DR ADEBOYEJO K.

BACTERIAL DISEASES AND LABORATORY DIAGNOSIS

Bacteria are ubiquitous. They play an important role in maintaining the environment in which we live.
Only a small percentage of the world’s bacteria cause infection and disease. These bacterial infections
have a large impact on public health. As a general rule, bacterial infections are easier to treat than viral
infections, since the armamentarium of antimicrobial agents with activity against bacteria is more
extensive. More so than with infectious diseases caused by viruses and parasites, however, bacterial
resistance to antimicrobials is a rapidly growing problem with potentially devastating consequences

Once a person is infected, clinically apparent disease may or may not be seen, and only in a small subset
of infections do we see clinically significant disease. Bacterial infections can be transmitted by a variety
of mechanisms. In order to be spread, a sufficient number of organisms must survive in the environment
and reach a susceptible host. Many bacteria have adapted to survive in water, soil, food, and elsewhere.
Some infect vectors such as animals or insects before being transmitted to another human.

All of the human organs are susceptible to bacterial infection. Each species of bacteria has a predilection
to infect certain organs and not others. For example, Neisseria meningitidis normally infects the
meninges (covering) of the central nervous system, causing meningitis, and can also infect the lungs,
causing pneumonia. It is not, however, a cause of skin infection. Staphylococcus aureus, which people
typically carry on their skin or mucus membranes, often causes skin and soft tissue infections, but also
spreads readily throughout the body via the bloodstream and can cause infection of the lungs,
abdomen, heart valves, and almost any other site. Some bacterial infections are discussed below:

Bacterial Pharyngitis

Bacterial pharyngitis can occur suddenly or following a viral pharyngitis. The sudden onset of throat
pain, fever, and anterior cervical lymphadenopathy without upper respiratory infection symptoms
(cough, rhinorrhea, or nasal congestion) is suggestive of a bacterial infection. The pharynx usually, but
not always, demonstrates enlarged tonsils with white exudates. Petechial hemorrhages are occasionally
observed on the soft palate. The Centor criteria for bacterial infection includes: (1) fever, (2) anterior
cervical lymphadenopathy, (3) enlarged tonsils with white exudates, and (4) absence of cough (5). If
three or more criteria are present, the diagnosis is presumed without cultures or rapid strep testing, and
empiric therapy should be administered.

Gonorrhea pharyngitis has a similar presentation to Group A beta-heamolytic streptococcus (GABHS)

pharyngitis, and is suggested by a history of unprotected oral sex and a non-winter-time bacterial
pharyngitis (i.e., when bacterial infection secondary to a viral pharyngitis is less common). Urethritis,
urethral discharge, or joint pain and swelling are also suggestive. Lymphadenopathy is less common;
though pharyngeal exudates are more pronounced.

Diphtheria is rare in an immunized population, though it is observed with greater frequency in

alcoholics. A history of nonimmunization or incomplete childhood immunization (e.g., immigrant
populations) and a gray pseudomembrane extending over the pharynx, nasal passages, or the tracheo-
broncial tree suggest this diagnosis. It is also associated with difficulty in swallowing and shortness of
breath.
Otitis Media

Acute otitis media is a middle ear effusion associated with the rapid onset of one or more signs or
symptoms of inflammation of the middle ear, such as otalgia, otorrhea, fever, or irritability. Otitis media
with effusion is a middle ear effusion without manifestations of an acute infection, and may occur as a
result of, or in the absence of, acute otitis media. Hearing loss may be present in both conditions. The
most common bacterial isolates of middle ear aspirates are Streptococcus pneumoniae, nontypable
Haemophilus influenzae, and Moraxella catarrhalis.

Meningitis

Bacterial meningitis remains a disease with significant morbidity and mortality. Major epidemiological
changes, including the decline of Haemophilus influenzae meningitis and the emergence of drug-
resistant S. pneumoniae, have changed the empiric therapy of patients with bacterial meningitis. S.
pneumoniae is now responsible for almost half of all episodes. Patients with bacterial meningitis present
with acute onset of fever, headache, stiff neck, nausea/vomiting, and mental status changes.

