicron

are implicated in serious intrapelvic infections such as pelvic inflammatory disease and ovarian abscesses.

B fragilis group species are the most common species recovered in some series of anaerobic bacteremia, and

these organisms are associated with a very high mortality rate. B fragilis is capable of elaborating numerous

virulence factors, which contribute to its pathogenicity and mortality in the host.

2. Prevotella—Prevotella species are gram-negative bacilli and may appear as slender rods or coccobacilli.

Most commonly isolated are P melaninogenica, Prevotella bivia, and Prevotella disiens. P melaninogenica and

similar species are

found in infections associated with the upper respiratory tract. P bivia and P disiens occur in the female genital

tract.

Prevotella species are found in brain and lung abscesses, in empyema, and in pelvic inflammatory disease and

tubo-ovarian abscesses. In these infections, the Prevotella are often associated with other anaerobic organisms

that are part of the normal microbiota; particularly Peptostreptococcus, anaerobic Gram-positive rods, and

Fusobacterium species as well as Gram-positive and Gram-negative facultative anaerobes that are part of the

normal microbiota.

3. Porphyromonas: The Porphyromonas species also are Gram-negative bacilli that are part of the normal oral

microbiota and occur at other anatomic sites as well. Porphyromonas species can be cultured from gingival and

periapical tooth infections and, more commonly, breast, axillary, perianal, and male genital infections.

4. Fusobacteria: There are approximately 13 Fusobacterium species, but most human infections are caused by

Fusobacterium necrophorum and Fusobacterium nucleatum. Both species differ in morphology and habitat as

well as the range of associated infections. F necrophorum is a very pleomorphic; long rod with round ends and

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tends to make bizarre forms. It is not a component of the healthy oral cavity. F necrophorum is quite virulent,

causing severe infections of the head and neck.

Bacterial Vaginosis

Bacterial vaginosis is a common vaginal condition of women of reproductive age. It is associated with

premature rupture of membranes and preterm labor and birth. Bacterial vaginosis has a complex microbiology;

one organism, Gardnerella vaginalis, has been most specifically associated with the disease process.

G vaginalis is a serologically distinct organism isolated from the normal female genitourinary tract and also

associated with vaginosis, so named because inflammatory cells are not present. In wet smears, this

“nonspecific” vaginitis, or bacterial vaginosis, yields “clue cells,” which are vaginal epithelial cells covered

with many Gram-variable bacilli (pleomorphic), and there is an absence of other common causes of vaginitis

such as trichomonads or yeasts. Vaginal discharge often has a distinct “fishy” odor and contains many

anaerobes in addition to G vaginalis. The pH of the vaginal secretions is greater than 4.5 (normal pH is <4.5).

The vaginosis attributed to this organism is suppressed by metronidazole, suggesting an association with

anaerobes. Oral metronidazole is generally curative.

Gram-Positive Anaerobes

A. Gram-Positive Bacilli

1. Actinomyces: The Actinomyces group includes several species that cause actinomycosis, of which

Actinomyces israelii and Actinomyces gerencseriae are the ones most commonly encountered. Several new,

recently described species that are not associated with actinomycosis have been associated with infections of

the groin, urogenital area, breast, and axilla and postoperative infections of the mandible, eye, and head and

neck.

On Gram stain, they vary considerably in length; they may be short and club shaped or long, thin, beaded

filaments. They may be branched or unbranched. Because they often grow slowly, prolonged incubation of the

culture may be necessary before laboratory confirmation of the clinical diagnosis of actinomycosis can be

made. Some strains produce colonies on agar that resemble molar teeth.

Figure shows colony of Actinomyces

Species after 72 hours growth on brain–heart infusion agar, which

usually yields colonies about 2 mm in diameter; they are often termed

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“Molar tooth” colonies

Some Actinomyces species are oxygen tolerant (aerotolerant) and grow in the presence of air; these strains may

be confused with Corynebacterium species. Actinomycosis is a chronic suppurative and granulomatous

infection that produces pyogenic lesions with interconnecting sinus tracts that contain granules composed of

microcolonies of the bacteria embedded in tissue elements.

