ПЕНЗЕНСКИЙ ГОСУДАРСТВЕННЫЙ УНИВЕРСИТЕТ

МЕДИЦИНСКИЙ ИНСТИТУТ
КАФЕДРА «МИКРОБИОЛОГИИ, ЭПИДЕМИОЛОГИИ И
ИНФЕКЦИОННЫХ БОЛЕЗНЕЙ»

А.С. Есаулов
Н.А. Правосудова
Л.В. Мельников

ЧАСТНАЯ БАКТЕРИОЛОГИЯ

Учебноe пособие

ПЕНЗА, 2017

1
 PENZA STATE UNIVERSITY
MEDICAL INSTITUTE
DEPARTMENT OF MICROBIOLOGY, EPIDEMIOLOGY AND
INFECTIOUS DISEASES

A.S. Esaulov
N.A. Pravosudova
L.V. Melnikov

SPECIAL BACTERIOLOGY

Tutorial

PENZA, 2017

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 УДК 579

Рецензенты:

доктор медицинских наук, профессор,

заведующая кафедрой инфекционных болезней
ГБОУ ДПО «Пензенский институт усовершенствования врачей»
И.П. Баранова

кандидат биологических наук, доцент кафедры

«Биотехнологии и техносферная безопасность»
Пензенского Государственного Технологического Университета
А.А. Кузьмин

Издание составлено в соответствии с программой для студентов

медицинских вузов «Частная бактериология» составлено в соответствии с
программой для студентов 2 – 3 курса по микробиологии, вирусологии.
Пособие посвящено вопросам частной микробиологии. Рассмотрены
свойства возбудителей бактериальных заболеваний человека, молекулярно-
биологические и микробиологические аспекты патогенности и вирулентности
бактерий, их токсины, формы и виды инфекции. Приведены современные
методы диагностики бактериальных инфекций.
Руководство рекомендовано для самостоятельной подготовки студентов
к практическим занятиям.

УДК 579

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Contents
ENTEROBACTERIACEAE..................................................................................... 5
VIBRIO, CAMPYLOBACTER, AND HELICOBACTER ................................... 18
PURULENT-SEPTIC INFECTION ....................................................................... 28
CLOSTRIDIUM...................................................................................................... 46
AIRBORNE BACTERIAL INFECTION ............................................................... 52
SEXUALLY TRANSMITTED DISEASES ........................................................... 65
ZOONOTIC DISEASES ......................................................................................... 71

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 ENTEROBACTERIACEAE

The Enterobacteriaceae are ubiquitous organisms, found worldwide in soil, water,

and vegetation and are part of the normal intestinal flora of most animals, including
humans. These bacteria cause a variety of human diseases, including one third of all
bacteremias, more than 70% of urinary tract infections (UTIs), and many intestinal
infections. Some organisms (e.g., Salmonellaserotype Typhi, Shigella species,
Yersinia pestis) are always associated with human disease, whereas others (e.g.,
Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis) are members of the
normal commensal flora that can cause opportunistic infections. A third group of
Enterobacteriaceae exists—those normally commensal organisms that become
pathogenic when they acquire virulence genes present on plasmids, bacteriophages,
or pathogenicity islands. Infections with the Enterobacteriaceae can originate from
an animal reservoir (e.g., most Salmonella species, Yersinia species), from a human
carrier (e.g.,Shigella species, Salmonella serotype Typhi), or through the
endogenous spread of organisms (e.g., spread of E. coli from the intestine to the
peritoneal cavity following perforation of the intestine).
Morphology and structure
The Enterobacteriaceae are among the largest bacteria, measuring 2 to 4 µm in
length and 0.4 to 0.6 µm in width, with parallel sides and rounded ends. Forms range
from large coccobacilli to elongated, filamentous rods. The organisms do not form
spores or demonstrate acid fastness.

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Gram stain of Escherichia coli.

The cell wall, cell membrane, and internal structures are morphologically similar for
all Enterobacteriaceae. Components of the cell wall and surface, which are antigenic,
have been extensively studied in some genera and form the basis of systems dividing
species into serotypes. The outer membrane lipopolysaccharide (LPS) is called the
O antigen. Its antigenic specificity is determined by the composition of the sugars
that form the long terminal polysaccharide side chains linked to the core
polysaccharide and lipid A. Cell surface polysaccharides may form a well-defined
capsule or an amorphous slime layer and are termed the K antigen (from the Danish
Kapsel, capsule). Motile strains have protein peritrichous flagella, which extend well
beyond the cell wall and are called the H antigen. Many of the Enterobacteriaceae
have surface pili, which are antigenic proteins but not yet part of any formal typing
scheme.

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Antigenic structure of Enterobacteriaceae.

Most Enterobacteriaceae are motile, with the exception of some common genera
(e.g., Klebsiella, Shigella, Yersinia). The motile strains are surrounded with flagella
(peritrichous). Many Enterobacteriaceae also possess fimbriae (also referred to as
pili), which have been subdivided into two general classes: chromosomally mediated
common fimbriae and sex pili that are encoded on conjugative plasmids. The
common fimbriae are important for the ability of bacteria to adhere to specific host
cell receptors, whereas the sex or conjugative pili facilitate genetic transfer between
bacteria.

Growth and metabolism

Enterobacteriaceae grow readily on simple media, often with only a single carbon
energy source. Growth is rapid under both aerobic and anaerobic conditions,
producing 2- to 5- µm colonies on agar media and diffuse turbidity in broth after 12
to 18 hours of incubation. All Enterobacteriaceae ferment glucose, reduce nitrates to
nitrites, and are oxidase negative.
Classification
Genus and species designations are based on phenotypic characteristics, such as
patterns of carbohydrate fermentation, and amino acid breakdown. The O, K, and H
antigens are used to further divide some species into multiple serotypes. These types
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are expressed with letter and number of the specific antigen, such as Escherichia
coli O157:H7, the cause of numerous food-borne outbreaks. These antigenic
designations have been established only for the most important species and are
limited to the structures at hand. For example, many species lack capsules and/or
flagella. In recent years, DNA and RNA homology data have been used to validate
these relationships and establish new ones. The genera containing the species most
virulent for humans are Escherichia, Shigella, Salmonella, Klebsiella, and Yersinia.
Other less common medically important genera are Enterobacter, Serratia, Proteus,
Morganella, and Providencia.
Pathogenesis and immunity
Numerous virulence factors have been identified in the members of the family
Enterobacteriaceae. Some are common to all genera, and others are unique to
specific virulent strains.
Common Virulence Factors Associated with Enterobacteriaceae
• Endotoxin
• Capsule
• Antigenic phase variation
• Type III secretion systems
• Sequestration of growth factors
• Resistance to serum killing
• Antimicrobial resistance

ESCHERICHIA
Escherichia coli is the most common and important member of the genus
Escherichia. This organism is associated with a variety of diseases, including
gastroenteritis and extraintestinal infections, such as UTIs, meningitis, and sepsis. A
multitude of strains are capable of causing disease, with some serotypes associated
with greater virulence (e.g., E. coli O157 is the most common cause of hemorrhagic
colitis and hemolytic uremic syndrome).
E. coli possesses a broad range of virulence factors. In addition to the general factors
possessed by all members of the family Enterobacteriaceae, Escherichia strains
possess specialized virulence factors that can be placed into two general categories:
adhesins and exotoxins.
Epidemiology
Large numbers of E. coli are present in the gastrointestinal tract. Although these
organisms can be opportunistic pathogens when the intestines are perforated and the
bacteria enter the peritoneal cavity, most E. coli that cause gastrointestinal and
extraintestinal disease do so because they have acquired specific virulence factors

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encoded on plasmids or in bacteriophage DNA. The effectiveness of E. coli as a
pathogen is illustrated by the fact the bacteria are (1) the most common gram-
negative rods isolated from patients with sepsis; (2) responsible for causing more
than 80% of all community-acquired UTIs, as well as many hospital-acquired
infections; and (3) a prominent cause of gastroenteritis. Most infections (with the
exception of neonatal meningitis and gastroenteritis) are endogenous; that is, the E.
coli that are part of the patient’s normal microbial flora are able to establish infection
when the patient’s defenses are compromised (e.g., through trauma or immune
suppression).

Incidence of Enterobacteriaceae associated with bacteremia.

Clinical Diseases
Gastroenteritis
The strains of E. coli that cause gastroenteritis are subdivided into five major groups:
enterotoxigenic (ETEC), enteropathogenic (EPEC), enteroaggregative (EAEC),
enterohemorrhagic (EHEC), and enteroinvasive E. coli (EIEC). The first three
groups primarily cause a secretory diarrhea involving the small intestine, while the
last two groups primarily involve the large intestine.

Escherichia coli in diarrheal disease (Bloch, 2006)

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Extraintestinal Infections
Urinary Tract Infection
Most gram-negative rods that produce UTIs originate in the colon, contaminate the
urethra, ascend into the bladder, and may migrate to the kidney or prostate. Although
most strains of E. coli can produce UTIs, disease is more common with certain
specific serogroups. These bacteria are particularly virulent because of their ability
to produce adhesins that bind to cells lining the bladder and upper urinary tract
(preventing the elimination of the bacteria in voided urine) and hemolysin HlyA that
lyses erythrocytes and other cell types (leading to cytokine release and stimulation
of an inflammatory response).
Neonatal Meningitis
E. coli and group B streptococci cause the majority of CNS infections in infants
younger than 1 month. Approximately 75% of the E. coli strains possess the K1
capsular antigen. This serogroup is also commonly present in the gastrointestinal
tracts of pregnant women and newborn infants. However, the reason this serogroup
has a predilection for causing disease in newborns is not understood.
Septicemia
Typically, septicemia caused by gram-negative rods, such as E. coli, originates from
infections in the urinary or gastrointestinal tract (e.g., intestinal leakage leading to
an intraabdominal infection). The mortality associated with E. coli septicemia is high
for patients in whom immunity is compromised or the primary infection is in the
abdomen or CNS.

SHIGELLA
The commonly used taxonomic classification of Shigella is simple, although
technically incorrect. Four species consisting of almost 50 O-antigen–based

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serogroups have been described: S. dysenteriae, Shigella flexneri, Shigella boydii,
and Shigella sonnei. However, analysis of DNA has determined that these four
species are actually biogroups within the species E. coli. Because it would be
confusing to refer to these bacteria as E. coli, their historical names have been
retained.
Pathogenesis
Infection is initiated by ingestion of Shigella (usually via fecal-oral contamination).
An early symptom, diarrhea (possibly elicited by enterotoxins and/or cytotoxin),
may occur as the organisms pass through the small intestine. The hallmarks of
shigellosis are bacterial invasion of the colonic epithelium and inflammatory colitis.
These are interdependent processes amplified by local release of cytokines and by
the infiltration of inflammatory elements. Colitis in the rectosigmoid mucosa, with
concomitant malabsorption, results in the characteristic sign of bacillary dysentery:
scanty, unformed stools tinged with blood and mucus.
Epidemiology
Humans are the only reservoir for Shigella. It is estimated that almost 450000 cases
of Shigella infections occur each year in the United States. This figure pales in
comparison with the estimated 150 million cases that occur annually worldwide. S.
sonnei is responsible for almost 85% of the developed countries infection, while S.
flexneri predominates in developing countries. Epidemics of S. dysenteriae, a
particularly virulent species, occur in Africa and Central America with case fatality
rates of 5% to 15%.
Shigellosis is primarily a pediatric disease with 60% of all infections in children
younger than 10 years. Endemic disease in adults is common in male homosexuals
and in household contacts of infected children. Epidemic outbreaks of disease occur
in daycare centers, nurseries, and custodial institutions. Shigellosis istransmitted
person-to-person by the fecal-oral route, primarily by people with contaminated
hands and less commonly in water or food. Because as few as 100 to 200 bacteria
can establish disease, shigellosis spreads rapidly in communities where sanitary
standards and the level of personal hygiene are low.
Clinical Diseases
Shigellosis is characterized by abdominal cramps, diarrhea, fever, and bloody stools.
The clinical signs and symptoms of the disease appear 1 to 3 days after the bacteria
are ingested. Shigellae initially colonize the small intestine and begin to multiply
within the first 12 hours. The first sign of infection (profuse, watery diarrhea without
histologic evidence of mucosal invasion) is mediated by an enterotoxin. However,
the cardinal feature of shigellosis is lower abdominal cramps and tenesmus (straining
to defecate), with abundant pus and blood in the stool. It results from invasion of the

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colonic mucosa by the bacteria. Abundant neutrophils, erythrocytes, and mucus are
found in the stool. Infection is generally self-limited, although antibiotic treatment
is recommended to reduce the risk of secondary spread to family members and other
contacts. Asymptomatic colonization of the organism in the colon develops in a
small number of patients and represents a persistent reservoir for infection.

YERSINIA
The best known human pathogens within the genus Yersinia are Y. pestis,
Yersinia enterocolitica, and Yersinia pseudotuberculosis. Y. pestis is a highly
virulent pathogen that causes the highly fatal systemic disease known as plague;
Y. enterocolitica and Y. pseudotuberculosis are primarily enteric pathogens that are
relatively uncommon and rarely cultured from blood.
Pathogenesis and Immunity
A common characteristic of the pathogenic Yersinia species is their ability to resist
phagocytic killing. The invasive process and its effect on the host cell are driven by
a large array of virulence factors that are deployed under complex genetic and
environmental regulation.
Some of the virulence factors produced by Yersinia are regulated in a system in
which expression responds to either temperature or free calcium (Ca 2+)
concentration. The physiologic temperature in a mammalian host is different from
that in an insect or the environment, and the intracellular calcium concentration is
markedly different from that of extracellular fluids. By sensing the environment,
Yersinia is able to express or suppress virulence factors at different stages of the
pathogenic process.
The biological outcome of this extraordinary multifactorial process is the enhanced
capacity of the pathogenic Yersinia to enter, replicate and to delay the cellular
immune response. This leads to the formation of microabscesses and destruction of
the cytoarchitecture of Peyer’s patches and the mesenteric lymph nodes. The
systemic symptoms seen with dissemination can largely be attributed to the effects
of endotoxin.
Epidemiology
All Yersinia infections are zoonotic, with humans the accidental hosts.
Y. enterocolitica is a common cause of enterocolitis in Scandinavian and other
northern European countries and in the colder areas of North America. Most studies
show that infections are more common during the cold months. Virulence with this
organism is associated with specific serogroups. The most common serogroups
found in Europe, Africa, Japan, and Canada are O3 and O9. Y. pseudotuberculosis
is a relatively uncommon cause of human disease.

