present.

The number of stool specimens submitted to the laboratory is controversial. It is recommended if more than one

specimen is submitted that it should be on different days

Specimen transport and handling

Stool held at room temperature should be cultured within 1 hour of collection.

Refrigeration of stool specimens is not recommended as some pathogens, especially Shigella species, are very

susceptible to lower temperatures and will die rapidly.

159

 Arranged by Sarah Mohssen

Section I– Microbiology By Dr. Mohammed Ayad

Specimens that cannot be sent immediately to the laboratory or processed shortly after collection should be

placed in an appropriate enteric transport media (Cary-Blair transport swab or Amie's transport swab).

Specimens containing barium, mineral oil or urine should be rejected

Stool direct examination

Leukocyte evaluation

1- Perform a Gram stain on the stool specimen to evaluate for the presence of leukocytes.

Direct Gram stain is not useful beyond its determination of the presence of leukocytes, as it will not

differentiate suspected pathogens from normal microbial flora.

Place a drop of feces on a slide, spread out, and allow to air dry. Stain slide using Gram’s stain procedure.

Evaluate slide under oil immersion for PMN’s

2- Latex agglutination for detection of lactoferrin released by fecal leukocytes in diarrheal stool specimens.

Not appropriate for detection of leukocytes in breastfeeding babies. Lactoferrin present in breast milk

Culture Setup

A. The purpose of a stool culture is to use selective and routine media to screen for the presence of stool

pathogens.

B. In many cases, full identification of a suspicious colony is not performed. Instead, screening tests are

performed to rule out potential stool pathogens. If suspicious colonies are noted then full identification

performed.

C. Routine stool cultures should always include testing for the presence of Salmonella species, Shigella

species, and Campylobacter jejuni

D. It is recommended that routine stool cultures include testing for the presence of Aeromonas species,

and Plesiomonas species. The CDC recommends routine screening of stool for E. coli strains that

produce a Shiga-like cytotoxin.

E. When required by the physician, media may also be included to detect Yersinia species or Vibrio

species. However, in areas where these pathogens are common including media for these organisms is

part of the routine culture

F. The CDC recommends testing to detect shiga-like toxin producing strains of E. coli in addition to

culture for Escherichia coli O157:H7.

G. It is no longer recommended to do culture alone. Clinical sites may perform culture and toxin testing

or toxin testing alone.

H. A macroscopic exam is reported with every culture

Pathogens commonly isolated in stool

a. Salmonella species

b. Shigella species

c. Campylobacter jejuni

d. Vibrio cholera

e. Vibrio parahaemolyticus

f. Yersinia enterocolitica

g. Clostridium difficile

160

 Arranged by Sarah Mohssen

Section I– Microbiology By Dr. Mohammed Ayad

h. Staphylococcus aureus

i. Aeromonas species

j. Plesiomonas species

k. Hemorrhagic Escherichia coli O157:H7

l. Shiga-like toxin producing strains of Escherichia coli

Normal flora commonly isolated in stool

a. Enteric organisms

b. Staphylococcus species

c. Streptococcus / Enterococccus species

d. Anaerobic organisms

Inoculate media

Use a swab to inoculate plates using representative areas of the specimen. Inoculate the media making the first

streak then use a sterile loop to streak for isolation. Non inhibitory media should always be inoculated first

Routine stool culture

a. Blood agar

b. MacConkey’s agar

c. Hektoen enteric (HE) agar or xylose-lysine deoxycholate (XLD) agar

d. Selective agar for Campylobacter (i.e., CVA, Campy BAP, Skirrow’s)

e. GN broth (or Selenite F) is a selectively enhances the growth of Salmonella and Shigella while suppressing

the growth of normal bowel flora. Also, used for the detection of shiga-like toxin producing strains of E. coli.

This will increase the chances of isolating these pathogens when they are present in small numbers.

Yersinia culture

1. Cefsulodin Novobiocin (CN) agar or Yersinia Selective agar

2. Phosphate buffered saline (PBS) broth – suppresses the growth of normal bowel flora allowing easier

detection of Yersinia species

Vibrio culture

a. TCBS agar

b. Alkaline peptone broth

Incubate media Temperature

a. Campy BAP: 42ºC

b. PBS broth: 4ºC

c. CN plate: room temperature

d. All other plates: 35ºC

Atmosphere

Campy BAP: microaerophilic (increased CO2)

b. BAP: either ambient air or CO2

c. All other plates: ambient air Time: 24- 48 hours

161

 Arranged by Sarah Mohssen

Section I– Microbiology By Dr. Mohammed Ayad

Culture Interpretation

A. Evaluation for Salmonella and Shigella

After 24 hours incubation, examine the HE and MAC plates for non-lactose fermenting colonies and H2S

producing colonies.

These colony types are suspicious for Salmonella species and Shigella species, but may also be normal enteric

flora; so biochemical screens must be performed to rule in or out the presence of these pathogens.

For colonies that are suspicious for Salmonella or Shigella perform KIA slants.