Figure shows granule of Actinomyces species in tissue with Brown and

Breen stain. Filaments of the branching

bacilli are visible at the periphery of the granule. Such granules

are commonly called “sulfur granules” because of their unstained

gross yellow color

Infection is initiated by trauma that introduces these endogenous bacteria into the mucosa. The organisms grow

in an anaerobic niche, induce a mixed inflammatory response, and spread with the formation of sinuses, which

contain the granules and may drain to the surface.

The infection causes swelling and may spread to neighboring organs, including the bones. Based on the site of

involvement, the three common forms are cervicofacial, thoracic, and abdominal actinomycosis. Cervicofacial

disease presents as a swollen, erythematosus process in the jaw area (known as “lumpy jaw”). With

progression, the mass becomes fluctuant, producing draining fistulas. The disease will extend to contiguous

tissue, bone, and lymph nodes of the head and neck. The symptoms of thoracic actinomycosis resemble those of

a subacute pulmonary infection and include a mild fever, cough, and purulent sputum. Eventually, lung tissue is

destroyed, sinus tracts may erupt through to the chest wall, and invasion of the ribs may occur.

Abdominal actinomycosis often follows a ruptured appendix or an ulcer. In the peritoneal cavity, the pathology

is the same, but any of several organs may be involved. Genital actinomycosis is a rare occurrence in women

that results from colonization of an intrauterine device with subsequent invasion.

Diagnosis can be made by examining pus from draining sinuses, sputum, or specimens of tissue for the

presence of sulfur granules. The granules are hard, lobulated, and composed of tissue and bacterial filaments,

which are club shaped at the periphery. Specimens should be cultured anaerobically on appropriate media.

Treatment requires prolonged administration of penicillin (6-12 months). Clindamycin or erythromycin is

effective in penicillin-allergic patients. Surgical excision and drainage may be required.

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2. Propionibacterium species are members of the normal microbiota of the skin, oral cavity, large intestine,

conjunctiva, and external ear canal. Their metabolic products include propionic acid, from which the genus

name derives. On Gram stain, they are highly pleomorphic; showing curved, clubbed, or pointed ends; long

forms with beaded uneven staining; and occasionally coccoid or spherical forms. Propionibacterium acnes,

often considered an opportunistic pathogen, causes the disease acne vulgaris and is associated with a variety of

inflammatory conditions.

It causes acne by producing lipases that split free fatty acids off from skin lipids. These fatty acids can produce

tissue inflammation that contributes to acne formation.

P acnes is frequently a cause of postsurgical wound infections, particularly those that involve insertion of

devices, such as prosthetic joint infections, particularly of the shoulder, central nervous system shunt infections,

osteomyelitis, endocarditis,

and endophthalmitis.

3. Clostridia

(In lecture of spore-forming gram-positive Bacilli: Bacillus and Clostridium Species)

B. Gram-Positive Cocci

The group of anaerobic gram-positive cocci has undergone significant taxonomic expansion. Many species

within the genus Peptostreptococcus have been reassigned to new genera such as Anaerococcus, Finegoldia,

and Peptoniphilus. The species contained within these genera, as well as Peptococcus niger, are important

members or the normal microbiota of the skin, oral cavity, upper respiratory tract, gastrointestinal tract, and

female genitourinary system. The members of this group are opportunistic pathogens and are most frequently

found in mixed infections particularly from specimens that have not been carefully procured. However, these

organisms have been associated with serious infections such as brain abscesses, pleuropulmonary infections,

necrotizing fasciitis, and other deep skin and soft tissue infections, intra-abdominal infections, and infections of

the female genital tract.

The polymicrobial anaerobic infections

Most anaerobic infections are associated with contamination of tissue by normal microbiota of the mucosa of

the mouth, pharynx, gastrointestinal tract, or genital tract. Typically, multiple species (five or six species or

more when standard culture conditions are used) are found, including both anaerobes and facultative anaerobes.