Pneumonia

Community-acquired pneumonia is an acute infection of the pulmonary parenchyma in a patient not

hospitalized or residing in a long-term care facility for greater than 14 days prior to the onset of
symptoms. Symptoms include cough, fever, myalgias, or dyspnea. Chest radiograph may reveal a
pulmonary infiltrate. Physical examination demonstrates findings consistent with pulmonary infection
such as egophony, tactile fremitus, or crackles. Infection can occur through heamatogenous spread (i.e.,
septic emboli from right sided endocarditis), inhalation of aerosolized infectious particles (as with
Mycobacterium tuberculosis, influenza or Legionella pneumophila), direct extension from surrounding
structures (as with a liver abscess or trauma), or, most commonly, by aspiration of oropharyngeal
secretions.

Infective endocarditis(IE)

Infective endocarditis refers to any infection involving the endocardium of the heart. It may involve
native or prosthetic valves, papillary muscles, or the myocardium itself. More commonly, IE affects the
left side of the heart; however, multiple valves may be infected simultaneously on each side of the
heart. Both age and gender influence the incidence of IE. More males acquire the disease than females,
with a ratio of almost 2:1. Because the incidence of acquired cardiac valvular lesions increases with age,
the mean incidence of IE is shifting toward the elderly. A wide variety of pathogens may cause IE,
including gram-positive, gram-negative, anaerobic bacteria, atypical organisms such as Legionella and
Rickettsia. Patients with acute IE appear septic, with fever, chills, tachycardia, and/or tachypnea. In
severe cases, septic shock with multiorgan system failure may occur. Endocarditis should be included on
the differential diagnosis in any case of sepsis, particularly in the presence of a cardiac murmur or in a
patient with risk factors.
Diarrhea

While the definition for acute diarrhea varies, it can be defined practically as an increased number of
stools of decreased form (from the normal) lasting less than 14 days. A decrease in consistency and an
increase in frequency of bowel movements to greater than or equal to three stools per day has often
been used as a definition for epidemiological investigations. Additional associated signs and symptoms
can include nausea, vomiting, abdominal pain and cramps, fever, bloody stools, tenesmus (constant
sensation of urge to move bowels), and fecal urgency. Diarrhea can be characterized as acute or chronic.
Diarrhea lasting as long as 14 days is considered persistent and that lasting more than a month is chronic
diarrhea. The majority of diarrhea is classified as infectious (>80%) which is divided into two syndromes:
inflammatory and noninflammatory. Inflammatory diarrhea is usually accompanied by abdominal pain,
tenesmus, bloody (or hemoccult positive) stool, and fever. Organisms in inflammatory diarrhea can
disrupt the mucosal lining of the colon (both macroscopically and microscopically). Noninflammatory
diarrhea is usually a milder syndrome of symptoms that can include nausea, vomiting, abdominal
cramping, and watery stools. Stools do not typically contain blood and polymorphonuclear cells
although dehydration can occur. Organisms and toxins affect the small intestine more than the colon
and typically do not disrupt the normal colonic mucosa. Although the greatest pathology is usually seen
with those agents that cause invasive, inflammatory disease, any infectious agent can be associated with
morbidity and mortality. Example of bacteria that causes inflammatory infectious diarrhea are
Campylobacter jejuni, Shigella, Nontyphi Salmonella, Enterohaemorrhagic and enteroinvasive E. coli.
However, those that are associated with noninflammatory are Vibro cholerae, Enterotoxigenic E coli,
Staph aureus, Bacillus cereus etc.