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Clinical Diseases
Approximately two thirds of all Y. enterocolitica infections are enterocolitis, as the
name implies. The gastroenteritis is typically associated with ingestion of
contaminated food products or water. After an incubation period of 1 to 10 days
(average, 4 to 6 days), the patient experiences disease characterized by diarrhea,
fever, and abdominal pain that last as long as 1 to 2 weeks. A chronic form of the
disease can also develop and persist for months. Disease involves the terminal ileum
and, if the mesenteric lymph nodes become enlarged, can mimic acute appendicitis.
Y. enterocolitica infection is most common in children, with pseudoappendicitis
posing a particular problem in this age group. Y. pseudotuberculosis can also
produce an enteric disease with the same clinical features. Other manifestations seen
in adults are septicemia, arthritis, intraabdominal abscess, hepatitis, and
osteomyelitis.
In 1987, Y. enterocolitica was first reported to cause blood transfusion–related
bacteremia and endotoxic shock. Because Yersinia organisms can grow at 4°C, this
organism can multiply to high concentrations in nutritionally rich blood products
that are stored in a refrigerator.

SALMONELLA
The taxonomic classification of the genus Salmonella is problematic. DNA
homology studies have revealed that most clinically significant isolates belong to
the species Salmonella enterica. More than 2500 unique serotypes have been
described for this single species; however, these serotypes are commonly listed as
individual species (e.g., Salmonella typhi, Salmonella choleraesuis, Salmonella
typhimurium, Salmonella enteritidis). These designations are incorrect—for
example, the correct nomenclature is Salmonella enterica, serovar. Typhi. In an
effort to prevent confusion and still retain the historical terms, individual serotypes
are now commonly written with the serotype name capitalized and not in italics. For
example, Salmonella enterica, serovar. Typhi is commonly designated as
Salmonella Typhi.

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An electron micrograph of Salmonella Typhi.

Pathogenesis and Immunity

After ingestion and passage through the stomach, salmonellae attach to the mucosa
of the small intestine and invade into the M (microfold) cells located in Peyer
patches, as well as into enterocytes. The bacteria remain in endocytic vacuoles,
where they replicate. The bacteria can also be transported across the cytoplasm and
released into the blood or lymphatic circulation. The inflammatory response
confines the infection to the gastrointestinal tract.
Epidemiology
Salmonella can colonize virtually all animals, including poultry, reptiles, livestock,
rodents, domestic animals, birds, and humans. Animal-to-animal spread and the use
of Salmonella-contaminated animal feeds maintain an animal reservoir. Serotypes
such as Salmonella Typhi and Salmonella Paratyphi are highly adapted to
humans and do not cause disease in nonhuman hosts. Other Salmonella serotypes
(e.g., Salmonella Choleraesuis) are adapted to animals and, when they infect
humans, can cause severe disease. In addition, in contrast with other Salmonella
serotypes, strains that are highly adapted to humans (i.e., Salmonella Typhi,
Salmonella Paratyphi) can survive in the gallbladder and establish chronic carriage.
Finally, many Salmonella strains have no host specificity and cause disease in both
human and nonhuman hosts.
Most infections result from the ingestion of contaminated food products and, in
children, from direct fecal-oral spread. The incidence of disease is greatest in
children younger than 5 years and adults older than 60 years who are infected during
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the summer and autumn months, when contaminated foods are consumed at outdoor
social gatherings. The most common sources of human infections are poultry, eggs,
dairy products, and foods prepared on contaminated work surfaces (e.g., cutting
boards where uncooked poultry was prepared). More than 1.4 million infections and
600 deaths occur each year. Salmonella Typhi infections occur when food or water
contaminated by infected food handlers is ingested. There is no animal reservoir. 21
million Salmonella Typhi infections and 200,000 deaths occur each year worldwide.
The risk of disease is highest in children living in poverty in a developing country.
The infectious dose for Salmonella Typhi infections is low, so person-to-person
spread is common. In contrast, a large inoculum (e.g., 106 to 108 bacteria) is required
for symptomatic disease to develop with most other Salmonella serotypes. The
organisms can multiply to this high density if contaminated food products are
improperly stored (e.g., left at room temperature). The infectious dose is lower for
people at high risk for disease because of age, immunosuppression or underlying
disease (leukemia, lymphoma, sickle cell disease), or reduced gastric acidity.
Clinical Diseases
The following four forms of Salmonella infection exist: gastroenteritis, septicemia,
enteric fever, and asymptomatic colonization.
Gastroenteritis
Gastroenteritis is the most common form of salmonellosis in the United States.
Symptoms generally appear 6 to 48 hours after the consumption of contaminated
food or water, with the initial presentation consisting of nausea, vomiting, and
nonbloody diarrhea. Fever, abdominal cramps, myalgias, and headache are also
common. Colonic involvement can be demonstrated in the acute form of the disease.
Symptoms can persist from 2 to 7 days before spontaneous resolution.
Septicemia
All Salmonella species can cause bacteremia, although infections with
Salmonella Typhi, Salmonella Paratyphi, and Salmonella Choleraesuis more
commonly lead to a bacteremic phase. The risk for Salmonella bacteremia is higher
in pediatric and geriatric patients and in immunocompromised patients (HIV
infections, sickle-cell disease, congenital immunodeficiencies). The clinical
presentation of Salmonella bacteremia is like that of other gram-negative
bacteremias; however, localized suppurative infections (e.g., osteomyelitis,
endocarditis, arthritis) can occur in as many as 10% of patients.
Enteric Fever
Salmonella Typhi produces a febrile illness called typhoid fever. A milder form of
this disease, referred to as paratyphoid fever, is produced by Salmonella Paratyphi
A, Salmonella Schottmuelleri (formerly Salmonella Paratyphi B), and

15
Salmonella Hirschfeldii (formerly Salmonella Paratyphi C). Other Salmonella
serotypes can rarely produce a similar syndrome. The bacteria responsible for enteric
fever pass through the cells lining the intestines and are engulfed by macrophages.
They replicate after being transported to the liver, spleen, and bone marrow. Ten to
14 days after ingestion of the bacteria, patients experience gradually increasing
fever, with nonspecific complaints of headache, myalgias, malaise, and anorexia.
These symptoms persist for 1 week or longer and are followed by gastrointestinal
symptoms. This cycle corresponds to an initial bacteremic phase that is followed by
colonization of the gallbladder and then reinfection of the intestines. Enteric fever is
a serious clinical disease and must be suspected in febrile patients who have recently
traveled to developing countries where disease is endemic.
Asymptomatic Colonization
The strains of Salmonella responsible for causing typhoid and paratyphoid fevers
are maintained by human colonization. Chronic colonization for more than 1 year
after symptomatic disease develops in 1% to 5% of patients, the gallbladder being
the reservoir in most patients. Chronic colonization with other species of Salmonella
occurs in less than 1% of patients and does not represent an important source of
human infection.

OTHER ENTEROBACTERIACEAE
Klebsiella
Members of the genus Klebsiella have a prominent capsule that is responsible for
the mucoid appearance of isolated colonies and the enhanced virulence of the
organisms in vivo. The most commonly isolated members of this genus are
K. pneumonia and Klebsiella oxytoca, which can cause community- or hospital-
acquired primary lobar pneumonia. Pneumonia caused by Klebsiella species
frequently involves the necrotic destruction of alveolar spaces, formation of cavities,
and the production of blood-tinged sputum. These bacteria also cause wound, soft-
tissue, and UTIs.
Proteus
P. mirabilis, the most common member of this genus, primarily produces infections
of the urinary tract (e.g., bladder infection or cystitis; kidney infection or
pyelonephritis). P. mirabilis produces large quantities of urease, which splits urea
into carbon dioxide and ammonia. This process raises the urine pH, precipitating
magnesium and calcium in the form of struvite and apatite crystals, respectively, and
results in the formation of renal (kidney) stones. The increased alkalinity of the urine
is also toxic to the uroepithelium.
Enterobacter, Citrobacter, Morganella, and Serratia

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Primary infections caused by Enterobacter, Citrobacter, Morganella, and Serratia
are rare in immunocompetent patients. They are more common causes of hospital-
acquired infections in neonates and immunocompromised patients. For example,
Citrobacter koseri has been recognized to have a predilection for causing meningitis
and brain abscesses in neonates. Antibiotic therapy for these genera can be
ineffective because the organisms are frequently resistant to multiple antibiotics.
Resistance is a particularly serious problem with Enterobacter species.

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 VIBRIO, CAMPYLOBACTER, AND HELICOBACTER

VIBRIO
The second major group of gram-negative, facultatively anaerobic, fermentative
rods are the genus Vibrio. These organisms were at one time classified together in
the family Vibrionaceae and were separated from the Enterobacteriaceae on the
basis of a positive oxidase reaction and the presence of polar flagella.
The genus Vibrio has undergone numerous changes in recent years, with a number
of less common species described or reclassified. Currently, the genus is composed
of more than 100 species of curved rods. A number of species are associated with
human disease, but three species are particularly important human pathogens:Vibrio
cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus.

Vibrio Species Associated with Human Disease

Species Sourse of infection Clinical desease
V. cholerae Water, food Gastroenteritis,
bacteremia
V. parahaemolyticus Shellfish, seawater Gastroenteritis, wound
infection, bacteremia
V. vulnificus Shellfish, seawater Bacteremia, wound
infection

Physiology and Structure

Vibrio species can grow on a variety of simple media within a broad temperature
range (from 14° C to 40° C). All species of Vibrio require sodium chloride (NaCl)
for growth. V. cholera can grow on most media without added salt, but most other
species (halophilic species) require the addition of NaCl. Vibrios tolerate a wide
range of pH (e.g., pH of 6.5 to 9.0) but are susceptible to stomach acids. If gastric
acid production is reduced or neutralized, patients are more susceptible to
Vibrio infections.
Most vibrios have polar flagella (important for motility), as well as various pili that
are important for virulence. For example, epidemic strains of V. cholerae, the
etiologic agent of cholera, have the toxinco-regulated pilus. The cell wall structure
of vibrios is also important. All strains possess lipopolysaccharides consisting of
lipid A (endotoxin), core polysaccharide, and an O polysaccharide side chain. The
O polysaccharide is used to subdivide Vibrio species into serogroups:There are 200
serogroups of V. cholera plus multiple serogroups of V. vulnificus plus
V. parahaemolyticus. The interest in this classification scheme is more than
academic—V. cholera O1 and O139 produce cholera toxin and are associated with
18
epidemics of cholera. Other strains of V. choleraegenerally do not produce cholera
toxin and do not cause epidemic disease. V. cholera serogroup O1 is further
subdivided into serotypes and biotypes. Three serotypes are recognized: Inaba,
Ogawa, and Hikojima. Strains can shift between serotype Inaba and serotype
Ogawa, with Hikojima a transitional state in which both Inaba and Ogawa antigens
are expressed. Two biotypes of V. cholera O1 are recognized: Classical and El Tor.
These biotypes are subdivided by differences in phenotypic and morphologic
properties. Seven worldwide pandemics of V. cholera infections have been
documented. V. cholera strains responsible for the sixth worldwide pandemic of
cholera were of the Classical biotype, whereas most strains responsible for the
current seventh pandemic are the El Tor biotype.

Diagram of the bacterium, V. cholerae

V. vulnificus and non-O1 V. cholera produce acidic polysaccharide capsules that are
important for disseminated infections. V. cholera O1 does not produce a capsule, so
infections with this organism do not spread beyond the confines of the intestine.
V. cholera and V. parahaemolyticus possess two circular chromosomes, each of
which carry essential genes for these bacteria. It is not known if other Vibrio species
have a similar genomic structure. Plasmids, including those encoding antimicrobial
resistance, are also commonly found in Vibrio species.

Pathogenesis
To produce disease, V. cholerae must reach the small intestine in sufficient numbers
to multiply and colonize. In healthy people, ingestion of large numbers of bacteria
is required to offset the acid barrier of the stomach. Colonization of the entire
intestinal tract from the jejunum to the colon by V. cholerae requires organism
19
adherence to the epithelial surface, most probably by surface pili. The outstanding
feature of V. cholera pathogenicity is the ability of virulent strains to secrete CT
(cholera toxin), which is responsible for the disease cholera. The water and
electrolyte shift from the cell to the intestinal lumen is the fundamental cause of the
watery diarrhea of cholera.
The fluid loss that results from the adenylate cyclase stimulation of cells depends on
the balance between the amount of bacterial growth, toxin production, fluid
secretion, and fluid absorption in the entire gastrointestinal tract. The outpouring of
fluid and electrolytes is greatest in the small intestine, where the secretory capacity
is high and absorptive capacity low. The diarrheal fluid can amount to many liters
per day, with approximately the same sodium content as plasma but two to five times
the potassium and bicarbonate concentrations. The intestinal mucosa remains
unaltered except for some hyperemia, because V. cholerae does not invade or
otherwise injure the enterocyte.

Epidemiology
Vibrio species, including V. cholerae, grow naturally in estuarine and marine
environments worldwide. All Vibrio species are able to survive and replicate in
contaminated waters with increased salinity. Pathogenic vibrios can also flourish in
waters with chitinous shellfish (e.g., oxysters, clams, mussels)—hence the
association between Vibrio infections and the consumption of shellfish.
Asymptomatically infected humans can also be an important reservoir for this
organism in areas where V. cholera disease is endemic.
Seven major pandemics of cholera have occurred since 1816, resulting in thousands
of deaths and major socioeconomic changes. Sporadic disease and epidemics
occurred before this time, but worldwide spread of the disease became possible with
intercontinental travel resulting from increased commerce and wars.
The seventh pandemic, which was caused by V. cholera O1 biotype El Tor, began
in Asia in 1961 and spread to Africa, Europe, and Oceania in the 1970s and 1980s.
In 1991, the pandemic strain spread to Peru and, subsequently, has caused disease in
most countries in South and Central America, as well as in the United States and
Canada. A second epidemic strain emerged in 1992 in India and rapidly spread
across Asia. This strain, V. cholera O139 Bengal, produces the cholera toxin and
shares other traits with V. cholera O1. This is the first non-O1 strain capable of
causing epidemic disease and is capable of producing disease in adults who were
previously infected with the O1 strain (showing that no protective immunity
is conferred).