Oropharyngeal, pleuropulmonary, abdominal, and female pelvic infections associated with contamination by

normal mucosal microbiota have a relatively equal distribution of anaerobes and facultative anaerobes as

causative agents; about 25% have anaerobes alone, about 25% have facultative anaerobes alone, and about

50% have both anaerobes and facultative anaerobes. Aerobic bacteria may also be present, but obligate aerobes

are much less common than anaerobes and facultative anaerobes.

Diagnosis of anaerobic infections

Clinical signs suggesting possible infection with anaerobes include the following:

1. Foul-smelling discharge (caused by short-chain fatty-acid products of anaerobic metabolism)

2. Infection in proximity to a mucosal surface (anaerobes are part of the normal microbiota)

3. Gas in tissues (production of CO2 and H2)

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4. Negative aerobic culture results

Diagnosis of anaerobic infection is made by anaerobic culture of properly obtained and transported specimens.

Anaerobes grow most readily on complex media such as trypticase soy agar base, blood agar, brucella agar,

brain–heart infusion agar, and each highly supplemented (e.g., with hemin, vitamin K1, blood). A selective

complex medium containing kanamycin is used in parallel. Kanamycin (similar to all aminoglycosides) does

not inhibit the growth of obligate anaerobes; thus, it permits them to proliferate without being overshadowed by

rapidly growing facultative anaerobes. Cultures are incubated at 35-37°C in an anaerobic atmosphere

containing CO2. Colony morphology, pigmentation, and fluorescence are helpful in identifying anaerobes.

Biochemical activities and production of short-chain fatty acids as measured by gas liquid chromatography are

used for laboratory confirmation.

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Lecture 11

(Part –Two)

Spore-forming gram-positive Bacilli: Bacillus and Clostridium Species

These bacilli are ubiquitous, and because they form spores, they can survive in the environment for many years.

Bacillus species are aerobes and the Clostridium species are anaerobes.

Figure shows the vegetative cells with spores

Many species of Bacillus and related genera, most do not cause disease and are not well characterized. There

are a few species, however, that cause important diseases in humans. Anthrax, a classical disease in the history

of microbiology, is caused by Bacillus anthracis.

Anthrax remains an important disease of animals and occasionally of humans. Because of its potent toxins, B

anthracis is a major potential agent of bioterrorism and biologic warfare.

Bacillus cereus and Bacillus thuringiensis cause food poisoning and occasionally eye or other localized

infections.

The genus Clostridium is extremely heterogeneous and more than 200 species have been described.

Clostridia cause several important toxin mediated diseases, including tetanus (Clostridium tetani), botulism

(Clostridium botulinum), and gas gangrene (Clostridium perfringens), and antibiotic-associated diarrhea and

pseudomembranous colitis (Clostridium difficile)

Bacillus species

The genus Bacillus includes large aerobic, gram-positive rods occurring in chains. The members of this genus

are closely related but differ both phenotypically and in terms of pathogenesis.

Pathogenic species possess virulence plasmids. Most members of this genus are saprophytic organisms

prevalent in soil, water, and air, and on vegetation (e.g., Bacillus subtilis).

Some are insect pathogens, such as B thuringiensis. This organism is also capable of causing disease in

humans. B cereus can grow in foods and cause food poisoning by producing either an enterotoxin (diarrhea) or

an emetic toxin (vomiting). Both B cereus and B thuringiensis may occasionally produce disease in

8

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immunocompromised humans (e.g., meningitis, endocarditis, endophthalmitis, conjunctivitis, or acute

gastroenteritis). B anthracis, which causes anthrax, is the principal pathogen of the genus.

The typical cells, measuring 3–4 μm, have square ends and are arranged in long chains; spores are located in

the center of the bacilli

Colonies of B anthracis are round and have a “cut glass” appearance in transmitted light. Hemolysis is

uncommon with B anthracis but common with B cereus and the saprophytic bacilli. Gelatin is liquefied, and

growth in gelatin stabs resembles an inverted fir tree.