Urinary tract infection

Urinary tract infection refers to the presence of infectious organisms anywhere along the genitourinary
tract. It can be classified as lower or upper tract infections. The clinical diagnosis of upper tract infection
is generally made when any of the symptoms of fever, chills, flank, or back pain are present. The
diagnosis of lower tract infection is made when these upper tract symptoms are absent and there are
symptoms of cystitis such as frequency and dysuria. In addition, there is the category of Asymptomatic
bacteriuria(ASB), which is defined as the presence of significant counts of bacteria in the urine with no
signs or symptoms of an upper or lower infection. Escherichia coli, remains the most common pathogen
for all manifestations of UTI and in all groups of patients. Staphylococcus saprophyticus, a coagulase-
negative gram-positive coccus, is the second most common pathogen in uncomplicated cystitis,
implicated in 10–15% of cases of UTI. Klebsiella, Serratia, Enterobacter, and Proteus species as well as
Pseudomonas aeruginosa, Staphylococcus aureus, group B and group D (enterococcus) Streptococcus
are rare causes of UTI in normal host.

Impetigo

Impetigo, most commonly seen in young children, is a superficial cutaneous infection involving the
epidermis. Two types of impetigo exist: nonbullous and bullous. Highly communicable, this infection can
spread easily over the body of the same individual or to others in closed living situations where
crowding and suboptimal hygiene exist. Potential complications include more extensive skin, soft-tissue,
or bone infection. Acute glomerulonephritis with hypertension, hematuria, and proteinuria can occur
approximately 10–21 days after this skin infection when certain M serotypes of group A streptococcus
(Streptococcus pyogenes) are implicated.
Cellulitis

Cellulitis involves the dermis and subcutaneous tissues, usually resulting from the inoculation of bacteria
through tiny breaks in the skin. These disruptions in the skin barrier can result from tinea pedis, burns,
venous insufficiency, lymphedema, traumatic wounds, thrombophlebitis, and ulcers. Host immune
defects that result from diabetes mellitus, malnutrition, HIV infection, immunosuppressive medications,
and intravenous-drug use may also contribute to the development of infection. Men are more
commonly infected, and the extremities (lower more so than upper) are the most commonly involved
areas. In general, uncomplicated infections refer to immunocompetent patients whose infections are
due to S. aureus or group A streptococcus. Complicated infections are more likely to include gram-
negative or anaerobic organisms and occur in the setting of comorbidities such as diabetes mellitus,
burns, chronic pressure ulcers, and postsurgical wounds.

Syphilis

Syphilis is caused by the spirochete, Treponema pallidum. The organism is a spiral shaped, obligate
human bacteria with no environmental or animal reservoirs. It is too small to be visualized with light
microscopy but is visible by dark-field or phase-contrast microscopy. In addition, this organism is one of
the few pathogens that has not been cultivated in vitro successfully and must be propagated in rabbit
testicles in order to perform experimental studies. T. pallidum is acquired by sexual contact and
perinatal transmission (congenital syphilis). Accidental direct inoculation of medical personnel and
transfusion-associated syphilis have been virtually eliminated due to universal precautions, serologic
screening of donated blood, and use of blood components that have been stored at 4°C (T. pallidum is
killed at this temperature).

Gonorrhea

Gonorrhea is caused by Neisseria gonorrhoeae and humans are the only known reservoir. Mucous
membranes lined with columnar, cuboidal, or noncornified genitourinary tract epithelial cells such as
those found in the urethra, cervical canal, epididymis, fallopian tubes, and the Bartholin glands are most
often infected with this organism. Other mucosal sites such as the pharynx, rectum, and conjunctiva
may also be infected and hematogenous dissemination to nonmucosal sites occurs. The initial phase of
gonococcal infection includes adherence of the organism to cells, which is mediated by pili and other
surface proteins. In addition, N. gonorrhoeae occasionally may evade the immune system and cause
disseminated disease.

Laboratory Diagnosis: Introduction

The laboratory diagnosis of infectious diseases involves three main approaches: one is the bacteriologic
approach in which the organism is identified by staining and culturing the organism; Secondly, is the
immunologic (serologic) approach in which the organism is identified by detection of antibodies against
the organism in the patient's serum; Thirdly, molecular (nucleic acid-based) tests.