20
It is estimated that 3 to 5 million cases of cholera and 100,000 deaths occur
worldwide each year. The most recent epidemics occurred in 2004 in Bangladesh
following flooding, in 2008 to 2009 in Zimbabwe, and in 2010 in Haiti following
the devastating earthquake. Cholera is spread by contaminated water and food rather
than direct person-to-person spread because a high inoculum (e.g., more than 108
organisms) is required to establish infection in a person with normal gastric acidity.
In a person with achlorhydria or hypochlorhydria, the infectious dose can be as low
as 103 to 105organisms. Cholera is usually seen in communities with poor sanitation.
Indeed, one outcome from the cholera pandemics was recognition of the role of
contaminated water in the spread of disease and the need to improve community
sanitation systems so that the disease could be controlled. Thus it is not surprising
to observe cholera outbreaks when natural disasters, such as the earthquake in Haiti,
compromise the control of sanitary wastes.
Infections caused by V. parahaemolyticus, V. vulnificus, and other pathogenic
vibrios result from the consumption of improperly cooked seafood, particularly
oysters, or exposure to contaminated seawater. V. parahaemolyticus is the most
common cause of bacterial gastroenteritis in Japan and Southeast Asia and is the
most common Vibrio species responsible for gastroenteritis in the United States.
V. vulnificus is not frequently isolated but is responsible for severe wound infections
and a high incidence of fatal outcomes. V. vulnificus is the most common cause of
Vibrio septicemia. Gastroenteritis caused by vibrios occurs throughout the year
because oysters are typically contaminated with abundant organisms year-round. In
contrast, septicemia and wound infections with Vibrio occur during the warm
months, when the organisms in seawater can multiply to high numbers.

Clinical Diseases
Vibrio cholerae
The majority of individuals exposed to toxigenic V. cholera O1 have asymptomatic
infections or self-limited diarrhea; however, some individuals develop severe,
rapidly fatal diarrhea. The clinical manifestations of cholera begin an average of 2
to 3 days after ingestion of the bacteria, with the abrupt onset of watery diarrhea and
vomiting. As more fluid is lost, the feces-streaked stool specimens become colorless
and odorless, free of protein, and speckled with mucus (“rice-water” stools). The
resulting severe fluid and electrolyte loss can lead to dehydration, painful muscle
cramps, metabolic acidosis (bicarbonate loss), and hypokalemia and hypovolemic
shock (potassium loss), with cardiac arrhythmia and renal failure. The mortality rate
is 60% in untreated patients but less than 1% in patients who are promptly treated
with replacement of lost fluids and electrolytes. Disease caused by V. cholera

21
O139can be as severe as disease caused by V. cholera O1. Other serotypes of V.
cholera (commonly called V. cholera non-O1) do not produce cholera toxin and are
usually responsible for mild watery diarrhea. These strains can also cause
extraintestinal infections, such as septicemia, particularly in patients with liver
disease or hematologic malignancies.
Vibrio parahaemolyticus
The severity of gastroenteritis caused by V. parahaemolyticus can range from a self-
limited diarrhea to a mild, cholera-like illness. In general, the disease develops after
a 5- to 72-hour incubation period (mean, 24 hours), with explosive, watery diarrhea.
No grossly evident blood or mucus is found in stool specimens except in severe
cases. Headache, abdominal cramps, nausea, vomiting, and low-grade fever may
persist for 72 hours or more. The patient usually experiences an uneventful recovery.
Wound infections with this organism can occur in people exposed to contaminated
seawater.
Vibrio vulnificus
V. vulnificus is a particularly virulent species of Vibrio responsible for more than
90% of the Vibrio-related deaths in the United States. The most common
presentations are primary septicemia after consumption of contaminated raw oysters
or rapidly progressive wound infection after exposure to contaminated seawater.
Patients with primary septicemia present with a sudden onset of fever and chills,
vomiting, diarrhea, and abdominal pain. Secondary skin lesions with tissue necrosis
are often present. The mortality in patients with V. vulnificus septicemia can be as
high as 50%. The wound infections are characterized by initial swelling, erythema,
and pain at the wound site, followed by the development of vesicles or bullae and
eventual tissue necrosis together with systemic signs of fever and chills. Mortality
associated with wound infections ranges from 20% to 30%. V. vulnificus infections
are most severe in patients with hepatic disease, hematopoietic disease, or chronic
renal failure and in those receiving immunosuppressive drugs.

Laboratory Diagnosis
Microscopy
Vibrio species are small (0.5 to 1.5 to 3 µm), curved, gram-negative rods. Large
numbers of organisms are typically present in the stools of patients with cholera, so
the direct microscopic examination of stool specimens can provide a rapid,
presumptive diagnosis in endemic cholera outbreaks. Examination of Gram-stained
wound specimens may also be useful in a setting suggestive of V. vulnificus infection
(e.g., exposure of susceptible individual to seafood or seawater).

22
Gram stain depicts flagellated vibrio comma bacteria.

Culture
Vibrio organisms survive poorly in an acidic or dry environment. Specimens must
be collected early in the disease and inoculated promptly onto culture media. If
culture will be delayed, the specimen should be mixed in a Cary-Blair transport
medium and refrigerated. Vibrios have low survival rates in buffered glycerol-saline,
the transport medium used for most enteric pathogens.
Vibrios grow on most media used in clinical laboratories for stool and wound
cultures, including blood agar and MacConkey agar. Special selective agar for
vibrios (e.g., thiosulfate citrate bile salts sucrose [TCBS] agar), as well as an
enrichment broth (e.g., alkaline peptone broth; pH 8.6), can also be used to recover
vibrios in specimens with a mixture of organisms (e.g., stools). Isolates are identified
with selective biochemical tests and serotyped using polyvalent antisera. In tests
23
performed to identify halophilic vibrios, the media for biochemical testing must be
supplemented with 1% NaCl.

Yellow coloured (sucrose fermenting) colonies of Vibrio cholerae on TCBS agar.

Treatment
Patients with cholera must be promptly treated with fluid and electrolyte
replacement before the resultant massive fluid loss leads to hypovolemic shock.
Antibiotic therapy, although of secondary value, can reduce toxin production and
clinical symptoms, as well as decrease transmission by the more rapid elimination
of the organism. A single dose of azithromycin is currently the drug of choice for
children and adults because macrolide resistance is relatively uncommon. A single
dose of doxycycline or ciprofloxacin in nonpregnant adults can be used as alternative
therapy if demonstrated to be active in vitro; however, resistance to the tetracycline
and fluoroquinolones is relatively common.
V. parahaemolyticus gastroenteritis is usually a self-limited disease, although
antibiotic therapy can be used in addition to fluid and electrolyte therapy in patients
with severe infections. V. vulnificus wound infections and septicemia must be
promptly treated with antibiotic therapy. The combination of minocycline or
doxycycline combined with ceftriaxone or cefotaxime appears to be the most
effective treatment.

Prevention and Control

People infected with V. cholerae can shed bacteria for the first few days of acute
illness and represent important sources of new infections. Although long-term
carriage of V. cholerae does not occur, vibrios are free living in estuarine and marine
24
reservoirs. Only improvements in sanitation can lead to effective control of the
disease. This involves adequate sewage management, the use of purification systems
to eliminate contamination of the water supply, and the implementation of
appropriate steps to prevent contamination of food.
None of the vaccines provide long-term protection. A killed vaccine consisting of
whole cells of V. cholerae O1 plus recombinant cholera toxin B subunit or a bivalent
killed vaccine of whole cells of V. cholerae O1 and O139 is recommended for short-
term protection of travelers in high-risk settings (e.g., exposure to untreated water
or care of ill patients) to endemic regions of the world. Antibiotic prophylaxis of
contacts to household patients with cholera can limit the spread but is generally
ineffective in communities where disease occurs.

CAMPYLOBACTER and HELICOBACTER

The classification of Campylobacter and Helicobacter has undergone many changes
since the bacteria were first discovered. However, molecular biology techniques
(e.g., sequence analysis of the 16S rRNA gene), characterization of cell wall proteins
and lipids, serologic characterization, and analysis of biochemical properties have
been used to resolve much of the taxonomic confusion. These genera are now
separated into two families: Campylobacteraceae, which includes the genera
Campylobacter, Arcobacter, and Sulfurospirillum, and Helicobacteraceae, which
includes Helicobacter and Wolinella. The members of both families are spiral, gram-
negative rods with (1) a low DNA guanine plus cytosine base ratio, (2) an inability
to ferment or oxidize carbohydrates, and (3) microaerophilic growth requirements
(i.e., growth only in the presence of a reduced oxygen level).Campylobacter and
Helicobacter are the most important human pathogens.

CAMPYLOBACTER
The genus Campylobacter consists of small (0.2 to 0.5 µm wide and 0.5 to 5.0 µm
long), comma-shaped, gram-negative rods that are motile by means of a polar
flagellum. Older cultures may appear coccoid. Most species are microaerobic,
requiring an atmosphere with decreased oxygen and increased carbon dioxide levels
for aerobic growth; thus special culture conditions are required for recovery of these
organisms from clinical specimens. A total of 32 species and 13 subspecies are now
recognized, many of which are associated with human disease, but only four species
are common human pathogens.

25
SEM image of Campylobacter jejuni with size bar.

The primary diseases caused by campylobacters are gastroenteritis and septicemia.

Campylobacter is the most common cause of bacterial gastroenteritis, with
Campylobacter jejuni responsible for most infections and Campylobacter coli
associated with 2% to 5% of the cases of Campylobacter gastroenteritis. The latter
is a more common cause of gastroenteritis in developing countries. The incidence of
gastroenteritis caused by Campylobacter upsaliensisьis unknown because the
organism is inhibited by the antibiotics used in isolation media for other
campylobacters; however, some have estimated that 10% of Campylobacter
gastroenteritis is caused by C. upsaliensis. A variety of other species are rare causes
of gastroenteritis or systemic infections. Unlike other Campylobacter species,
Campylobacter fetus is primarily responsible for causing systemic infections, such
as bacteremia, septic thrombophlebitis, arthritis, septic abortion, and meningitis.

HELICOBACTER
Spiral, gram-negative rods resembling campylobacters were found in 1983. The
organisms were originally classified as Campylobacter but were subsequently
reclassified as a new genus, Helicobacter. Helicobacters were subsequently
subdivided into species that primarily colonize the stomach (gastric helicobacters)
and those that colonize the intestines (enterohepatic helicobacters). The most
important helicobacter species is Helicobacter pylori, a gastric helicobacter
associated with gastritis, peptic ulcers, gastric adenocarcinoma, and gastricmucosa–

26
associated lymphoid tissue (MALT) B-cell lymphomas. The most important
enterohepatic helicobacters associated with human disease are Helicobacter cinaedi
and Helicobacter fennelliae, which have been isolated from homosexual men with
proctitis, proctocolitis, or enteritis. Helicobacters are also isolated from the stomachs
and intestines of many other mammals (e.g., monkeys, dogs, cats, cheetahs, ferrets,
mice, rats).

H. pylori crossing mucus layer stomach

27
 PURULENT-SEPTIC INFECTION
Skin infections spread rapidly by contact, especially in enclosed populations or
where sanitation is poor and humidity high. Staphylococcus aureus and
Streptococcus pyogenes are the commonest.

Disease patterns
Cellulitis
• Affects all layers of the skin.
• Causes include S. pyogenes, S. aureus, Pasteurella multocida or, rarely, marine
vibrios or Gram-negative bacilli.
• Organisms invade via skin abrasions, insect bites or wounds.
• Empirical flucloxacillin should be given until culture results are available. Severe
disease should be treated with intravenous antibiotics, including benzylpenicillin
and flucloxacillin.
Necrotizing fasciitis
• A rapidly progressive infection that spreads to involve the skin and subcutaneous
layers.
• It is caused by mixed aerobic and anaerobic organisms or pure S. pyogenes.
• The condition is characterized by pain, fever and shock, and the infected area may
be discoloured.
• Progression is rapid, leading to death in a very short time.
• Surgical resection of infected tissue is critical, supplemented with antibiotics
targeted against streptococci, staphylococci, Gramnegative bacilli and obligate
anaerobes (e.g. benzylpenicillin, a third-generation cephalosporin and
metronidazole).
Erythrasma
This superficial infection of the flexures is caused by Corynebacterium
minutissimum – the lesions fluoresce under Wood’s light. The organism may be
cultured; treatment is with erythromycin or tetracycline.
Erysipelas
• A well-demarcated streptococcal infection confined to the dermis.
• The condition is found on the face or shins.
• It is hot and red on examination.
• A modest increase in the peripheral white blood cells and fever occur.
• Treatment is with oral amoxicillin or flucloxacillin given intravenously in severe
cases.
Erysipeloid

28
• Zoonosis found in pig handlers and fisherman caused by Erysipelothrix
rhusiopathiae.
• It may be self-limiting but treatment with oral penicillin or tetracycline speeds the
response and is needed in rare septicaemic cases.
Burns
• Burns are susceptible to bacterial colonization.
• Typical organisms are Pseudomonas aeruginosa, S. aureus, S. pyogenes and, less
commonly, coliforms.
• Antibiotic resistance is an increasing problem.
Complications include loss of skin grafts and secondary bacteraemia.
Paronychia
This is a common infection in community practice. The cuticle is damaged, which
allows invasion with organisms such as S. aureus. There is pain and swelling,
followed by a small abscess. The abscess may be drained and antibiotics can be
given (e.g. flucloxacillin).
Manifestations of systemic infections
The skin is a large organ that is a window onto systemic infection.
Examples of this include:
• the petechial rash of meningococcal septicaemia;
• ecthyma gangrenosum of Pseudomonas septicaemia;
• the splinter haemorrhages of endocarditis;
• skin infarctions due to staphylococcal septicaemia;
• rash as part of a systemic infection (e.g. chickenpox and measles);
• the primary site of herpes simplex infection;
• the different skin manifestations of toxin-mediated systemic disease: toxic shock
syndrome due to Staphylococcus aureus (generalized and palmar rash); scarlet fever
due to !-haemolytic streptococci (rash with circumoral pallor, scalded skin and
desquamation in neonates).

29
 GRAM-POSITIVE COCCI
The gram-positive cocci are a heterogeneous collection of bacteria. Features that
they have in common are their spherical shape, their Gram-stain reaction, and an
absence of endospores. The presence or absence of catalase activity is a simple test
that is used to subdivide the various genera. Catalases are enzymes that
convert hydrogen peroxide into water and oxygen gas. If a drop of a peroxide
solution is placed on a catalase-producing bacterial colony, bubbles appear when the
oxygen gas is formed. The aerobic catalase-positive genera (e.g., Staphylococcus,
Micrococcus, and related organisms) are discussed in this chapter; the aerobic
catalase-negative genera (Streptococcus, Enterococcus, and related organisms).