If screen is positive perform biochemical ID panels such as API 20 E.

Colonies that appear to be Salmonella species or Shigella species are confirmed by performing serological

agglutination tests. If enrichment broth (i.e., GN broth or Selenite F) is used, they are subcultured to HE at 24

hours.

Suspicious colonies from the enrichment broth subculture plates are screened biochemically.

A susceptibility test is performed only on any confirmed colonies of Salmonella and Shigella species when

requested

Treatment with antibiotics is not recommended for Salmonella species because it may induce a carrier state in

the patient. In most case treatment of the clinical symptoms such as dehydration is sufficient.

If no suspicious colonies are found or if all biochemical screens are negative, the report is sent out as: “No

Salmonella or Shigella isolated”

B. Campylobacter jejuni

Campy plates are examined at 48 hours and 72 hours. Colonies growing on the original Campy BAP are tested

for oxidase. Any oxidase positive colonies are Gram stained to look for the typical curved rods of

Campylobacter species.

Oxidase positive curved gram-negative rods should be further tested to confirm the presence of Campylobacter

species. A positive Sodium Hippurate test will confirm the species Campylobacter jejuni.

C. Aeromonas and Plesiomonas

After 24 hours incubation, examine the BAP for large gray colonies that are gram-negative rods in

predominance.

Do an oxidase on each different colony type of gram-negative rods. If a colony type is oxidase positive and has

a smooth morphology, perform a spot indole test. An oxidase positive indole positive gram-negative rod is

suspicious for Aeromonas species or Plesiomonas species.

Confirm identification of suspicious organisms with various biochemical ID panels.

Aeromonas species are slightly beta hemolytic on the BAP but Plesiomonas species are nonhemolytic.

Do not use the MAC to screen for Aeromonas or Plesiomonas as these organisms can be lactose variable

D. Yersinia

After 24 hours incubation, examine the CN plate for dark red colonies with a “bull’s eye” center surrounded by

transparent border colonies that indicate mannitol fermentation and is suspicious for Yersinia.

Confirm identification of suspicious organisms with various biochemical ID panels.

CN plates are held for 72 hours at room temperature.

E. Vibrio

162

 Arranged by Sarah Mohssen

Section I– Microbiology By Dr. Mohammed Ayad

After 8-24 hours of incubation, subculture alkaline peptone broth to a TCBS agar. Examine the TCBS agar for

yellow colonies that indicate sucrose fermentation. Screen suspicious colonies biochemically.

F. Staphylococcus aureus or Yeast

After 24 hours incubation, examine the BAP for Staphylococcus aureus or yeast. If organism is in moderate to

many amounts or as the predominant organism, work up the organism

G. Escherichia coli O157:H7

Setup stool culture with a MacConkey’s agar with 1% D-sorbitol (instead of lactose).

After 24 hours of incubation, examine Mac-Sorbitol plate for colorless colonies (E. coli O157:H7 is sorbitol

negative and most other normal flora strains of E. coli are sorbitol positive).

Figure shows E coli O157:H7 on a sorbitol-MacConkey’s agar plate Arrow indicates distinctive colorless

Confirm identification of suspicious organisms with various biochemical ID panels and O157:H7 antisera.

Shiga-like toxin producing strains of E. coli carry out EIA or molecular testing is performed to detect

these strains.

Serological Testing for Salmonella and Shigella

A. All isolates that biochemically resemble either Salmonella species or Shigella species must be confirmed by

serological methods following site specific procedures.

B. For suspected Shigella species, test the following somatic (“O”) antigens:

1. Antigen A = Shigella dysenteriae

2. Antigen B = Shigella flexneri

3. Antigen C = Shigella boydii

4. Antigen D = Shigella sonnei

163

 Arranged by Sarah Mohssen

Section I– Microbiology By Dr. Mohammed Ayad

C. For suspected Salmonella species, test the following somatic (“O”) antigens:

1. Polyvalent A-E, Vi serum

2. Polyvalent F-I serum

Clostridium difficile

A. Clostridium difficile is an anaerobic gram-positive spore-forming rod that causes antibiotic-associated

pseudomembranous enterocolitis.

Since C. difficile is found as part of the normal fecal flora in many individuals, isolation of the organism does

not prove the presence of disease. In cases of C. difficile diarrhea, the patient’s normal fecal flora is suppressed

by prolonged antibiotic therapy allowing the C. difficile to multiply and produce a toxin

B. Detection of the presence of the toxin is used to diagnose the disease. Toxin may be detected by tissue

culture assay, a latex agglutination test, or an ELISA test.

Stool specimens collected for toxin studies should be refrigerated until testing to preserve the toxin that is

rapidly labile at higher temperatures.

164

 Arranged by Sarah Mohssen

Section I– Microbiology By Dr. Mohammed Ayad

Lecture 16

Diagnostic Microbiology

Urinary tract infections (UTIs)

Urinary tract infections (UTIs) are among the most common bacterial infections and account for a significant part of the workload

in clinical microbiology laboratories.