Bacteriologic Tests

In the bacteriologic approach to the diagnosis of infectious diseases, several important steps precede
the actual laboratory work, namely, (1) choosing the appropriate specimen to examine, which requires
an understanding of the pathogenesis of the infection; (2) obtaining the specimen properly to avoid
contamination from the normal flora; (3) transporting the specimen promptly to the laboratory or
storing it correctly; and (4) providing essential information to guide the laboratory personnel.

Bacteriologic tests typically begin with staining the patient's specimen and observing the organism in the
microscope. This is followed by culturing the organism, typically on blood agar, then performing various
tests to identify the causative organism. Obtaining a pure culture of the bacteria is essential to accurate
diagnosis.

 Blood cultures are useful in cases of sepsis and other diseases in which the organism is often
found in the bloodstream, such as endocarditis, meningitis, pneumonia, and osteomyelitis. It is
important to obtain at least three 10-mL blood samples in a 24-hour period, because the
number of organisms can be small and their presence intermittent. The site for venipuncture
must be cleansed with 2% iodine to prevent contamination by members of the flora of the skin,
usually Staphylococcus epidermidis. The blood obtained is added to 100 mL of a rich growth
medium such as brain–heart infusion broth. Whether one or two bottles are inoculated varies
among hospitals. If two bottles are used, one is kept under anaerobic conditions and the other is
not. If one bottle is used, the low oxygen tension at the bottom of the bottle permits anaerobes
to grow. Blood cultures are checked for turbidity or for CO2 production daily for 7 days or longer.
If growth occurs, Gram stain, subculture, and antibiotic sensitivity tests are performed. If no
growth is observed after 1 or 2 days, blind subculturing onto other media may reveal organisms.
Cultures should be held for 14 days when infective endocarditis, or infection by slow-growing
bacteria, e.g., Brucella, is suspected.
 Throat cultures are most useful to diagnose pharyngitis caused by Streptococcus pyogenes
(strep throat), but they are also used to diagnose diphtheria, and gonococcal pharyngitis. When
the specimen is being obtained, the swab should touch not only the posterior pharynx but both
tonsils or tonsillar fossae as well. The material on the swab is inoculated onto a blood agar plate
and streaked to obtain single colonies. If colonies of beta-hemolytic streptococci are found after
24 hours of incubation at 35°C, a bacitracin disk is used to determine whether the organism is
likely to be a group A streptococcus. If growth is inhibited around the disk, it is a group A
streptococcus; if not, it is a nongroup A beta-hemolytic streptococcus.
 Sputum cultures are used primarily when pneumonia or tuberculosis is suspected. The most
frequent cause of community-acquired pneumonia is S. pneumoniae, whereas S. aureus and
gram-negative rods, such as K. pneumoniae and P. aeruginosa, are common causes of hospital-
acquired pneumonias. It is important that the specimen for culture really be sputum, not saliva.
Examination of a gram-stained smear of the specimen frequently reveals whether the specimen
is satisfactory. A reliable specimen has more than 25 leukocytes and fewer than 10 epithelial
cells per 100 x field. An unreliable sample can be misleading and should be rejected by the
laboratory. If the patient cannot cough and the need for a microbiologic diagnosis is strong,
induction of sputum, transtracheal aspirate, bronchial lavage, or lung biopsy may be necessary.
Because these procedures bypass the normal flora of the upper airway, they are more likely to
provide an accurate microbiologic diagnosis. A preliminary assessment of the cause of the
pneumonia can be made by Gram stain if large numbers of typical organisms are seen.
 Spinal fluid cultures are most useful in suspected cases of meningitis. These cultures are often
negative in encephalitis, brain abscess, and subdural empyema.
 Stool cultures are useful primarily when the complaint is bloody diarrhea (dysentery,
enterocolitis) rather than watery diarrhea, which is often caused by either enterotoxins or
viruses. For culture of Salmonella and Shigella, a selective, differential medium such as
MacConkey or eosin-methylene blue (EMB) agar is used. These media are selective because they
allow gram-negative rods to grow but inhibit many gram-positive organisms. Their differential
properties are based on the fact that Salmonella and Shigella do not ferment lactose, whereas
many other enteric gram-negative rods do. If non-lactose-fermenting colonies are found, a triple
sugar iron (TSI) agar slant is used to distinguish Salmonella from Shigella. Some species of
Proteus resemble Salmonella on TSI agar but can be distinguished because they produce the
enzyme urease, whereas Salmonella does not. The organism is further identified as either a
Salmonella or a Shigella species by the use of specific antisera to the organism's cell wall O
antigen in an agglutination test. This is usually done in hospital laboratories, but precise
identification of the species is performed in public health laboratories. Campylobacter jejuni is
cultured on antibiotic-containing media, e.g., Skirrow's agar, at 42°C in an atmosphere
containing 5% O2 and 10% CO2. It grows well under these conditions, unlike many other