STAPHYLOCOCCUS
The genus name Staphylococcus refers to the fact that these gram-positive cocci
grow in a pattern resembling a cluster of grapes; however, organisms in clinical
specimens may also appear as single cells, pairs, or short chains. Most staphylococci
are 0.5 to 1.5 µm in diameter, non-motile, and able to grow in a variety of
30
conditions—aerobically, and anaerobically, in the presence of a high concentration
of salt (e.g., 10% sodium chloride) and at temperatures ranging from 18° C to 40°
C. These bacteria are present on the skin and mucous membranes of humans. The
genus currently consists of 45 species and 24 subspecies, many of which are found
on humans. Some species are commonly found in very specific niches. For example,
Staphylococcus aureus colonizes the anterior nares, Staphylococcus capitis is found
where sebaceous glands are present (e.g., forehead), and Staphylococcus
haemolyticus and Staphylococcus hominis are found in areas where apocrine glands
are present (e.g., axilla). Staphylococci are important pathogens in humans, causing
a wide spectrum of life-threatening systemic diseases, including infections of the
skin, soft tissues, bones, and urinary tract; and opportunistic infections. The species
most commonly associated with human diseases are S. aureus (the most virulent and
best-known member of the genus), Staphylococcus epidermidis, S. haemolyticus,
Staphylococcus lugdunensis, and Staphylococcus saprophyticus. Methicillin-
resistant S. aureus (MRSA) is notorious for producing serious infections in
hospitalized patients and outside the hospital in previously healthy children and
adults. S. aureus colonies can have a yellow or gold color because of the carotenoid
pigments that form during their growth, hence the species name. It is also the most
common species in humans that produces the enzyme-coagulase and, therefore, this
property is a useful diagnostic test. When a colony of S. aureus is suspended in
plasma, coagulase binds to a serum factor, and this complex converts fibrinogen to
fibrin, resulting in the formation of a clot. Other staphylococcal species that do not
produce coagulase are referred to collectively as coagulase-negative staphylococci.

31
SEM of S. aureus

Physiology and Structure

The outermost layer of the cell wall of many staphylococci is covered with a
polysaccharide capsule. The capsule protects the bacteria by inhibiting phagocytosis
of the organisms by polymorphonuclear leukocytes (PMNs). A loose-bound, water-
soluble film (slime layer or biofilm) consisting of monosaccharides, proteins, and
small peptides is produced by most staphylococci in varying amounts. This
extracellular substance binds the bacteria to tissues and foreign bodies, such as
catheters, grafts, prosthetic valves and joints, and shunts, and it is particularly
important for the survival of relatively avirulent coagulase-negative staphylococci.
Half of the cell wall by weight is peptidoglycan, a feature common to gram-positive
bacteria. The peptidoglycan has endotoxin-like activity.
Teichoic acids are the other major component of the cell wall. Teichoic acids are
species-specific, phosphate-containing polymers that are bound covalently to N-
acetylmuramic acid residues of the peptidoglycan layer or to the lipids in the
cytoplasmic membrane (lipoteichoic acids). Although the teichoic acids are poor
immunogens, a specific antibody response is stimulated when they are bound to
peptidoglycan.
A large collection of surface proteins have been identified in S. aureus that are
important for adherence to host matrix proteins bound to host tissues (e.g.,
fibronectin, fibrinogen, elastin, collagen). Most of these surface adhesion proteins
are covalently bound to the cell wall peptidoglycan in staphylococci and have been

32
designated MSCRAMM (microbial surface components recognizing adhesive
matrix molecules) proteins. The best characterized MSCRAMM proteins are
staphylococcal protein A, fibronectin-binding proteins A and B, and clumping factor
proteins A and B. The clumping factor proteins (also called coagulase) bind
fibrinogen and convert it to insoluble fibrin, causing the staphylococci to clump or
aggregate. Detection of these proteins is the primary identification test for S. aureus.

Epidemiology of Staphylococcus aureus infections

Because S. aureus is a major cause of nosocomial and community-acquired
infections, it is necessary to determine the relatedness of isolates collected during
the investigation of an outbreak. Typing systems must be reproducible,
discriminatory, and easy to interpret and to use. The traditional method for typing
S. aureus is phage-typing. This method is based on a phenotypic marker with poor
reproducibility. Also, it does not type many isolates (20% in a recent survey at the
Center for Disease Control and Prevention), and it requires maintenance of a large
number of phage stocks and propagating strains and consequently can be performed
only by specialist reference laboratories.
Many molecular typing methods have been applied to the epidemiological analysis
of S. aureus, in particular, of methicillin-resistant strains (MRSA). Plasmid analysis
has been used extensively with success, but suffers the disadvantage that plasmids
can easily be lost and acquired and are thus inherently unreliable. Methods designed
to recognize restriction fragment length polymorphisms (RFLP) using a variety of
gene probes, including rRNA genes (ribotyping), have had limited success in the
epidemiology of MRSA. In this technique the choice of restriction enzyme used to
cleave the genomic DNA, as well as the probes, is crucial. Random primer PCR
offers potential for discriminating between strains but a suitable primer has yet to be
identified for S. aureus. The method currently regarded as the most reliable is pulsed
field gel electrophoresis, where genomic DNA is cut with a restriction enzyme that
generates large fragments of 50-700 kb.

Pathogenesis of S. aureus infections

S. aureus expresses many cell surface-associated and extracellular proteins that are
potential virulence factors. For the majority of diseases caused by this organism,
pathogenesis is multifactorial. Thus it is difficult to determine precisely the role of
any given factor. This also reflects the inadequacies of many animal models for
staphylococcal diseases.
However, there are correlations between strains isolated from particular diseases and
expression of particular factors, which suggests their importance in pathogenesis.

33
With some toxins, symptoms of a human disease can be reproduced in animals with
pure proteins. The application of molecular biology has led to recent advances in the
understanding of pathogenesis of staphylococcal diseases. Genes encoding potential
virulence factors have been cloned and sequenced and proteins purified. This has
facilitated studies at the molecular level on their modes of action, both in in vitro
and in model systems. In addition, genes encoding putative virulence factors have
been inactivated, and the virulence of the mutants compared to the wild-type strain
in animal models. Any diminution in virulence implicates the missing factor. If
virulence is restored when the gene is returned to the mutant then “Molecular Koch's
Postulates” have been fulfilled. Several virulence factors of S. aureus have been
confirmed by this approach

Clinical manifestations of S. aureus

S. aureus is notorious for causing boils, furuncles, styes, impetigo and other
superficial skin infections in humans. It may also cause more serious infections,
particularly in persons debilitated by chronic illness, traumatic injury, burns or
immunosuppression. These infections include pneumonia, deep abscesses,
osteomyelitis, endocarditis, phlebitis, mastitis and meningitis, and are often
associated with hospitalized patients rather than healthy individuals in the
community. S. aureus and S. epidermidis are common causes of infections
associated with indwelling devices such as joint prostheses, cardiovascular devices
and artificial heart valves.

34
Pathogenesis of staphylococcal infections.

Infections associated with indwelling devices

35
Laboratory Diagnosis
Staphylococci are Gram-positive cocci about 0.5 – 1.0 μm in diameter. They grow
in clusters, pairs and occasionally in short chains. The clusters arise because
staphylococci divide in two planes. The configuration of the cocci helps to
distinguish micrococci and staphylococci from streptococci, which usually grow in
chains. Observations must be made on cultures grown in broth, because streptococci
grown on solid medium may appear as clumps. Several fields should be examined
before deciding whether clumps or chains are present.

Catalase Test
The catalase test is important in distinguishing streptococci (catalase-negative)
staphylococci which are catalase positive. The test is performed by flooding an agar
slant or broth culture with several drops of 3% hydrogen peroxide. Catalase-positive
cultures bubble at once. The test should not be done on blood agar because blood
itself will produce bubbles.

Isolation and Identification

The presence of staphylococci in a lesion might first be suspected after examination
of a direct Gram stain. However, small numbers of bacteria in blood preclude
microscopic examination and require culturing first.
The organism is isolated by streaking material from the clinical specimen (or from
a blood culture) onto solid media such as blood agar, tryptic soy agar or heart
infusion agar. Specimens likely to be contaminated with other microorganisms can
be plated on mannitol salt agar containing 7.5% sodium chloride, which allows the
halo-tolerant staphylococci to grow. Ideally a Gram stain of the colony should be
performed and tests made for catalase and coagulase production, allowing the
coagulase-positive S. aureus to be identified quickly. Another very useful test for S
aureus is the production of thermostable deoxyribonuclease. S. aureus can be
confirmed by testing colonies for agglutination with latex particles coated with
immunoglobulin G and fibrinogen which bind protein A and the clumping factor,
respectively, on the bacterial cell surface. These are available from commercial
suppliers (e.g., Staphaurex). The most recent latex test (Pastaurex) incorporates
monoclonal antibodies to serotype 5 and 8 capsular polysaccharide in order to reduce
the number of false negatives. (Some recent clinical isolates of S. aureus lack
production of coagulase and/or clumping factor, which can make identification
difficult.)
The association of S. epidermidis (and to a lesser extent of other coagulase-negative
staphylococci) with nosocomial infections associated with indwelling devices means

36
that isolation of these bacteria from blood is likely to be important and not due to
chance contamination, particularly if successive blood cultures are positive.
Nowadays, identification of S. epidermidis and other species of Staphylococcus is
performed using commercial biotype identification kits, such as API Staph Ident,
API Staph-Trac, Vitek GPI Card and Microscan Pos Combo. These comprise
preformed strips containing test substrates.

Resistance of Staphylococci to Antimicrobial Drugs

Hospital strains of S. aureus are often resistant to many different antibiotics. Indeed
strains resistant to all clinically useful drugs, apart from the glycopeptides
vancomycin and teicoplanin, have been described. The term MRSA refers to
methicillin resistance and most methicillin-resistant strains are also multiply
resistant. Plasmid-ASSOCIATED vancomycin resistance has been detected in some
enterococci and the resistance determinant has been transferred from enterococci toS
aureus in the laboratory and may occur naturally. S. epidermidis nosocomial isolates
are also often resistant to several antibiotics including methicillin. In addition, S.
aureus expresses resistance to antiseptics and disinfectants, such as quaternary
ammonium compounds, which may aid its survival in the hospital environment.
Since the beginning of the antibiotic era S. aureus has responded to the introduction
of new drugs by rapidly acquiring resistance by a variety of genetic mechanisms
including (1) acquisition of extrachromosomal plasmids or additional genetic
information in the chromosome via transposons or other types of DNA insertion and
(2) by mutations in chromosomal genes
Many plasmid-encoded determinants have recently become inserted into the
chromosome at a site associated with the methicillin resistance determinant. There
may be an advantage to the organism having resistance determinants in the
chromosome because they will be more stable. There are essentially four
mechanisms of resistance to antibiotics in bacteria: (1) enzymatic inactivation of the
drug, (2) alterations to the drug target to prevent binding, (3) accelerated drug efflux
to prevent toxic concentrations accumulating in the cell, and (4) a by-pass
mechanism whereby an alternative drug-resistant version of the target is expressed.

Treatment
Most boils and superficial staphylococcal abscesses resolve spontaneously without
antimicrobial therapy. Those that are more extensive, deeper, or in vital organs
require a combination of surgical drainage and antimicrobics for optimal outcome.
Penicillins and cephalosporins are active against S. aureus cell wall peptidoglycan
and vary in their susceptibility to inactivation by staphylococcal _-lactamases.

37
Although penicillin G is the treatment of choice for susceptible strains, the
penicillinase-resistant penicillins (methicillin, nafcillin, oxacillin) and first-
generation cephalosporins are more commonly used because of resistance. For
strains resistant to these agents or patients with _-lactam hypersensitivity, the
alternatives are vancomycin, clindamycin, or erythromycin. Synergy between cell
wall–active antibiotics and the aminoglycosides is present when the staphylococcus
is sensitive to both types of agents. Such combinations are often used in severe
systemic infections when effective and rapid bactericidal action is needed,
particularly in compromised hosts.

Prevention and Control

Staphylococcus aureus spreads by airborne transmission and via the hands of
healthcare workers. Patients colonized or infected with MRSA or GRSA should be
isolated in a side room with wound and enteric precautions. Staff may become
carriers and disseminate the organism widely in the hospital environment. Carriage
may be eradicated by using topical mupirocin and chlorhexidine.

OTHER STAPHYLOCOCCUS

Staphylococcus epidermidis
Staphylococcus epidermidis is the most important of the coagulasenegative
staphylococci (CoNS). Once dismissed as contaminants, these organisms are now
recognized as pathogens if conditions favour their multiplication.

Staphylococcus haemolyticus
Less common than S. epidermidis, Staphylococcus haemolyticus causes a similar
disease pattern. It differs from S. epidermidis in that it causes haemolysis on blood
agar. More importantly, it is naturally resistant to teicoplanin; significant infections
require vancomycin therapy.

Staphylococcus saprophyticus
These CoNS are a common cause of urinary tract infection in young women. They
can be rapidly distinguished from other species by their resistance to novobiocin.

38
39
 STREPTOCOCCUS
The main characteristics of streptococci include the following.
• They are Gram-positive cocci arranged in pairs and chains.
• They are fastidious facultative anaerobes.
• They require rich blood-containing media.
• They may be cultured from the site of infection (throat, wound, etc.) or in
blood cultures.
• Colonies are distinguished by haemolysis: complete (β) or incomplete (α) and none
(γ).

Classification
At the turn of the 20th century, a classification based on hemolysis and biochemical
tests was sufficient to associate some streptococcal species with infections in
humans and animals. Rebecca Lancefield, who demonstrated carbohydrate antigens
in cell-wall extracts of the _-hemolytic streptococci, put this taxonomy on a sounder
basis. Her studies formed a classification by serogroups (eg, A, B, C), each of which
is generally correlated with an established species. Later it was discovered that some
nonhemolytic streptococci had the same cell wall antigens. Over the years it has
become clear that possession of one of the Lancefield antigens defines a particularly
virulent segment of the streptococcal genus regardless of hemolytic patterns. These
are called the pyogenic streptococci, and in medical circles they are now better
known by their Lancefield letter than the older species name. Pediatricians instantly
recognize GBS as an acronym for group B streptococcus but may be confused by
use of the proper name, Streptococcus agalactiae.
For practical purposes, the type of hemolysis and certain biochemical reactions
remain valuable for the initial recognition and presumptive classification of
streptococci, and as an indication of what subsequent taxonomic tests to perform.
Thus, _-hemolysis indicates that the strain has one of the Lancefield group antigens,
but some Lancefield positive strains or groups may be _-hemolytic or even
nonhemolytic. The streptococci will be considered as follows: (1) pyogenic
streptococci (Lancefield groups); (2) pneumococci; (3) viridans and other
streptococci.