Enteric bacteria (in particular, Escherichia coli) remain the most frequent cause of UTIs, although the distribution of pathogens

that cause UTIs is changing.

More important is the increase in resistance to some antimicrobial agents, particularly the resistance to trimethoprimsulfamethoxazole seen in E. coli. Physicians distinguish UTIs from other diseases that have similar clinical presentations with use

of a small number of tests, none of which, if used individually, have adequate sensitivity and specificity. Among the diagnostic

tests, urinalysis is useful mainly for excluding bacteriuria. Urine culture may not be necessary as part of the evaluation of

outpatients with uncomplicated UTIs, but it is necessary for outpatients who have recurrent UTIs, experience treatment failures,

or have complicated UTIs, as well as for inpatients that develop UTIs.

Urinary tract infections (UTIs) are among the most common bacterial infections. It has been estimated that symptomatic UTIs

result in as many as 7 million visits to outpatient clinics, 1 million visits to emergency departments, and 100,000 hospitalizations

annually. UTIs have become the most common hospital-acquired infection, accounting for as many as 35% of nosocomial

infections, and they are the second most common cause of bacteremia in hospitalized patients.

UTIs are challenging, not only because of the large number of infections that occur each year, but also because the diagnosis of

UTI is not always straightforward. Physicians must distinguish UTI from other diseases that have a similar clinical presentation,

some UTIs are asymptomatic or present with atypical signs and symptoms, and the diagnosis of UTIs in neutropenic patients

(who do not typically have pyuria) may require different diagnostic criteria than those used for the general patient population.

Because of these factors, physicians frequently rely on a small number of imperfect laboratory tests to augment clinical

impressions; even when clinical diagnosis are unequivocal, physicians may order laboratory tests to identify the cause of the

infection and/or to provide isolates for antimicrobial susceptibility testing. It therefore comes as no surprise that the laboratory

examination of urine specimens accounts for a large part of the workload in many hospital-based laboratories.

In fact, in many clinical laboratories, urine cultures are the most common type of culture, accounting for 24%–40% of submitted

cultures; as many as 80% of these urine cultures are submitted from the outpatient setting.

Causes of UTIS

The etiological agents of community-acquired and hospital-acquired UTIs differ. Enteric bacteria (in particular, Escherichia coli)

have been and remain the most frequent cause of UTI, although there is some evidence that the percentage of UTIs caused by E.

coli is decreasing. In contrast, significant changes in the causes of nosocomial UTI have been reported since 1980; from 1980

through 1991, the percentage of UTIs caused by E. coli, Proteus species, and Pseudomonas species decreased, whereas the

percentage of UTIs caused by yeasts, group B streptococci, and Klebsiella pneumoniae increased.

165

 Arranged by Sarah Mohssen

Section I– Microbiology By Dr. Mohammed Ayad

Different changes in the causative agents of UTI, with a decrease in the percentage of UTIs caused by Enterobacter species, but

with an increase in the percentage of UTIs caused by Acinetobacter species and Pseudomonas aeruginosa. Candida albicans is

the most common cause of funguria, followed by Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida krusei,

and other yeasts.

Specimen collection

Suprapubic aspiration is the best method to avoid contamination of specimens with bacteria in the distal urethra. This collection

method is used infrequently because it is not indicated clinically (except in rare circumstances), it is invasive and uncomfortable,

and it requires too much time and too many resources to be practical. Collection of urine by use of a single catheter (straight

catheter technique) is the next best technique for obtaining urine specimens with minimal contamination, but, again, it is not

indicated clinically for most patients because it is too labor intensive and costly for routine use and it is invasive.

It has added disadvantages, because the process of inserting a catheter through the urethra can introduce bacteria into the bladder

(and thereby cause UTI), and rare complications have been reported.

Most urine specimens are obtained from adult patients via the clean-catch midstream technique. This technique has the following

advantages: it is neither invasive nor uncomfortable, it is simple and inexpensive, it can be performed in almost any clinical

setting, there is no risk of introducing bacteria into the bladder by catheterization, and there is no risk of complications.

Colony counts from urine specimens collected by this method correlate reasonably well with those of specimens collected via

suprapubic aspiration or straight catheterization.

The obvious disadvantage of this technique is that the urine sample passes through the distal urethra and can become

contaminated with commensal bacteria. Simple procedures that have been developed to decrease the contamination rate include

cleansing of skin and mucous membranes adjacent to the urethral orifice before micturition, allowing the first part of the urine

stream to pass into the toilet, and collecting urine for culture from the midstream.

Although the clean-catch midstream method is accepted and used widely, the available evidence suggests that the cleansing

procedures may not decrease urine contamination rates significantly and, therefore, may be unnecessary as a routine. There may

be difficulties with proper collection of samples from elderly patients, as well as from those patients who have physical or other

types of impairments, which adds to the importance of collecting specimens properly to avoid contamination.