Anthrax is primarily a disease of herbivores—goats, sheep, cattle, horses, and so on; other animals (e.g., rats)

are relatively resistant to the infection.

Humans become infected incidentally by contact with infected animals or their products. In animals, the portal

of entry is the mouth and the gastrointestinal tract. Spores from contaminated soil find easy access when

ingested with spiny or irritating vegetation. In humans, the infection is usually acquired by the entry of spores

through injured skin (cutaneous anthrax) or rarely the mucous membranes (gastrointestinal anthrax) or by

inhalation of spores into the lung (inhalation anthrax). A fourth category of the disease, injection anthrax, has

caused outbreaks among persons who inject heroin that has been contaminated with anthrax spores. The spores

germinate in the tissue at the site of entry, and growth of the vegetative organisms results in formation of a

gelatinous edema and congestion. Bacilli spread via lymphatics to the bloodstream, and they multiply freely in

the blood and tissues shortly before and after the animal’s death.

Anthrax toxins are made up of three proteins, protective antigen (PA), edema factor (EF), and lethal factor

(LF). PA is a protein that binds to specific cell receptors, and after proteolytic activation, it forms a membrane

channel that mediates entry of EF and LF into the cell. EF is an adenylate cyclase; with PA, it forms a toxin

known as edema toxin. Edema toxin is responsible for cell and tissue edema. LF plus PA form lethal toxin,

which is a major virulence factor and cause of death in infected animals and humans.

When injected into laboratory animals (e.g., rats), the lethal toxin can quickly kill the animals by impairing both

innate and adaptive immunity, allowing organism proliferation and cell death.

In inhalation anthrax (woolsorters’ disease), the spores from the dust of wool, hair, or hides are inhaled;

phagocytosed in the lungs; and transported by the lymphatic drainage to the mediastinal lymph nodes, where

germination occurs. This is followed by toxin production and the development of hemorrhagic mediastinitis and

sepsis.

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Figure shows Bacillus anthracis in broth culture

In humans, approximately 95% of cases are cutaneous anthrax, and 5% are inhalation. Gastrointestinal anthrax

is very rare; it has been reported from Africa, Asia, and the United States when people have eaten meat from

infected animals. The bioterrorism events in the fall of 2001 resulted in 22 cases of anthrax 11 inhalation and

11 cutaneous. Five of the patients with inhalation anthrax died.

Cutaneous anthrax generally occurs on exposed surfaces of the arms or hands followed in frequency by the face

and neck. A pruritic papule develops 1–7 days after entry of the organisms or spores through a scratch. Initially,

it resembles an insect bite. The papule rapidly changes into a vesicle or small ring of vesicles that coalesce, and

a necrotic ulcer develops. The lesions typically are 1–3 cm in diameter and have a characteristic central black

eschar. Marked edema occurs. Lymphangitis, lymphadenopathy, and systemic signs and symptoms of fever,

malaise, and headache may occur.

After 7–10 days, the eschar is fully developed. Eventually, it dries, loosens, and separates; healing is by

granulation and leaves a scar. It may take many weeks for the lesion to heal and the edema to subside.

Antibiotic therapy does not appear to change the natural progression of the disease but prevents dissemination.

In as many as 20% of patients, cutaneous anthrax can lead to sepsis, the consequences of systemic infection

including meningitis and death.

The incubation period in inhalation anthrax may be as long as 6 weeks. The early clinical manifestations are

associated with marked hemorrhagic necrosis and edema of the mediastinum. Substernal pain may be

prominent, and there is pronounced mediastinal widening visible on chest radiographs. Hemorrhagic pleural

effusions follow involvement of the pleura; cough is secondary to the effects on the trachea.

Sepsis occurs, and there may be hematogenous spread to the gastrointestinal tract, causing bowel ulceration, or

to the meninges, causing hemorrhagic meningitis. The fatality rate in inhalation anthrax is high in the setting of

known exposure; it is higher when the diagnosis is not initially suspected.