Streptococcus pyogenes
Streptococcus pyogenes is carried asymptomatically in the pharynx of 5–30% of the
population, more commonly in children. It is transmitted by the aerosol route and by
contact.

40
Pathogenesis
Streptococcus pyogenes is surrounded by the M protein antigen, which reduces
leucocyte phagocytosis. Antibodies to M proteins are protective but only against
infection with the same M type and there are multiple M types. They adhere via
fibronectin receptors.
Other features are:
• toxins are important in their pathogenicity:
° erythrogenic toxin associated with scarlet fever;
° pyrogenic exotoxins A, B and C associated with toxic shock;
• production of S. pyogenes cell envelope proteinase (SpyCEP), which degrades IL-
8 and other cytokines, thereby retarding neutrophil activation;
• ability to invade and survive intracellularly making them difficult to eradicate with
penicillin;
• production of degrading enzymes (immunoglobulin proteases, hyaluronidase and
collagenases).

Clinical manifestations
Streptococcus pyogenes, which is an important cause of mortality worldwide, has
the following associations:

41
1 Invasive disease characterized by rapid onset, local tissue destruction and rapid
spread within the tissues. Systemic toxicity is common and associated with toxin
production. Syndromes include:
• pharyngitis – the most common bacterial cause;
• skin infection – erysipelas, impetigo, cellulitis, wound infections and rarely,
necrotizing fasciitis or pneumonia;
• puerperal sepsis;
• complications such as septicaemia and metastatic infections (e.g. osteomyelitis);
• severe toxicity:
° erythrogenic toxin causes scarlet fever;
° pyrogenic toxin-producing strains are associated with streptococcal shock
and have a high mortality due to multiple organ failure.
2 Postinfectious immune-mediated diseases which include rheumatic fever,
glomerulonephritis and erythema nodosum, are thought to be immune-mediated
because antibodies to bacterial structures cross-react with host tissues. Rheumatic
fever, now uncommon in developed countries, is a major cause of long-term
morbidity and mortality, particularly in areas of poverty and malnutrition.

Prevention and control

Streptococcus pyogenes can spread rapidly in surgical and obstetric wards; infected
or colonized patients should be isolated in a side room until 48 h after initiation of
effective antibiotics. Prompt treatment prevents secondary immune disease (e.g.
rheumatic fever). Benzylpenicillin is the treatment of choice and resistance has never
been reported. Amoxicillin may be used for oral therapy in less severe infections.
Macrolides are an alternative for patients with allergy.

Streptococcus agalactiae
Streptococcus agalactiae (group B streptococcus) is a commensal in the gut and
female genital tract. It causes:
• early perinatal pneumonia or septicaemia;
• later perinatal meningitis;
• puerperal sepsis.
The polysaccharide antiphagocytic capsule is the main pathogenicity determinant.
Prophylactic therapy to prevent neonatal disease can be given to those mothers in
labour who are febrile, known to be colonized or who have previously had an
affected child. There is currently no vaccine available.

Clinical manifestations and diagnosis

42
Infected neonates may initially lack the classical clinical signs of sepsis, such as
fever and the bulging fontanelle of meningitis. A chest X-ray may demonstrate
pneumonia, and specimens of blood, CSF, amniotic fluid and gastric aspirate should
be cultured. Antigen detection tests are available and can be applied to body fluids
for rapid diagnosis.

Treatment and prevention

Neonatal group B streptococcal sepsis requires empirical therapy that includes a
penicillin and an aminoglycoside. Perinatal penicillin can prevent invasive infection
but should be targeted at babies who are at high risk.

Enterococcus spp.
Enterococci possess a group D carbohydrate cell wall antigen and can exhibit all
three types of haemolysis. They are principally commensals of the bowel but may
cause disease if they become established at other sites. Of more than 12 species,
Enterococcus faecalis and E. faecium are the most common members to act as
human pathogens, causing urinary tract infection, wound infection and endocarditis.
Enterococci are emerging as hospital pathogens, with some species (e.g. E. faecium)
now resistant to commonly used antibiotics. Most strains are sensitive to
ampicillin/amoxicillin; however, resistance levels are increasing. Some enterococci
have developed resistance to glycopeptides by the acquisition of an alternative
peptidoglycan transpeptidation enzyme (the van A or van C system) that alters the
usual Ala–DAla cross-linkage so it is not glycopeptide susceptible. Such strains may
require therapy with drugs such as linezolid, daptomycin or pristinamycin.

PSEUDOMONAS
Most Pseudomonas spp. are environmental organisms that can cause opportunistic
infections in a healthcare environment.

43
 Pseudomonas aeruginosa
This organism is widespread in the environment, but rare in the flora of healthy
individuals. Its carriage increases with hospitalization. Moist places such as sink-
traps, drains and flower vases can harbour Pseudomonas.

Pathogenesis
• Produces cytotoxins and proteases (e.g. exotoxins A and S, haemolysins
and elastase).
• Isolates from patients with cystic fibrosis produce a polysaccharide alginate that
protects from opsonization, phagocytosis and antibiotics in microcolonies.
• Alginate, pili and outer membrane protein mediate adherence.
• Alginate production is associated with hypersusceptibility to antibiotics,
lipopolysaccharide deficiency, non-motility and reduced exotoxin production.

Clinical syndromes
• Chronic pulmonary infection in cystic fibrosis.
• Septicaemia, which has a high mortality and is a particular threat to
neutropenic patients.
• Rapidly progressive corneal infection and otitis externa.
• Colonization of burns, followed by septicaemia.
44
• Ecthyma gangrenosum, a destructive skin complication of bacteraemia.
• Osteomyelitis, septic arthritis and meningitis.

Laboratory diagnosis
• Culture on selective media containing cetrimide, irgasin and naladixic acid.
• Identification by biochemical testing.
• Typing by pulse-field gel electrophoresis or multilocus sequence typing (MLST).

Treatment and preventoin

Organisms are often resistant; therefore, treatment is guided by susceptibilities.
Vaccines incorporating somatic antigens from multiple P. aeruginosa serotypes
have been developed and proved immunogenic in humans. The primary candidates
for such preparations are patients with burn injuries, CF, or immunosuppression.
Although some protection has been demonstrated, these preparations are still
experimental.

45
 CLOSTRIDIUM
Clostridium spp. are anaerobic spore-forming organisms that are able to survive well
in the environment. Their normal habitats are soil, water and the intestinal tract of
humans and animals. Only a few of the 80 species are human pathogens. Toxin
production is the main pathogenicity mechanism.

CLOSTRIDIUM PERFRINGENS
Clostridium perfringens is the organism most commonly associated with gas
gangrene, but C. septicum, C. novyi, C. histolyticum and C. sordellii can also be
implicated.
C. perfringens is a large, Gram-positive, nonmotile rod with square ends. It grows
overnight on blood agar medium under anaerobic conditions, producing colonies
surrounded by a double zone of hemolysis.C. perfringens produces multiple
exotoxins that have different pathogenic significance in different animal species and
serve as the basis for classification of the five types (A to E). Type A is by far the
most important in humans and is found consistently in the colon and often in soil.
The most important exotoxin is the α-toxin, a phospholipase that hydrolyzes lecithin
and sphingomyelin, thus disrupting the cell membranes of various host cells,
including erythrocytes, leukocytes, and muscle cells. The θ-toxin alters capillary
permeability and is toxic to heart muscle.
C. perfringens produces a wide range of wound and soft tissue infections, many of
which are no different from those caused by other opportunistic bacteria. The most
dreaded of these, gas gangrene, begins as a wound infection but progresses to shock
46
and death in a matter of hours. Another form of C. perfringens –caused disease, food
poisoning, is characterized by diarrhea without fever or vomiting.

Epidemiology
Gas gangrene develops in traumatic wounds with muscle damage when they are
contaminated with dirt, clothing, or other foreign material containing C. perfringens
or another species of histotoxic clostridia. The clostridia can come from the patient’s
own intestinal ora or spores in the en vironment. Compound fractures, bullet
wounds, or the kind of trauma seen in wartime are prototypes for this infection. A
signi cant delay between the injury and definitive surgical management is an
additional requirement. These conditions are more likely to occur in peacetime in a
hiking accident in a remote area rather than in an automobile accident on a freeway.

Pathogenesis
If the oxidation–reduction potential in a wound is sufficiently low, C. perfringens
spores can germinate and can multiply, elaborating _-toxin. The process passes
along the muscle bundles, producing rapidly spreading edema and necrosis as well
as conditions that are more favorable for growth of the bacteria. Very few leukocytes
are present in the myonecrotic tissue. As the disease progresses, increased vascular
permeability and systemic absorption of the toxin and inflammatory mediators leads
to shock. θ-toxin and oxygen deprivation due to the metabolic activities of C.
perfringens are probable contributors. The basis for the profound systemic effects is
not known, but toxin absorption seems probable, because fatal cases occur without
bacteremia.

Manifestations
Gas gangrene usually begins 1 to 4 days after the injury but may start within 10
hours. The earliest reported finding is severe pain at the site of the wound
accompanied by a sense of heaviness or pressure. The disease then progresses
rapidly with edema, tenderness, and pallor, which is followed by discoloration and
hemorrhagic bullae. The gas is apparent as crepitance in the tissue, but this is a late
sign. Systemic findings are those of shock with intravascular hemolysis,
hypotension, and renal failure leading to coma and death. Patients are often
remarkably alert until the terminal stages.

Diagnosis
Diagnosis is based ultimately on clinical observations. Bacteriologic studies are
adjunctive. It is quite common, for example, to isolate C. perfringens from

47
contaminated wounds of patients who have no evidence of clostridial disease. The
organism can also be isolated from the postpartum uterine cervix of healthy women
or from those with only mild fever. Occasionally, C. perfringens is even isolated
from blood cultures of patients who do not develop serious clostridial infection.

Treatment and prevention

Treatment of gas gangrene and endometritis must be initiated immediately because
these conditions are almost always fatal if untreated. Excision of all devitalized
tissue is of paramount importance, because it denies the organism the anaerobic
conditions required for further multiplication and toxin production. This often entails
wide resection of muscle groups, hysterectomy, and even amputation of limbs.
Administration of massive doses of penicillin is an important adjunctive procedure.
Because nonclostridial anaerobes and members of the Enterobacteriaceae frequently
contaminate injury sites, clindamycin and broad-spectrum cephalosporins are often
added to the antibiotic regimen. Placement of patients in a hyperbaric oxygen
chamber, which increases the tissue level of dissolved oxygen, has been shown to
slow the spread of disease, probably by inhibiting bacterial growth and toxin
production and by neutralizing the activity of θ-toxin.
The most effective method for prevention of gas gangrene is the surgical
debridement of traumatic injuries as soon as possible. Thorough cleansing, removal
of dead tissue and foreign bodies, and drainage of hematoma limit organism
multiplication and toxin production. Antimicrobic prophylaxis is indicated but
cannot replace surgical debridement, because the antimicrobics may fail to reach the
organism in devascularized tissues.

48
 CLOSTRIDIUM TETANI
C. tetani is a slim, Gram-positive rod, which may stain Gram negative in very young
or old cultures. It forms spores readily in nature and in culture, yielding a typical
round terminal spore that gives the organism a drumstick appearance before the
residual vegetative cell disintegrates. The organism is flagellate and motile. C. tetani
requires strict anaerobic conditions. Its identity is suggested by cultural and
biochemical characteristics, but definite identification depends on demonstrating its
neurotoxic exotoxin. C. tetani spores remain viable in soil or culture for many years.
It is resistant to most disinfectants and withstands boiling for several minutes. The
most important product of C. tetani is its neurotoxic exotoxin, tetanospasmin or
tetanus toxin.

Epidemiology
The spores of C. tetani exist in many soils, especially those that have been treated
with manure, and the organism is sometimes found in the lower intestinal tract of
humans and animals. The spores are introduced into wounds contaminated with soil
or foreign bodies. The wounds are often quite small, (eg, a puncture wound with a
splinter). In many developing countries, the majority of tetanus cases occur in
recently delivered infants when the umbilical cord is severed or bandaged in a
nonsterile manner. Similarly, tetanus may follow an unskilled abortion, scarification
rituals, female circumcision, and even surgery performed with nonsterile
instruments or dressings.

Pathogenesis
The usual predisposing factor for tetanus is an area of very low oxidation–reduction
potential in which tetanus spores can germinate, such as a large splinter, an area of
necrosis from introduction of soil, or necrosis after injection of contaminated illicit
drugs. Infection with facultative or other anaerobic organisms can contribute to the
development of an appropriate anaerobic nidus for spore germination. Tetanus
bacilli multiply locally and neither damage nor invade adjacent tissues.
Tetanospasmin is elaborated at the site of infection and enters the presynaptic
terminals of lower motor neurons, reaching the central nervous system (CNS)
mainly by exploiting the retrograde axonal transport system in the nerves. In the
spinal cord, it acts at the level of the anterior horn cells, where its blockage of
postsynaptic inhibition of spinal motor reflexes produces spasmodic contractions of
both protagonist and antagonist muscles. This process takes place initially in the area
of the causative lesion but may extend up and down the spinal cord. Minor stimuli,
such as a sound or a draft, can provoke generalized spasms.

49
Manifestations
The incubation period of the disease is from 4 days to several weeks. The shorter
incubation period is usually associated with wounds in areas supplied by the cranial
motor nerves, probably because of a shorter transmission route for the toxin to the
CNS. In general, shorter incubation periods are associated with more severe disease.
The diagnosis is clinical; neither culture nor toxin testing are useful. Although
tetanus may be localized to muscles innervated by nerves in the region of the
infection, it is usually more generalized. The masseter muscles are often the first to
be affected, resulting in inability to open the mouth properly (trismus); this effect
accounts for the use of the term lockjaw to describe the disease. As other muscles
become affected, intermittent spasms can become generalized to include muscles of
respiration and swallowing. In extreme cases, massive contractions of the back
muscles (opisthotonos) develop.
Untreated patients with tetanus retain consciousness and are aware of their plight, in
which small stimuli can trigger massive contractions. In fatal cases, death results
from exhaustion and respiratory failure. Untreated, the mortality caused by the
generalized disease varies from 15 to more than 60%, according to the lesion,
incubation period, and age of the patient. Mortality is highest in neonates and in
elderly patients.