Correct processing and handling of urine specimens, as well as correct interpretation of test results, is dependent on the method

used to collect the specimen. It is, therefore, of obvious importance for clinicians to specify the method of collection on the test

requisition. Other information that should be included on the test requisition includes the date and time of specimen collection,

patient demographic information, and any clinically relevant information (e.g., whether the patient was treated with antimicrobial

agents or whether anatomic abnormalities, stones, or an indwelling urinary catheter were present).

Specimen transportation

Urine specimens must plated within 2 hours after collection and if plated after delayed for 24 hours then plated so the culture

results were compared to determine whether delays in plating resulted in an increase in colony counts. In each of the situation,

some of the cultures that were delayed showed increases in the number of colony forming units (CFU) per ml to >105 CFU/ml;

thereby leading to false-positive results.

166

 Arranged by Sarah Mohssen

Section I– Microbiology By Dr. Mohammed Ayad

It should be noted that recent studies were performed for interpreting quantitative urine cultures which regarded colony counts of

102

-103 CFU/ml were used to define probable infection in specific patients. On the basis of these results, it is currently

recommended that urine specimens should be plated within 2 h after collection unless specimens have been refrigerated or kept in

a preservative.

Specimen processing

Routine urine cultures should be plated using calibrated loops for the semi-quantitative method. This method has the advantage of

providing information regarding the number of CFU/ml, also providing isolated colonies for identification and susceptibility

testing. The types of media used for routine cultures should be limited to blood agar and MacConkey's agar.

For urine specimens obtained from outpatients, it is not necessary to routinely inoculate a medium that is selective for grampositive bacteria, because nearly all UTIs in outpatients are caused by aerobic and facultative gram-negative bacteria.

Even in patient populations in which Staphylococcus saprophyticus is a common cause of UTIs, it is not necessary to use selective

media and urine specimens which obtained from hospitalized patients are likely to contain Enterococci, which have emerged as

the second most common cause of nosocomial infections.

Urine cultures should be incubated overnight at 35°C-37°C in ambient air before being read. There is no added benefit to

incubating routine urine cultures for 48 h, provided that specimens are incubated for a full 24 h and that urine specimens

containing <104

uropathogens or specimens from patients with suspected funguria are incubated for 48 hours.

Most pathogenic yeasts grow well on blood agar plates, so it is unnecessary to use selective fungal media for urine cultures, even

for samples obtained from patients with suspected funguria. Selective fungal media can be used in those rare instances in which

there is a high clinical probability that a UTI is caused by a more fastidious yeast or mold. Urine specimens obtained from patients

with suspected mycobacterial UTIs should be processed and plated to the appropriate mycobacterial media

Detection of bacteriuria by urine microscopy

Bacteriuria can be detected microscopically using Gram staining of uncentrifuged urine specimens, Gram staining of centrifuged

specimens, or direct observation of bacteria in urine specimens. Gram stain of uncentrifuged urine specimens is a simple method.

A volume of urine is applied to a glass microscope slide, allowed to air dry, stained with Gram stain, and examined

microscopically.

The performance characteristics of the test are not well-defined, because different criteria have been used to define a positive test

result. In one study, the test was found to be sensitive for the detection of ⩾105 CFU/ml but insensitive for the detection of lower

numbers of bacteria.

The urine Gram stain test has the important advantage of providing immediate information as to the nature of the infecting

bacterium or yeast and thereby guiding the physician in selecting empiric antimicrobial therapy. This is of importance in some

settings, but the Gram stain test has 3 disadvantages that limit its usefulness in most clinical settings:

1- It is an insensitive test, being reliably positive only if the concentration of bacteria in the urine is ⩾105 CFU/ml; infections

with bacterial concentrations of 102

-103 CFU/ml may not be detected by this test.

167

 Arranged by Sarah Mohssen

Section I– Microbiology By Dr. Mohammed Ayad

2- The test is too labor intensive for it to be practical for most clinical microbiology laboratories to offer it on more than a select

basis.

3- Because it may not detect bacteria at concentrations of 102

-103 CFU/ml, it should not be used in the outpatient setting for

patients with uncomplicated UTIs.

Because of these limitations, its use should be limited to patients with cases of acute pyelonephritis, patients with invasive UTIs,

or other patients for whom it is important to have immediate information as to the nature of the infecting pathogen.

Detection of bacteriuria by nitrite test

Bacteriuria can be detected chemically when bacteria produce nitrite from nitrate. The biochemical reaction that is detected by the

nitrite test is associated with members of the family Enterobacteriaceae (the pathogens most commonly responsible for UTIs),

but the usefulness of the test is limited because nitrite production is not associated with urinary-tract pathogens such as S.

saprophyticus, Pseudomonas species, or Enterococci.

Another limitation to the test is that it requires testing a specimen of the first urine produced in the morning, as ⩾4 h are required

for bacteria to convert nitrate to nitrite at levels that are reliably detectable.

Detection of pyuria by urine microscopy

Pyuria can be detected and quantified microscopically by measuring the urinary leukocyte excretion rate, counting leukocytes

with a hemocytometer, counting leukocytes in urine specimens using Gram staining, or counting leukocytes in a centrifuged

specimen.