intestinal pathogens. Although the techniques are available, stool cultures are infrequently
performed for organisms such as Yersinia enterocolitica, Vibrio parahaemolyticus, and
enteropathic or toxigenic E. coli. Despite the presence of large numbers of anaerobes in feces,
they are rarely pathogens in the intestinal tract, and anaerobic cultures of stool specimens are
therefore unnecessary.
 Urine cultures are used to determine the cause of either pyelonephritis or cystitis. Urine in the
bladder of a healthy person is sterile, but it acquires organisms of the normal flora as it passes
through the distal portion of the urethra. To avoid these organisms, a midstream specimen,
voided after washing the external orifice, is used for urine cultures. In special situations,
suprapubic aspiration or catheterization may be required to obtain a specimen. Because urine is
a good culture medium, it is essential that the cultures be done within 1 hour after collection or
stored in a refrigerator at 4°C for no more than 18 hours. It is commonly accepted that a
bacterial count of at least 100,000/mL must be found to conclude that significant bacteriuria is
present (in asymptomatic persons). There is evidence that as few as 100/mL are significant in
symptomatic patients. For this determination to be made, quantitative or semiquantitative
cultures must be performed. There are several techniques. (1) A calibrated loop that holds 0.001
mL of urine can be used to streak the culture. (2) Serial 10-fold dilutions can be made and
samples from the dilutions streaked. (3) A screening procedure suitable for the physician's office
involves an agar-covered "paddle" that is dipped into the urine. After the paddle is incubated,
the density of the colonies is compared with standard charts to obtain an estimate of the
concentration of bacteria.
 Genital tract cultures are performed primarily on specimens from individuals with an abnormal
discharge or on specimens from asymptomatic contacts of a person with a sexually transmitted
disease. One of the most important pathogens in the genital tract is Neisseria gonorrhoeae. The
laboratory diagnosis of gonorrhea is made by microscopic examination of a gram-stained smear
and by culture of the organism. Specimens are obtained by swabbing the urethral canal (for
men), the cervix (for women), or the anal canal (for men and women). A urethral discharge from
the penis is frequently used. Because N. gonorrhoeae is very delicate, the specimen should be
inoculated directly onto a Thayer-Martin chocolate agar plate or onto a special transport
 medium (e.g., Trans-grow). Gram-negative diplococci found intracellularly within neutrophils on
a smear of a urethral discharge from a man have over 90% probability of being N. gonorrhoeae.
Because smears are less reliable when made from swabs of the endocervix and anal canal,
cultures are necessary. The finding of only extracellular diplococci suggests that these neisseriae
may be members of the normal flora and that the patient may have nongonococcal urethritis.
Nongonococcal urethritis and cervicitis are also extremely common infections. The most
frequent cause is Chlamydia trachomatis, which cannot grow on artificial medium but must be
grown in living cells. For this purpose, cultures of human cells or the yolk sacs of embryonated
eggs are used. The finding of typical intracytoplasmic inclusions when using Giemsa stain or
fluorescent antibody is diagnostic. Because of the difficulty of culturing C. trachomatis,
nonbacteriologic methods, such as enzyme-linked immunosorbent assay (ELISA) to detect
chlamydial antigens in exudates or urine or DNA probe assays to detect chlamydial nucleic acids,
are now often used to diagnose sexually transmitted diseases caused by this organism. Because
Treponema pallidum, the agent of syphilis, cannot be cultured, diagnosis is made by microscopy
and serology. The presence of motile spirochetes with typical morphologic features seen by
darkfield microscopy of the fluid from a painless genital lesion is sufficient for the diagnosis. The
serologic tests fall into two groups: the nontreponemal antibody tests such as the Venereal
Disease Research Laboratory (VDRL) or rapid plasma reagin (RPR) test, and the treponemal
antibody tests such as the fluorescent treponemal antibody-absorption (FTA-ABS) test.
 A great variety of organisms are involved in wound and abscess infections. The bacteria most
frequently isolated differ according to anatomic site and predisposing factors. Abscesses of the
brain, lungs, and abdomen are frequently caused by anaerobes such as Bacteroides fragilis and
gram-positive cocci such as S. aureus and S. pyogenes. Traumatic open-wound infections are
caused primarily by members of the soil flora such as Clostridium perfringens; surgical-wound
infections are usually due to S. aureus. Infections of dog or cat bites are commonly due to
Pasteurella multocida, whereas human bites primarily involve the mouth anaerobes. Because
anaerobes are frequently involved in these types of infection, it is important to place the
specimen in anaerobic collection tubes and transport it promptly to the laboratory. Because
many of these infections are due to multiple organisms, including mixtures of anaerobes and
nonanaerobes, it is important to culture the specimen on several different media under
different atmospheric conditions. The Gram stain can provide valuable information regarding
the range of organisms under consideration.