Treatment
Specific treatment of tetanus involves neutralization of any unbound toxin with large
doses of human tetanus immune globulin (HTIG), which is derived from the blood
of volunteers hyperimmunized with toxoid. Most important in treatment are
nonspecific supportive measures, including maintenance of a quiet dark
environment, sedation, and provision of an adequate airway. Benzodiazepines are
also used to indirectly antagonize the effects of the toxin. The value of antimicrobics
is not clear. Because toxin binding is irreversible, recovery requires the generation
of new axonal terminals.

Prevention
Routine active immunization with tetanus toxoid, combined with diphtheria toxoid
and pertussis vaccine (DTaP) for primary immunization in childhood and DT for
adults, can completely prevent the disease. It has reduced the incidence of tetanus in
the United States to less than 50 reported cases per year. Five doses of DT are
recommended, to be given at the ages of 2, 4, 6, and 18 months, and once again
between the ages of 4 and 6 years. Thereafter a booster of adult-type tetanus

50
diphtheria toxoid should be given every 10 years. Unfortunately, routine childhood
immunization is not administratively and economically feasible in many less well-
developed countries, where as many as a million cases of tetanus occur annually. In
such settings, immunization efforts have been focused on pregnant women, because
transplacental transfer of antibodies to the fetus also prevents the highly lethal
neonatal tetanus.
Unimmunized subjects with tetanus-prone wounds should be given passive
immunity with a prophylactic dose of HTIG as soon as possible. This immunization
provides immediate protection. Those who have had a full primary series of
immunizations and appropriate boosters are given toxoid for tetanus-prone wounds
if they have not been immunized within the previous 10 years in the case of clean
minor wounds or 5 years for more contaminated wounds. If immunization is
incomplete or the wound has been neglected and poses a serious risk of disease,
HTIG is also appropriate. Penicillin therapy is a prophylactic adjunct in serious or
neglected wounds but in no way alters the need for specific prophylaxis.

51
 AIRBORNE BACTERIAL INFECTION
The lower respiratory tract is sterile. However the upper respiratory tract, the nose
and throat are colonized by many organisms. These organism are: Staphylococci,
Streptococci, Pneumococci, Haemophilus and Neisseria.
Normal defenses against infection:
1. Arrangement of nose . there is no direct entry of air
2. Broncho constriction : helps the organisms to be trapped
3. Cough reflex : expels the microbes out side
4. Mucociliary blanket: traps the organisms
5. Mucosal factors: kill the organisms
a. Non specific
i. Lysozyme : Cell wall of gram positive organism are lysed
ii. Influenza virus inhibitors :
iii. Resident macrophages : kill the organism
b. Specific
i. Secretory IgA antibody : gives first line of defense
Predisposing factors for respiratory tract infections:
1. Ciliated epithelial cell damage due to
a. Viruses
b. Chemicals
c. Smoking
2. Fluid accumulation
3. Decreased activity of macrophages
All these factors help in the establishment of the microbes in the respiratory tract.
Respiratory infections can be conveniently classified into Upper respiratory and
Lower respiratory infections.

52
 Respiratory infections

URI LRI

Sinusitis Trachiitis
Pharyngitis Bronchitis
Laryngitis Tracheo bronchitis
Epiglotitis Alveolitis
Pneumonia

STREPTOCOCCUS PNEUMONIAE
Streptococcus pneumoniae (or pneumococcus) is a Gram-positive coccus seen in
pairs, which is typically α-haemolytic, but can be variable.
• tissue damaging enzymes (e.g. neuraminidase, hyaluronidase).

Epidemiology
Humans are the only host of S. pneumoniae. Carriage, which is usually
asymptomatic, is most common in the young or smokers and is associated with
overcrowding. Serotypes vary with country, time and subject group.
Children under 1 year of age are vulnerable to acute pneumonia. Complement
deficiency, agammaglobulinaemia, HIV infection, smoking, alcoholism and
splenectomy predispose to severe infection. The bacteria are able to adhere to
pneumocytes and invade the bloodstream by hijacking the platelet-aggregating
factor receptor pathway and produce complement-mediated damage to the alveolus
through the action of pneumolysin.

53
Pathogenesis
Streptococcus pneumoniae has a polysaccharide capsule that protects it from
phagocytosis. There are over 90 highly antigenic capsular serotypes and antibodies
to specific types are protective.
Pathogenicity features include:
• pro-inflammatory cell wall components (e.g. C-polysaccharide, F-antigen);
• IgA2 protease;
• pneumolysin, a cytotoxin that stimulates immune responses;
• adhesins that bind to cell surface carbohydrates (e.g. choline binding protein A,
pneumococcal surface protein A [PspA]).

Clinical manifestations
• Acute otitis media, sinusitis and acute pneumonia are the most common infections.
• Pneumococci cause between 50 and 75% of cases of communityacquired
pneumonia, up to 25–30% of which may develop bacteraemia.
• Bacteraemia is an important complication with a high mortality, despite treatment.
• Direct or haematogenous spread can give rise to meningitis, which has a high
mortality and is associated with brain damage. This is now the commonest cause of
meningitis in adults over 40 and the second commonest cause in children from
populations that have been vaccinated against Haemophilus influenzae type b (Hib).
• Pneumococcus rarely causes cellulitis, abscesses, peritonitis and endocarditis.
• The mortality and incidence of sequelae are high.
54
Antibiotic susceptibility and treatment
Once universally susceptible to penicillin, significant numbers of S. pneumoniae
have developed resistance through a genetically modified penicillin-binding protein
gene , and penicillin-resistant clones have spread internationally. S. pneumonia is
also susceptible to erythromycin, cephalosporins, tetracycline, rifampicin and
chloramphenicol, but multiple drug resistance is growing. Penicillin is the treatment
of choice for respiratory infection but third generation cephalosporins are used for
meningitis if it is caused by less sensitive strains. Where high-level penicillin
resistance occurs, a glycopeptide (usually vancomycin) should be added.

Prevention and control

A conjugate vaccine incorporating up to 13 capsular serotypes has been introduced
and is highly immunogenic in young children. Whilst this has led to a decline in
invasive pneumococcal disease in children and adults, there is some evidence that
serotypes not included in the vaccine are increasing.

Other streptococci
Streptococcus agalactiae (group B) Neonatal pneumonia, septicaemia,
meningitis, rarely adult infection
Streptococcus equi, S. equisimilis, Endocarditis, skin infection, pharyngitis
S. dysgalactiae, S. zooepidemicus
(group C)
Enterococci (group D) E. faecalis, UTI, endocarditis, intra-abdominal
faecium,E. bovis polymicrobial infection, infection in
immunocompromised, ! resistance to
glycopeptides
S. milleri group
S. anginosus-intermedius- Metastatic abscesses, periodontal sepsis
constellatus
S. oralis, S. sanguis, S. mitis Endocarditis
S. mutans Dental caries

55
 NEISSERIA MENINGITIDES
Meningococci are usually quiescent members of the nasopharyngeal flora but may
produce fulminant infection of the bloodstream and/or central nervous system
(CNS). There is little warning; localized infections that precede systemic spread are
rarely recognized. The major disease is an acute, purulent meningitis with fever,
headache, seizures, and mental signs secondary to inflammation and increased
intracranial pressure. Even when the CNS is not involved, N. meningitides infections
have a marked tendency to be accompanied by rash, purpura, thrombocytopenia, and
other manifestations associated with endotoxemia. This bacterium causes one of the
few infections in which patients may progress from normal health to death in less
than a day.

Pathogenesis and clinical features

• An antiphagocytic polysaccharide capsule that allows survival in the bloodstream.
• Lipo-oligosaccharide activates complement and stimulates cytokine release, which
leads to shock and disseminated intravascular coagulation (DIC).
• The organism hijacks the !2-adrenoreceptor to cross the brain vascular
endothelium.
• Meningococci cross mucosal epithelium by endocytosis.
Meningococcal meningitis is characterized by fever, neck stiffness and reduced
consciousness. The petechial rash, a sign of septicaemia, may be present without
other signs of meningitis. Septic or reactive arthritis may develop.

Epidemiology
Carriage of Neisseria meningitidis (meningococcus) is common; actual disease only
develops in a few individuals. Infection is most common in the winter, with
epidemics occurring every 10–12 years. In Africa, severe epidemics of group A
infection occur in the ‘meningitis belt’ where the incidence can rise to 1000 cases
per 100 000 each year. Most invasive infections are caused by serogroups A, B or
C. Group B infection is now the commonest, as the incidence of group C infection
has reduced in communities where vaccination has become routine. A group A
vaccination programme is being implemented in Africa.

Diagnosis and treatment

The diagnosis is usually made clinically and confirmed by culture of blood, aspirate
from the rash and CSF. Rapid antigen detection or NAAT on CSF and blood are
sensitive and reliable.

56
Infection is life-threatening and rapidly progressive; treatment should not await
laboratory confirmation or hospitalization. Intravenous benzylpenicillin
(intramuscular in the community setting) is the antibiotic of choice, but there have
been reports of meningococci with reduced susceptibility in other countries and
cefotaxime is an alternative. Treatment does not eradicate carriage so the patient
should be given ‘prophylaxis’ following recovery.

57
58
Prevention
• A protein-conjugated serogroup C vaccine is more than 90% efficient and has
vastly reduced the incidence where it has been introduced.
• An effective vaccine against serogroup B is not available, although a vaccine based
on membrane proteins specific for epidemic strains has shown promise.
• Close contacts of patients with meningococcal meningitis should be given
‘prophylaxis’ with rifampicin or ciprofloxacin.

BORDETELLA PERTUSSIS
Bordetella pertussis and B. parapertussis can cause whooping cough. In the absence
of an adequate vaccination campaign, epidemics of whooping cough occur in
children every 4 years. Asymptomatic or unrecognized infection in adolescents and
young adults maintains the cycle of infection in the human population.

59
Pathogenesis
Bordetella pertussis express fimbriae that aid their adhesion and produce a number
of exotoxins that include pertussis toxin, adenyl cyclase and tracheal cytotoxin.
There is a complex interaction with the cells of the respiratory tract that produces
thickened bronchial secretions and paroxysmal cough. Complications include:
• secondary respiratory tract infection;
• apnoea following coughing spasms;
• raised intracranial pressure.

Clinical manifestations
A 2-week, cold-like illness occurs before the characteristic cough is heard –
repeated, prolonged coughing fits followed by an inspiratory whoop that may be
absent in very young children and adults. Coughing may be associated with vomiting
and subconjunctival haemorrhage; this phase can last for up to 3 months. Infection
can be complicated by secondary pneumonia and otitis media.

Laboratory diagnosis
Specimens for culture are obtained using a pernasal swab, but the organism is
difficult to isolate and NAATs are more likely to achieve a diagnosis.

Treatment
Erythromycin is thought to decrease infectivity and shorten symptoms if given early
during the catarrhal phase. Symptomatic support and early treatment of secondary
infections is the mainstay of treatment.

Prevention and control

An acellular vaccine is given as part of the childhood vaccination scheme.

CORYNEBACTERIUM DIPHTHERIA
Diphtheria is a disease caused by the local and systemic effects of diphtheria toxin,
a potent inhibitor of protein synthesis. The local disease is a severe pharyngitis
typically accompanied by a plaquelike pseudomembrane in the throat and trachea.
The life-threatening aspects of diphtheria are due to the absorption of the toxin
across the pharyngeal mucosa and its circulation in the bloodstream. Multiple organs
are affected, but the most important is the heart, where the toxin produces an acute
myocarditis.

Pathogenesis
60
Diphtheria is caused by Corynebacterium diphtheriae that contain a bacteriophage
encoding diphtheria toxin. The toxin kills cells by interrupting protein synthesis,
acting on the myocardium to cause myocarditis and on the peripheral nervous system
to cause neuropathy and paralysis. The severity of infection is directly related to the
degree of toxin production. Cutaneous infection is often asymptomatic.
Corynebacterium diphtheriae is transmitted by the respiratory route or following
direct contact with cutaneous lesions.

Clinical manifestations and management

Infection of the skin, nasopharynx or larynx may occur; the severity of disease is
related to the extent of the infection. A rare cause of sore throat, it causes
inflammation and necrosis giving a green–black ‘pseudomembrane’ on the posterior
wall of the pharynx, which can cause respiratory obstruction. Management is based
on isolation and treatment with antitoxin and erythromycin. Intensive care support
may be required.

Laboratory diagnosis
Corynebacterium diphtheriae is isolated using specialist media (e.g. Hoyle’s) and
identified by biochemical tests and confirmed by 16s rRNA sequencing. The toxin
gene is detected by nucleic acid amplification test (NAAT).

Prevention and control

Diphtheria is prevented by childhood vaccination with a toxoid. Immunity is long-
lasting but boosters for adults at extra risk may be required (e.g. laboratory staff).

61
Contacts of cases must be identified and given antibiotic prophylaxis, vaccination
and/or specific antitoxin.

MYCOBACTERIUM TUBERCULOSIS
Tuberculosis is a systemic infection manifested only by evidence of an immune
response in most exposed individuals. In some infected persons, the disease either
progresses or, more commonly, reactivates after an asymptomatic period (years).The
most common reactivation form is a chronic pneumonia with fever, cough, bloody
sputum, and weight loss. Spread outside of the lung also occurs and is particularly
devastating when it reaches the central nervous system. The natural history follows
a course of chronic wasting to death aptly called “consumption” in the past.

Epidemiology and pathogenesis

Tuberculosis is spread from person to person by the airborne route. In childhood, the
lung is the first site of infection and most infections resolve with local scarring (the
primary complex). In about 5% the infection is not controlled and it spreads from
the primary focus throughout the body (miliary spread). This may resolve
spontaneously or develop into other localized infections (e.g. meningitis,
osteomyelitis). Disease may emerge if immunity wanes or in later life (10% lifetime
risk). Immunity depends on effective T-cell function so if compromised, individuals
are more likely to develop symptomatic disease.
Mycobacterium tuberculosis is ingested by macrophages but escapes from the
phagolysosome to multiply in the cytoplasm. The intense immune response causes
local tissue destruction (cavitation in the lung) and cytokine-mediated systemic
effects (fever and weight loss). Many antigens have been identified as possible
virulence determinants, including lipoarabinomannan (stimulates cytokines) and
superoxide dismutase (promotes intramacrophage survival).