The advantages to urine microscopy are that leukocytes, leukocyte casts, and other cellular elements are observed directly. One

disadvantage to urine microscopy is that leukocytes deteriorate quickly in urine that is not fresh or that has not been adequately

preserved. Because of these disadvantages, urine microscopy should be limited to patients in whom pyelonephritis or other more

serious infections are suspected.

The most accurate microscopic method for quantitating pyuria is to measure the urinary leukocyte excretion rate. Patients with

symptomatic UTIs have urinary leukocyte excretion rates of ⩾400,000 leukocytes/hour. The test is impractical for clinical use,

however, making it necessary for laboratories to use other methods. A simple and inexpensive alternative is to count urine

leukocytes with a hemocytometer. Comparison of hemocytometer counts with urinary leukocyte excretion rates has shown that a

hemocytometer count of ⩾10 leukocytes/mm3

correlates with a urinary leukocyte excretion rate of ⩾400,000 leukocytes/h.

Moreover, the correlation of hemocytometer counts with urine colony counts has shown that patients with symptomatic UTIs and

bacterial concentrations of >105 CFU/ml have urine leukocyte counts of ⩾10 leukocytes/mm3

. Although using a hemocytometer

to count leukocytes is easier than measuring urinary leukocyte excretion rates, it is impractical for clinical laboratories to use a

hemocytometer to count leukocytes on a routine basis. The most practical microscopic method involves counting the number of

leukocytes in the sediment of centrifuged urine specimens; this method is inaccurate because of inadequate standardization of the

method. For these reasons, and to facilitate the processing of large numbers of specimens, most laboratories use rapid tests for

leukocyte esterase as a surrogate for microscopic leukocyte counts.

Detection of pyuria by leukocyte esterase tests

168

 Arranged by Sarah Mohssen

Section I– Microbiology By Dr. Mohammed Ayad

Leukocyte esterase tests are based on the hydrolysis of ester substrates by proteins with esterolytic activity; human neutrophils

produce as many as 10 proteins with esterolytic activity. These proteins react with ester substrates to produce alcohols and acids

that then react with other chemicals to produce a color change that is proportional to the amount of esterase in the specimen.

These tests have the advantage of detecting both esterases in intact leukocytes and esterases released after cell lysis; therefore,

even specimens that have not been preserved properly may yield a positive test result.

Leukocyte esterase tests can yield false-positive test results when the urine is contaminated with bacteria present in vaginal fluid;

when the specimen contains eosinophils or Trichomonas species, both of which can act as sources of esterases; and when

oxidizing agents or formalin react with the test strips to generate false-positive test results.

Leukocyte esterase tests may show a decrease in positive test results when the specimen has an elevated specific gravity and/or

elevated levels of protein and glucose; when boric acid preservatives are present; when large amounts of ascorbic or oxalic acid

are present; and when the patient has received antimicrobial agents, such as Cephalothin, Cephalexin, or Tetracycline.

Routine bacterial urine cultures

Urine culture may not be necessary as part of the evaluation of outpatients with uncomplicated UTIs. However, urine cultures are

necessary for outpatients who have recurrent UTIs, experience treatment failures, or have complicated UTIs.

Urine cultures are also necessary for inpatients that develop UTIs. The bacterial culture remains an important test in the diagnosis

of UTI, not only because it helps to document infection, but also because it is necessary for determination of the identity of the

infecting microorganism(s) and for antimicrobial susceptibility testing. This is particularly true because of the increased incidence

of antimicrobial resistance.

The most commonly used criterion for defining significant bacteriuria is the presence of ⩾105 CFU per milliliter of urine. This

criterion was established only for women with acute pyelonephritis or women who were asymptomatic but had multiple urine

cultures that yielded this number of bacteria; however, the criterion is often applied to other patient populations. Most patients

with UTIs, however, do not fall into either category, and 30%-50% of patients with acute urethral syndrome will have colony

counts of <105 CFU/ml. For this reason, many laboratories have opted to use lower colony counts as a criterion for interpreting

and reporting results. One common criterion is a colony count of 104 CFU/ml, which would be expected to increase the sensitivity

of the test without making the test impractical for clinicians and laboratories to use.

Catheterized patients (who may have low concentrations of bacteria that can progress to higher concentrations) and many patients

with infections of the lower urinary tract have colony counts much lower than 105 CFU/ml if the specimens are obtained via

suprapubic aspirate or catheterization. Accordingly, the most appropriate diagnostic criterion for urine culture specimens obtained

via suprapubic aspirate or catheterization is a bacterial concentration of ⩾102 CFU/ml.

Follow-up cultures are recommended for patients with infections that do not respond to therapy, patients who have recurrent

UTIs, patients who have anatomic or functional abnormalities of the urinary tract, or patients who continue to have unexplained

abnormal urinalysis findings.