Immunologic (Serologic) Tests

Immunologic (serologic) tests can determine whether antibodies are present in the patient's serum as
well as detect the antigens of the organism in tissues or body fluids. In these tests, the antigens of the
causative organism can be detected by using specific antibody often labeled with a dye such as
fluorescein (fluorescent antibody tests) or ELIZA in which a specific antibody to which an easily assayed
enzyme has been linked is used to detect the presence of the homologous antigen. The presence of
antibody in the patient's serum can be detected using antigens derived from the organism. In some
tests, the patient's serum contains antibodies that react with an antigen that is not derived from the
causative organism, such as the VDRL test in which beef heart cardiolipin reacts with antibodies in the
serum of patients with syphilis.
In many tests in which antibodies are detected in the patient's serum, an acute and convalescent serum
sample is obtained and at least a fourfold increase in titer between the acute and convalescent samples
must be found for a diagnosis to be made. The reason these criteria are used is that the presence of
antibodies in a single sample could be from a prior infection, so a significant (fourfold or greater)
increase in titer is used to indicate that this is a current infection. IgM antibody can also be used as an
indicator of current infection.

Nucleic Acid-Based Methods

There are three types of nucleic acid-based tests used in the diagnosis of bacterial diseases: nucleic acid
amplification tests, nucleic acid probes, and nucleic acid sequence analysis. Nucleic acid-based tests are
highly specific, quite sensitive (especially the amplification tests), and much faster than culturing the
organism. These tests are especially useful for those bacteria that are difficult to culture such as
Chlamydia and Mycobacterium species.

Nucleic acid amplification tests utilize the PCR (polymerase chain reaction) or other amplifying process
to increase the number of bacteria-specific DNA or RNA molecules so the sensitivity of the test is
significantly higher than that of unamplified tests. Many bacteria can be identified using these tests but
they are especially useful in detecting Chlamydia trachomatis and Neisseria gonorrhoeae in urine
samples in STD clinics.

Tests that use nucleic acid probes are designed to detect bacterial DNA or RNA directly (without
amplification) using a labeled DNA or RNA probe that will hybridize specifically to the bacterial nucleic
acid. These tests are simpler to perform than the amplification tests but are less sensitive.

Nucleic acid sequence analysis is used to identify bacteria based on the base sequence of the organism's
ribosomal RNA. An organism that has never been cultured, Tropheryma whippelii, was identified using
this approach.