Clinical manifestations
Mycobacterium tuberculosis may affect every organ of the body mimicking both
inflammatory and malignant diseases.
• Pulmonary: chronic cough, haemoptysis, fever and weight loss, or recurrent
bacterial pneumonia. If untreated, it follows a chronic, deteriorating course.
• Meningitis: fever and deteriorating level of consciousness.
• Renal: signs of local infection, fever and weight loss, complicated by ureteric
fibrosis and hydronephrosis.

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• Bone: the lumbosacral spine is a common site of infection. Complicated by
vertebral collapse and nerve compression. Pus may spread under the psoas sheath to
appear in the groin (psoas abscess).
• Joints: infects large joints causing destructive arthritis.
• Abdominal: mesenteric lymphadenopathy and chronic peritonitis may present as
fever, weight loss, ascites and intestinal malabsorption.
• Miliary disease: may occur without evidence of active lung infection.

Laboratory diagnosis
A wide range of specimens can be examined, using a variety of techniques.
• Direct staining with Ziehl–Neelsen’s method or auramine.
• Culture on lipid-rich (egg-containing) medium with malachite green (Lowenstein–
Jensen or L–J medium) to suppress other organisms.
• Automated growth detection, which can speed isolation.
• Susceptibility is usually tested in automated systems (e.g. Mycobacterial Growth
Indicator Test [MGIT]).
• Nucleic acid amplification tests (NAATs) that include detection of drug resistance
mutations allow rapid identification of patients with multidrug resistance TB
(MDRTB) so they can be isolated and appropriately treated.
• Typing by identification of the number of copies of 23 repetitive DNA elements.
• Immunity is determined by measuring the !-interferon response to specific antigens
(Interferon Gamma Release Assay [IGRA]).

Treatment and prevention

The standard regimen for pulmonary infection is rifampicin and isoniazid for 6
months, with ethambutol and pyrazinamide for the first 2 months. Regimens for
other sites are similar, taking into account drug penetration (e.g. into CSF). There is
a rising trend of MDRTB. Features that predispose a patient to this include:
• previous incomplete treatment;
• being a known contact of an MDR patient;
• residence in a country where MDRTB is common;
• failure to respond to an adequate regimen.
Treatment of MDRTB is complex, requiring a combination of second-line agents,
such as aminoglycosides, fluoroquinolones, ethionamide or cycloserine, guided by
susceptibility tests.
Vaccination with attenuated bacille Calmette–Guerin (BCG) strain may protect
against miliary spread, but trials in some countries have shown no benefit. Patients

63
at high risk of developing tuberculosis may be given prophylaxis with isoniazid and
rifampicin.

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 SEXUALLY TRANSMITTED DISEASES
Sexually transmitted diseases are the most common communicable diseases after
common cold. They are transmitted through sexual contact. They can be classified
into the following:
1. Bacterial
2. Viral
3. Parasitic
4 Fungal diseases.
TREPONEMA PALLIDUM
Treponema pallidum causes syphilis, which may be transmitted sexually or
congenitally. The incidence is now increasing worldwide, having been falling for
many years. The related organisms T. pertenue and T. carateum cause yaws and
pinta respectively. They are spread by contact, usually in childhood. Once common
in the tropics, they are now rare as the result of an eradication campaign.

Clinical manifestations
Treponema pallidum penetrate intact skin or mucosa disseminating throughout the
body to cause disease in four stages:
1 Primary chancre (painless ulcer with a rubbery edge and regional
lymphadenopathy).
2 Secondary (an acute febrile illness with a generalized non-itchy scaling rash that
typically involves the palms, associated with lymphadenopathy).
3 Latent phase, which may last for many years.
4 Tertiary (systemic lesions become symptomatic, e.g. aortitis, posterior cord
degeneration and dementia).
The characteristic syphilitic lesions (gummas), which consist of necrosis and
obliterative endarteritis with fibroblastic proliferation and lymphocyte infiltration,
are found throughout the body.

Diagnosis
Microscopy
T. pallidum can be seen by darkfield microscopy in primary and secondary lesions,
but the execution of this procedure requires experience and attention to detail. The
suspect lesion must be cleaned and abraded to produce a serous transudate from
below the surface of the ulcer base. This material can be captured in a capillary tube
or placed directly on a microscope slide if a darkfield setup is close at hand. The
microscopist must observe the corkscrew morphology and characteristic motility to
make a diagnosis. A negative examination does not exclude syphilis, because to be

65
readily seen, the fluid must contain thousands of treponemes per milliliter. Darkfield
microscopy of oral and anal lesions is not recommended because of the risk of
misinterpretation of other spirochetes present in the normal flora. Direct fluorescent
antibody methods have been developed but are available only in certain centers.
Serologic Tests
Most cases of syphilis are diagnosed serologically using serologic tests that detect
antibodies directed at either lipid or specific treponemal antigens. The former are
called nontreponemal tests, and the latter are referred to as treponemal tests. Their
use in screening, diagnosis, and therapeutic evaluation of syphilis has been refined
over many decades.
Nontreponemal Tests
Nontreponemal tests measure antibody directed against cardiolipin, a lipid complex
so called because one component was originally extracted from beef heart.
Anticardiolipin antibody is called reagin, and the tests which detect it depend on
immune flocculation of cardiolipin in the presence of other lipids. The most common
nontreponemal tests are the rapid plasma regain (RPR) and the Venereal Disease
Research Laboratory (VDRL). They become positive in the early stages of the
primary lesion and, with the possible exception of some patients with advanced
human immunodeficiency virus (HIV) infection, are uniformly positive during the
secondary stage. They slowly wane in the later stages of the disease. In
neurosyphilis, VDRL tests on CSF may be positive when the serum VDRL has
reverted to negative. Nontreponemal tests are nonspecific; they may become positive
in a variety of autoimmune diseases or in diseases involving substantial tissue or
liver destruction, such as lupus erythematosus, viral hepatitis, infectious
mononucleosis, and malaria. False-positive results can also occur occasionally in
pregnancy and in patients with HIV infection.
Sensitivity and low cost make nontreponemal tests preferred for screening, but if
positive, they must be confirmed by one of the more specific treponemal tests
described below. They are also valuable for following treatment because the height
of the antibody titer is directly related to activity of disease. With successful
antibiotic therap y nontreponemal serologies slowly revert to negative.
Treponemal Tests
Treponemal tests detect antibody specific to T. pallidum such as an indirect
immunofluorescent procedure called the fluorescent treponemal antibody (FTA-
ABS) which uses spirochetes fixed to slides. The “ABS” refers to an absorption step
that removes nonspecific antispirochetal antibodies often found in normal serum.
Another method, the microhemagglutination test for T. pallidum (MHA-TP), uses

66
antigens attached to the surface of erythrocytes, which then agglutinate in the
presence of specific antibody.
Treponemal tests are considerably more specific than the cardiolipin-based
nontreponemal tests. Their primary role in diagnosis is to confirm positive RPR and
VDRL results obtained in the evaluation of a patient suspect for syphilis or in
screening programs.
They are not useful for screening or following therapy because, once positive, they
usually remain so for life. Thus, if nontreponemal tests can be thought of as the
measure of active syphilis, treponemal tests are the indelible print of sin. Until
recently, it was believed that a negative treponemal test excluded the possibility of
prior syphilis. The use of serologic tests in the diagnosis of congenital syphilis is
complicated by the presence of IgG antibodies in infants, who acquire it
transplacentally from their mothers. If available, treponemal IgM tests are useful in
establishing the presence of an acute infection in infants.

Treatment
• Penicillin (or either azithromycin or tetracyclines if the patient is allergic).
• An acute febrile response (the Jarisch–Herxheimer reaction) may develop in some
patients after the first dose of antibiotics.
• Careful serological follow-up is essential to confirm cure and/or detect early central
nervous system involvement.

NEISSERIA GONORRHOEAE
Neisseria gonorrhoeae are gram negative diplococci, kidney shaped, the concave
side face each other, nonmotile. Grows on enriched medium in presence of 5-10%
CO2, is most common in individuals between 15 and 35 years of age. It is almost
exclusively spread by sexual contact.

Pathogenesis
The organism adheres to the genitourinary epithelium via pili, then invades the
epithelial layer and provokes a local acute inflammatory response. Variation in the
proteins of the pili means that infection does not provide protection against re-
infection, therefore infections with another strain of different antigenic structure are
possible.

67
Clinical features
• Acute painful urethritis and urethral discharge.
• Female infection (cervicitis) is often asymptomatic or associated with vaginal
discharge.
68
• Pelvic inflammatory disease (PID) may develop.
• Pharyngeal infection causes sore throat.
• Rectal infection causes a purulent proctitis.
• Infection can be complicated by bacteraemia, septic or reactive arthritis of the large
joints, or pustular rashes.
• Late complications include female infertility and male urethral stricture.

Diagnosis
Gram Smear
The presence of multiple pairs of bean-shaped, Gram-negative diplococci within a
neutrophil is highly characteristic of gonorrhea when the smear is from a genital site.
The direct Gram smear is more than 95% sensitive and specific in symptomatic men.
Unfortunately, it is only 50 to 70% sensitive in women, and its specificity is
complicated by the presence of other bacteria in the female genital flora that may
have a similar morphology. Experience is required in reading smears, particularly in
women. Although a positive Gram smear is generally accepted as diagnostic in men,
it should not be used as the sole source for diagnosis when the findings are
unexpected or have social (divorce) or legal (rape, child abuse) implications.
Culture
Attention to detail is necessary for isolation of the gonococcus, because it is a fragile
organism that is often mixed with hardier members of the normal flora. Success
requires proper selection of culture sites, protection of specimens from
environmental exposure, culture on appropriate media, and definitive laboratory
identification. In men, the best specimen is urethral exudate or urethral scrapings
(obtained with a loop or special swab). In women, cervical swabs are preferred over
urethral or vaginal specimens. The highest diagnostic yield in women is with the
combination of a cervical and an anal canal culture, because some patients with
rectal gonorrhea have negative cervical cultures. Throat or rectal cultures in men are
needed only if indicated by the patient’s sexual practices.
Swabs may be streaked directly onto culture medium or transmitted to the laboratory
in a suitable transport medium if the delay is not more than 4 hours. Laboratory
requests must specify the suspicion of gonorrhea, so that media that satisfy the
nutritional requirements of the gonococcus and inhibit competing normal flora can
be seeded. The most common medium is Martin–Lewis agar, an enriched selective
chocolate agar. The exact formulation has changed over the years, but includes
antimicrobics active against Grampositive bacteria (vancomycin), Gram-negative
bacteria (colistin, trimethoprim), and fungi (nystatin, anisomycin) at concentrations
that do not inhibit N. gonorrhoeae.

69
Colonies appear after 1 to 2 days of incubation in carbon dioxide at 35°C. They may
be identified as Neisseria by demonstration of typical Gram stain morphology and a
positive oxidase test. Classically, speciation is by carbohydrate degradation pattern,
but this approach has been replaced by immunologic procedures
(immunofluorescence, coagglutination, enzyme immunoassay) using monoclonal
antibodies to unique antigens such as protein I. Neisseria species other than
N. gonorrhoeae are unusual in genital specimens, but speciation is the only way to
be certain of the diagnosis.
Direct Detection
Much effort has been directed at developing immunoassay and nucleic acid
hybridization methods that detect gonococci in clinical specimens without culture.
Such methods could have particular importance for screening populations where
culture is impractical. Of these only the DNA amplification methods have the
sensitivity to substitute for culture. The main barrier to their broader use is cost,
which may be overcome by combining them with Chlamydia detection that targets
the same clinical population.
Serology
Attempts to develop a serologic test for gonorrhea have not yet achieved the needed
sensitivity and specificity. A test that would detect the disease in asymptomatic
patients would be very useful in control of this disease.

Treatment and prevention

Treatment must be given before susceptibility results are available and is based on
the known susceptibility patterns found at the clinic, as emergence of resistance is a
problem. Ceftriaxone, spectinomycin or fluoroquinolones may be used. Gonorrhoea
can be prevented by avoiding sexual contact with individuals at high risk and using
effective barrier contraception. Contacts of infected individuals should be traced and
treated. At present vaccine development is precluded by the antigenic variation that
occurs within the pili.

CHLAMYDIAL DISEASE
There are many serotypes in Chlamydia. Some of them causegenital infections.
Lymphogranuloma venereum is one of chlamydial sexually transmitted diseases.
First a vesicle develops and the lesion ulcerates in the genitals. The inguinal lymph
nodes enlarge, suppurate and release pus through multiple sinus tracts. If not
treated it will lead to other complications. Sulfonamides and tetracycline are used
for the treatment.

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 ZOONOTIC DISEASES
Association between man and animals are there since the beginning of earth. Man’s
association with animals became more intimate that certain diseases are transmitted
from animals to man and vice a versa. Zoonotic diseases are transmissible between
man and animals. Zoonotic diseases spread by contact with infected animals by
handling them or living in their premises. Transmission is through direct contact or
aerosol spread. Drinking milk from infected animals is another way of getting
zoonotic infections. Insects could transfer mechanically or biologically different
zoonotic diseases. These diseases are classified as follows:
Zoonoses Animals ↔ Man
Anthrapozoonoses Animals → Man (Rabies)
Zooanthroponoses Man → Animals( Diphtheria)
Cyclozoonoses Animal ↔ Man (Hydatidosis, Taeniosis)
Metazoonoses Animals → Man ( through insect vector: Malaria)
Saprozoonoses Animals → Man (through soil)

BRUCELLA
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Brucella melitensis, B. abortus and B. suis have goats, cattle and pigs, respectively,
as their main hosts. They are aerobic or capnophilic and require serum-containing
medium to grow.

Epidemiology
Brucella infection spreads to humans through direct contact with domesticated
animals or their products (e.g. unpasteurized milk). Vets, farmers and abattoir
workers are at increased risk of infection.

Pathogenesis
Brucellae are able to survive inside the cells of the reticuloendothelial system using
superoxide dismutase and nucleotide-like substances to inhibit the intracellular
killing mechanisms of their host.

Clinical manifestations
• Intermittent, high fever in the early stages of infection, giving rise to its old name
‘undulant fever’.
• Myalgia, arthralgia and lumbosacral tenderness.
• Complications of acute infection that include septic arthritis, osteomyelitis and
epididymo-orchitis.
• A chronic infection that may develop without treatment, which may resolve or
continue to give symptoms, often accompanied by psychiatric complaints, for many
years.

Laboratory diagnosis
Culture of blood and bone marrow is diagnostic, although culture is less likely to be
positive in chronic disease. Incubation, in a high containment facility, must be
continued for up to 3 weeks. Serodiagnosis is by enzyme immunoassay (EIA) to
detect both IgG and IgM.