Interpretation of urine culture results

169

 Arranged by Sarah Mohssen

Section I– Microbiology By Dr. Mohammed Ayad

Microbiologists need to interpret the microbiologic relevance of growth on culture plates to determine whether further

identification and antimicrobial susceptibility testing are necessary. Most culture results can be interpreted readily; no growth and

gross contamination are both unambiguous results, as are pure cultures of common pathogens growing in a quantity of >105 CFU

per milliliter of urine.

The interpretation of cultures that yield pure growth in lower quantities is also clear for specimens obtained via suprapubic

aspiration or straight catheterization. On the other hand, interpretation of urine cultures that yield mixed flora in varying quantities

can be difficult. Test reports for cultures that yield mixed flora in varying quantities should specify the microorganisms that were

recovered, the quantity of each microorganism, and the probable clinical importance of each isolate.

Antimicrobial susceptibility testing

Each laboratory should have guidelines by which pathogens are tested for antimicrobial susceptibility. These guidelines should be

developed and antimicrobial susceptibility tests should be performed and reported according to the most recent version of the

NCCLS guidelines. Bacterial or fungal isolates of uncertain clinical importance should not be tested for antimicrobial

susceptibility for purposes of routine patient care

Most patients with uncomplicated acute cystitis have cases that are clinically straightforward, and they may not require any

laboratory testing beyond urinalysis. For a significant number of patients, however, the clinical history and physical findings

alone may be insufficient to make a definitive diagnosis of UTI.

For those patients and for patients with complicated UTIs, laboratory tests are necessary to make the diagnosis and to provide

specific information regarding the identity and the antimicrobial susceptibility pattern of pathogens. Both the laboratory diagnosis

and the clinical diagnosis of laboratory test results must be made in light of the method of collection used; clinicians should

specify the method of collection on test requisition forms.

Of the available laboratory tests, urinalysis is helpful primarily as a means of excluding bacteriuria, but it is not a surrogate for

culture. Although cultures identify pathogens, the accurate interpretation of culture results requires clinical information that is

usually available only to the clinician.

170

 Arranged by Sarah Mohssen

Section I– Microbiology By Nada Sajet

Bloodstream Infections :

 Invasion of the bloodstream by microorganisms constitutes one of the most serious situations in

infectious disease. Microorganisms present in the circulating blood—whether continuously, intermittently, or

transiently—are a threat to every organ in the body.

 The suffix emia is derived from the Greek word meaning “blood” and refers to the presence of a substance in

the blood; bacteremia refers to the presence of bacteria in the blood, fungemia refers to the presence of fungi

in the bloodstream, and septicemia indicates bacteria are present in the blood, producing an infection and

reproducing within the bloodstream.

 Microbial invasion of the bloodstream resulting from any organism can have serious immediate

consequences, including shock, multiple organ failure, disseminated intravascular coagulation (DIC), and death.

Approximately 200,000 cases of bacteremia and fungemia occur annually, with mortality

rates ranging from 20% to 50%. Timely detection and identification of blood-borne pathogens are two of the

most important functions of the microbiology laboratory.

Pathogens of all four major groups of microbes—bacteria, fungi, viruses, and parasites—may be found

circulating in blood during the course of many diseases. Positive blood cultures may help provide a clinical

diagnosis, as well as a specific etiologic diagnosis.

General considerations:

The successful recovery of microorganisms from blood by the laboratory depends on many, often complex,

factors: the type of bacteremia, the specimen collection method, the blood volume, the number and timing of

blood cultures, the interpretation of results, and the type of patient population being served by the laboratory.

 All of these parameters must be considered in the development of the blood culture protocol within the

laboratory in order to maximize the detection and recovery of microorganisms and ensure quality patient care.

Etiology:

As previously mentioned, all major groups of microbes can be present in the bloodstream during the course of

many diseases.

Bacteria:

The organisms most commonly isolated from blood are gram-positive cocci, including coagulase-negative

staphylococci, Staphylococcus aureus, and Enterococcus spp., and other organisms likely to be inhabitants of

the hospital environment that colonize the skin, oropharynx, and gastrointestinal tract of patients. Some of the

most common, clinically significant bacteria isolated from

blood cultures are listed in Table 1. (Table 1 Organisms Commonly Isolated from Blood Cultures)

Organisms Commonly Isolated from Blood Cultures:

Staphylococcus aureus

Escherichia coli

Coagulase-negative staphylococci

Enterococcus spp.

171

 Arranged by Sarah Mohssen

Section I– Microbiology By Nada Sajet

Candida albicans

Pseudomonas aeruginosa

Klebsiella pneumoniae

Viridans streptococci

Streptococcus pneumoniae

Enterobacter cloacae

Proteus spp.

Beta-hemolytic streptococci

Anaerobic bacteria: Bacteroides and Clostridium spp.

 In general, the number of fungi and coagulase-negative staphylococc has increased, whereas the number of

clinically significant anaerobic isolates has decreased since the early 2000s.

Of importance, the laboratory isolation of certain bacterial species from blood can indicate the presence of an

underlying, occult, or undiagnosed neoplasm. Alterations in local conditions at the site of the neoplasm

allowing bacteria to proliferate and seed the bloodstream have been suggested as a potential mechanism for the

association between bacteremia and cancer. Another possible mechanism is reduced killing of bacterial cells by

the host phagocytes. Organisms associated with neoplastic disease include Clostridium septicum and other

uncommonly isolated clostridial species, Streptococcus galldyticus, Aeromonas hydrophila, Plesiomonas

shigelloides, and Campylobacter spp. Finally, if Streptococcus anginosis group bacteria are isolated from

blood, the possibility of an abscess should be considered.

Fungi:

 Fungemia (the presence of fungi in blood) is usually a serious condition, occurring primarily in

immunosuppressed patients and in those with serious or terminal illness. Candida albicans is by far the most

common species, but Malassezia furfur can often be isolated in patients, particularly neonates, receiving lipidsupplemented parenteral nutrition. Candida spp. account for approximately 8% to 10% of all nosocomial

bloodstream infections. Except for Histoplasma, which multiply in leukocytes

(white blood cells), fungi do not invade blood cells, but their presence in the blood usually indicates a focus of

infection elsewhere in the body.

 Fungi in the bloodstream can disseminate (be carried) to all organs of the host, where they may grow, invade

normal tissue, and produce toxic products. Fungi gain entrance to the circulatory system via loss of integrity of

the gastrointestinal or other mucosa; through damaged skin; from primary sites of infection, such as the lung or

other organs; or by means of intravascular catheters. Systemic fungal infections begin as pneumonia and may

disseminate from the lungs, which serve as the portal of entry. Arthroconidia of Coccidioides immitis and

microconidia of Histoplasma capsulatum and Blastomyces dermatitidis are ingested by alveolar macrophages

in the lung.

 These macrophages carry the fungi to nearby lymph nodes, usually the hilar nodes. The fungi multiply within

the node tissue and ultimately are released into the circulating blood, from which they are capable of seeding

172

 Arranged by Sarah Mohssen

Section I– Microbiology By Nada Sajet

other organs or are destroyed by the body’s defenses. Molds are particularly insensitive to host defenses such as

antibody and phagocytic cells because of their large size and their sterol containing cell wall structure.

Parasites:

 Eukaryotic parasites may be found transiently in the bloodstream as they migrate to other tissues or organs.

Their presence, however, cannot be considered consistent with a state of good health. For example, tachyzoites

of the parasite Toxoplasma gondii may be found in circulating blood. They invade cells within lymph nodes

and other organs, including the lungs, liver, heart, brain, and eyes. The resulting cellular destruction accounts

for the manifestations of toxoplasmosis. Also, microfilariae are

seen in peripheral blood during infection with Dipetalonema, Mansonella, Loa loa, Wuchereria, or Brugia.

Malarial parasites invade host erythrocytes and hepatic parenchymal cells. The significant anemia and

subsequent tissue hypoxia (reduction in oxygen levels) may result from destruction of red blood cells by the

parasite. Vascular trapping of normal erythrocytes by the infected red blood cells, which are less flexible and

tend to clog small capillaries, is a major cause of morbidity. The host’s immunologic response is to remove the

parasites and damaged red blood cells; the immune response may also

have deleterious effects. Parasites in the bloodstream are usually detected by direct visualization. Those

parasites for which traditional diagnosis is dependent on observation of the organism

in peripheral blood smears include Plasmodium, Trypanosoma, and Babesia. Patients with malaria or filariasis

may display a periodicity in their episodes of fever that allows the physician to time the collection of blood for

microscopic examination intended for optimal detection. Rapid serological methods and molecular methods are

currently used to detect malaria, babesiosis, and trypanosomiasis.

Viruses:

 Although many viruses do circulate in the peripheral blood at some stage of disease, the primary pathology

relates to infection of the target organ or cells. Those viruses that preferentially infect blood cells are EpsteinBarr virus (invades lymphocytes), cytomegalovirus (invades monocytes, polymorphonuclear cells, and

lymphocytes), and human immunodeficiency virus (HIV)

(involves only certain T lymphocytes and perhaps macrophages) and other human retroviruses that attack

lymphocytes. The pathogenesis of viral diseases of the blood is the same as that for viral diseases of any organ;

by diverting the cellular machinery to create new viral components

or by other means, the virus may prevent the host cell from performing its normal function. The cell

may be destroyed or damaged by viral replication, and immunologic responses of the host may also contribute

to the pathogenesis. Although many viral diseases have a viremic stage,

recovery of virus particles or detection of circulating viruses is used in the diagnosis of only a few diseases.