Treatment and prevention

Optimal treatment is with tetracycline for 1 month. Streptomycin should be added
for patients with complications. Transmission by food can be prevented by
pasteurization. Appropriate animal husbandry techniques can reduce the risk of
occupational infection. An animal vaccine is available but is not sufficiently safe for
human use. Animal control measures have eradicated brucellosis from farms in
many countries.

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 YERSINIA PESTIS
Y. pestis is a nonmotile, non–spore-forming, Gram-negative bacillus with a tendency
toward pleomorphism and bipolar staining.
Plague, an infection of rodents transmitted to humans by the bite of infected fleas, is
the most explosively virulent disease known. Most cases begin with a painful
swollen lymph node (bubo) from which the bacteria rapidly spread to the
bloodstream. Plague pneumonia (Black Death) is produced by seeding from the
bloodstream or from another patient with pneumonia. All forms cause a toxic picture
with shock and death within a few days. No other disease regularly kills previously
healthy persons so rapidly.

Clinical manifestations
The incubation period for bubonic plague is 2 to 7 days after the flea bite. Onset is
marked by fever and the painful bubo, usually in the groin (bubo is from the Greek
boubon for “groin”) or, less often, in the axilla. Without treatment, 50 to 75% of
patients progress to bacteremia and die in Gram-negative septic shock within hours
or days of development of the bubo. About 5% of victims develop pneumonic plague
with mucoid, then bloody sputum. Primary pneumonic plague has a shorter
incubation period (2 to 3 days) and begins with only fever, malaise, and a feeling of
tightness in the chest. Cough, production of sputum, dyspnea, and cyanosis develop
later in the course. Death on the second or third day of illness is common, and there
are no survivors without specific therapy. A terminal cyanosis seen with pneumonic
plague is responsible for the term Black Death. Even today, plague pneumonia is
almost always fatal if appropriate treatment is delayed more than a day from the
onset.

Diagnosis
Gram smears of aspirates from the bubo typically reveal bipolar-staining Gram-
negative bacilli. An immunofluorescence technique is available in public health
laboratories for immediate identification of smears or cultures. Y. pestis is readily
isolated on the media used for other members of the Enterobacteriaceae (blood agar,
MacConkey agar), although growth may require more than 24 hours of incubation.
The appropriate specimens are bubo aspirate, blood, and sputum. Laboratories must
be notified of the suspicion of plague to avoid delay in the bacteriologic diagnosis
and to guard against laboratory infection.

Treatment

73
Streptomycin is the treatment of choice for both bubonic and pneumonic plague,
because its effectiveness has been proven. Tetracycline, chloramphenicol, and
trimethoprim–sulfamethoxazole are alternatives. Timely treatment reduces the
mortality of bubonic plague below 10%.

Prevention
Urban plague has been prevented by rat control and general public health measures
such as use of insecticides. Sylvatic plague is virtually impossible to eliminate
because of the size and dispersion of the multiple rodent reservoirs. Disease can be
prevented by avoidance of sick or dead rodents and rabbits. Eradication of fleas on
domestic pets, which have been known to transport infected fleas from wild rodents
to humans, is recommended in endemic areas. The continued presence of fully
virulent plague in its sylvatic cycle poses a risk of extension to the urban cycle and
epidemic disease in the event of major disaster or social breakdown.
Chemoprophylaxis with tetracycline is recommended for those who have had close
contact with a case of pneumonic plague. It is also used for the household contacts
of a case of bubonic plague, because they may have had the same flea contact. A
formalin-killed plague vaccine once used for those in high-risk occupations is no
longer available.

FRANCISELLA TULARENSIS
Francisella tularensis is a small, facultative, coccobacillary, Gram-negative rod
with much the same morphology as Brucella. It is one of the few bacterial species
of medical importance that does not grow on the usual enriched media. This
characteristic is due to a special requirement for sulfhydryl compounds, and growth
occurs best on a cysteine–glucose blood agar medium incubated aerobically. On
primary isolation, 2 to 10 days of incubation is required for appearance of the tiny
transparent colonies. The species is antigenically homogeneous.
Tularemia is a disease of wild mammals caused by F. tularensis. Humans become
infected by direct contact with infected animals or through the bite of a vector (tick
or deer fly). The illness is characterized by high fever and severe constitutional
symptoms. The epidemiology of tularemia and many features of the clinical
infection are similar to those of plague.

Clinical manifestations
After an incubation period of 2 to 5 days, tularemia may follow a number of courses,
depending on the site of inoculation and extent of spread. All begin with the acute
onset of fever, chills, and malaise. In the ulceroglandular form, a local papule at the
74
inoculation site becomes necrotic and ulcerative. Regional lymph nodes become
swollen and painful. The oculoglandular form, which follows conjunctival
inoculation, is similar except that the local lesion is a painful purulent conjunctivitis.
Ingestion of large numbers of F. tularensis (>108) leads to typhoidal tularemia, with
abdominal manifestations and a prolonged febrile course similar to that of typhoid
fever. Inhalation of the organisms can result in pneumonic tularemia or a more
generalized infection similar to the typhoidal form. Like plague pneumonia,
tularemic pneumonia may also develop through seeding of the lungs by bacteremic
spread of one of the other forms. Any form of tularemia may progress to a systemic
infection with lesions in multiple organs.
Without treatment, mortality ranges from 5 to 30%, depending on the type of
infection. Ulceroglandular tularemia, the most common form, generally carries the
lowest risk of a fatal outcome. In the US surveillance study mentioned earlier, the
mortality was 2%.

Diagnosis
Because tularemia is uncommon and F. tularensis has unique growth requirements,
the diagnosis is easily overlooked. Although some strains grow on chocolate agar,
laboratories must be alerted to the suspicion of tularemia so that specialized media
can be prepared and precautions taken against the considerable risk of laboratory
infection. An immunofluorescent reagent is available in reference laboratories for
use directly on smears from clinical material. Because of the difficulty and risk of
cultural techniques, many cases are diagnosed by serologic tests. Agglutinating
antibodies are usually present in titers of 1:40 by the second week of illness,
increasing to 1:320 or greater after 3 to 4 weeks. Unless previous exposure is known,
single high antibody titers are considered diagnostic.

Treatment and prevention

Streptomycin is the drug of choice in all forms of tularemia, although recent
experience indicates that gentamicin may be just as effective. Tetracycline and
chloramphenicol have also been effective, but relapses are more common than with
streptomycin. Prevention mainly involves the use of rubber gloves and eye
protection when handling potentially infected wild mammals. Prompt removal of
ticks is also important. A live attenuated vaccine exists, but it is used only in
laboratory workers and those individuals who cannot avoid contact with infected
animals.

BACILLUS ANTHRACIS
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The genus Bacillus includes many species of aerobic or facultative, spore-forming,
Gram-positive rods. With the exception of one species, B. anthracis, they are low-
virulence saprophytes widespread in air, soil, water, dust, and animal products.
B. anthracis causes the zoonosis anthrax, a disease of animals that is occasionally
transmitted to humans.

Epidemiology
Anthrax is primarily a disease of herbivores such as horses, sheep, and cattle who
acquire it from spores of B. anthracis contaminating their pastures. Humans become
infected through contact with these animals or their products in a way that allows
the spores to be inoculated through the skin, ingested, or inhaled.

Pathogenesis
When spores of B. anthracis reach the rich environment of human tissues, they
germinate and multiply in the vegetative state. The antiphagocytic properties of the
capsule aid in survival, eventually allowing production of large enough amounts of
the exotoxins to cause disease. The tripartite nature of the anthrax exotoxin complex
must play an important role but the timing and relative importance of the
components are not known. The adenylate cyclase activity is believed to correlate
with the striking edema seen at infected sites.

Clinical manifestations
Cutaneous anthrax usually begins 2 to 5 days after inoculation of spores into an
exposed part of the body, typically the forearm or hand. The initial lesion is an
erythematous papule, which may be mistaken for an insect bite. This papule usually
progresses through vesicular and ulcerative stages in 7 to 10 days to form a black
eschar (scab) surrounded by edema. This lesion complex is known as the “malignant
pustule,” although it is neither malignant nor a pustule. Associated systemic
symptoms are usually mild, and the lesion typically heals very slowly after the eschar
separates. Less commonly, the disease progresses with massive local edema,
toxemia, and bacteremia; it has a fatal outcome if untreated.
Pulmonary anthrax is contracted by inhalation of spores. Historically, this occurred
when contaminated hides, hair, wool, and the like are handled in a confined space
(woolsorter’s disease) or following laboratory accidents. Today it is the form we
would expect from the dissemination of a spore aerosol in biologic warfare. In the
pulmonary syndrome, 1 to 5 days of nonspecific malaise, mild fever, and
nonproductive cough lead to progressive respiratory distress and cyanosis. Massive
spread to the bloodstream and CNS follow rapidly. Mediastinal edema was a

76
prominent finding in the postal workers. If untreated, progression to a fatal outcome
is usually very rapid once bacteremia has developed.

Diagnosis
Culture of skin lesions, sputum, blood, and CSF are the primary means of anthrax
diagnosis. Given some suspicion on epidemiologic grounds, Gram stains of sputum
or other biologic fluids showing large numbers of Gram-positive bacilli can indicate
the diagnosis. In September of 2001, diagnosis of the first case in Florida was
speeded by an infectious disease specialist who knew such rods were extremely rare
in the spinal fluid. Such bacilli are also unusual in sputum. B. anthracis and other
Bacillus species are not difficult to grow. In fact, clinical laboratories frequently
isolate the nonanthrax species as environmental contaminants. The saprophytic
species are usually β-hemolytic and motile; these features can be used to exclude B.
anthracis. Blood cultures are positive in most cases of pulmonary anthrax.

Treatment
Antimicrobial treatment has little effect on the course of cutaneous anthrax but does
protect against dissemination. Almost all strains of B. anthracis are susceptible to
penicillin, which remains the treatment of choice for all forms of anthrax.
Doxycycline or ciprofloxacin are alternatives and are also recommended for
chemoprophylaxis in the case of known or suspected exposure.

Prevention
The most important preventive measures are those that eradicate animal anthrax and
limit imports from endemic areas. Vaccines are also useful. Pasteur’s vaccine used
a live strain attenuated by repeated subculture that resulted in the loss of a plasmid
encoding toxin production. A similar live vaccine is still effective for animals, but
inactivated human vaccines have a less certain efficacy. The vaccine used by the US
military is prepared from filtrates of a nonencapsulated B. anthracis strain that
produces the protective antigen component of the toxin complex. Its acceptance is
complicated by fears that the architects of biological warfare may have crafted
strains for which this vaccine is not protective. Proof of the efficacy of the vaccine
in humans is neither practical nor ethical.

77
 Questions for the colloquium number 2.

1. Acute intestinal infections and their causative agents. General characteristics

of the family of enteric bacteria, the role in human pathology.
2. The causative agent of colibacillosis. Characteristics. The role of E. coli in
health and disease. Laboratory diagnosis.
3. The causative agent of salmonellosis. Microbiological characterization of
causative agent. Laboratory diagnosis. Prevention, treatment.
4. The causative agent of typhoid. Characteristics. Laboratory diagnosis.
Specific prevention and treatment.
5. The causative agent of shigellosis. Characteristics. Laboratory diagnosis.
Specific prevention and treatment.
6. The causative agent of intestinal yersiniosis. Characteristics. Laboratory
diagnosis. Prevention.
7. The causative agent of cholera. Characteristics. Laboratory diagnosis.
Specific prevention and treatment.
8. The causative agent of food poisoning. Laboratory diagnosis. Opportunistic
bacteria and their role in the pathology.
9. Campylobacter. Characteristics. Laboratory diagnosis.
10.Staphylococci. Characteristics. Laboratory diagnosis. Specific prevention
and treatment.
11.Streptococci. Characteristics. Laboratory diagnosis.
12.Tetanus. Characteristics. Laboratory diagnosis. Specific prevention and
treatment.
13.Botulism. Characteristics. Laboratory diagnosis. Specific prevention and
treatment.
14.The causative agent of anaerobic gas infection. Characteristics. Laboratory
diagnosis. Specific prevention and treatment.
15.Airborne bacterial infection. Characteristic.
16.The causative agent of tuberculosis. Characteristics. Laboratory diagnostics.
Specific prevention and treatment.
17.The causative agent of leprosy. Characteristics. Laboratory diagnosis.
Prevention and treatment.
18.The causative agent of diphtheria. Characteristics. Laboratory diagnosis.
Specific prevention and treatment.
19.The causative agent of pertussis. Characteristics. Laboratory diagnosis.
Specific prevention and treatment.

78
20. Pseudomonas aeruginosa. Characteristics. Laboratory diagnosis. Prevention
and treatment.
21.Pneumococcus. Characteristics. Laboratory diagnosis. Prevention and
treatment.
22.Meningococcus (Neisseria meningitides). Characteristics. Laboratory
diagnosis. Specific prevention and treatment.
23.Gonococcus (Neisseria gonorrhoeae). Characteristics. Laboratory diagnosis.
Prevention and treatment.
24.The causative agent of syphilis. Characteristics. Laboratory diagnosis.
Prevention and treatment.
25.The anthrax. Characteristics. Laboratory diagnosis. Specific prevention and
treatment.
26.The causative agent of tularemia. Characteristics. Laboratory diagnosis.
Specific prevention and treatment.
27.The causative agent of plague. Characteristics. Laboratory diagnosis.
Specific prevention and treatment.
28.The causative agent of brucellosis. Characteristics. Laboratory diagnosis.
Specific prevention and treatment.

79
 References:
1. Cunningham F., Leveno K., Bloom S., Spong C. Y., Dashe J. Williams
Obstetrics, 24e. – McGraw-Hill, New York, 2014.
2. Ford M. Medical microbiology. – Oxford University Press, 2014.
3. Gillespie S. H. Medical microbiology illustrated. – Butterworth-Heinemann,
2014.
4. Goering R. V., Mims C. A., Dockrell H., Zuckerman M., Chiodini P. L., Roitt
I. Mims' Medical Microbiology, With STUDENT CONSULT Online Access,
5: Mims' Medical Microbiology. – Elsevier Health Sciences, 2013.
5. Levinson W. Review of medical microbiology and immunology. – McGraw-
Hill Education, New York, 2014.
6. Murray P. R., Rosenthal K. S., Pfaller M. A. Medical microbiology. –
Elsevier Health Sciences, Amsterdam, 2015.

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