Types of bacteremia:

 Bacteremia may be transient, continuous, or intermittent. Most people have experienced transient bacteremia;

teething infants and people having dental procedures have had oral flora gain entry to the bloodstream through

breaks in the gums. Other conditions in which bacteria are only transiently present in the bloodstream include

manipulation of infected tissues, devices or instrumentation inserted through contaminated mucosal surfaces,

173

 Arranged by Sarah Mohssen

Section I– Microbiology By Nada Sajet

and surgery involving nonsterile sites. These circumstances may also lead to significant septicemia, although

normally the bacteria are cleared from the blood by scavenging

leukocytes, resulting in no infection. Septicemia can occur when the bacteria multiply more rapidly than the

immune system is capable of killing and removing the organism. In septic shock, bacterial endocarditis, and

other endovascular infections, organisms are released into the bloodstream at a fairly constant rate (continuous

bacteremia). Also, during the early stages of specific infections, including typhoid fever, brucellosis, and

leptospirosis, bacteria are continuously present in the bloodstream.

In most other infections, such as in patients with undrained abscesses, bacteria can be found intermittently in

the bloodstream. Of note, the causative agents of meningitis, pneumonia, pyogenic arthritis, and osteomyelitis

are often recovered from blood during the early course of these diseases. In the case of transient seeding of the

blood from a sequestered focus of infection, such as an

abscess, bacteria are released into the blood approximately 45 minutes before a febrile episode.

The symptoms of septicemia are fever, chills, and malaise; these are caused by the presence of the invading

microorganism and the toxins produced by these microorganisms. The older the patient is, the greater the risk

and the rate of mortality as a result of septicemia.

Types of blood stream infections:

The two major categories of bloodstream infections are intravascular (those that originate within the

cardiovascular system) and extravascular (those that result from bacteria entering the blood circulation through

the lymphatic system from another site of infection). Of note, other organisms, such as fungi, may also cause

intravascular or extravascular infections. However, because bacteria account for the majority of significant

vascular infections, these types of bloodstream infections are discussed in more detail. Factors contributing to

the initiation of bloodstream infections are immunosuppressive agents, widespread use of broad-spectrum

antibiotics that suppress the normal flora and allow the emergence

of resistant strains of bacteria, invasive procedures allowing bacteria access to the interior of the host, more

extensive surgical procedures, and prolonged survival of debilitated and seriously ill patients.

1) Intravascular Infections:

 Intravascular infections include infective endocarditis, mycotic aneurysm, suppurative thrombophlebitis, and

intravenous (IV), catheter-associated bacteremia. Because these infections are within the vascular system,

organisms are present in the bloodstream at a fairly constant rate (i.e., a continuous bacteremia). These

infections in the cardiovascular system are extremely serious and considered life threatening.

Infective Endocarditis. The development of infective endocarditis (infection of the endocardium most

commonly caused by bacteria) is believed to involve several independent events. Cardiac abnormalities, such as

congenital valvular diseases that lead to turbulence in blood flow or direct trauma from IV catheters, can

damage cardiac endothelium. This damage to the endothelial

surface results in the deposition of platelets and fibrin. If bacteria transiently gain access to the bloodstream

(this can occur after an innocuous procedure such as brushing the teeth) after alteration of the capillary

endothelial cells, the organisms may stick to and then colonize the damaged cardiac endothelial cell surface.

174

 Arranged by Sarah Mohssen

Section I– Microbiology By Nada Sajet

After colonization, the surface will rapidly be covered with a protective layer of fibrin and platelets. This

protective environment is favorable to further bacterial multiplication. This web of platelets, fibrin,

inflammatory cells, and entrapped organisms is called a vegetation (Figure -1).

 The resulting vegetations ultimately seed bacteria into the blood at a slow but constant rate. The primary

causes of infective endocarditis are the viridans streptococci, comprising several species (Box 68-2). These

organisms are normal inhabitants of the oral cavity, often gaining entrance to the bloodstream as a result of

gingivitis, periodontitis, or dental manipulation. Heart valves, especially those previously damaged, present

convenient surfaces for attachment of these bacteria. Streptococcus sanguis and Streptococcus mutans are

frequently isolated in streptococcal endocarditis.

Gram-negative bacilli, known as the AACEK group, Aggregatibacter aphrophilus, Actinobacillus

actinomycetemcomitans Cardiobacterium hominis, Eikenella corrodens, and Kingellakingae, can also be

associated with endocarditis.

With the ever-increasing use of IV catheters, arterial lines, and vascular prostheses, organisms considered

normal or hospital-acquired inhabitants of the human skin are able to gain access to the bloodstream and attach

to various surfaces, including heart valves and vascular endothelium. It has been estimated that more than

200,000 nosocomial infections (bloodstream) occur annually in the United States in adults and children. The

majority of these infections are caused by the use of intravascular catheters. Staphylococcus epidermidis and

other coagulase-negative staphylococci have been increasingly implicated as the cause of infection associated

with intravascular catheters. S. epidermidis is the most common etiologic agent identified in prosthetic valve

endocarditis, with S. aureus being the second most common. S. aureus is an important cause of septicemia

without endocarditis and is found in association with other foci, such as abscesses, wound infections, and

pneumonia, as well as sepsis related to indwelling intravascular catheters.

Figure 1 Vegetations of bacterial endocarditis. Arrow indicates the vegetations.

(Courtesy Celeste N. Powers, MD, PhD, Virginia Commonwealth

University Medical Center, Medical College of Virginia Campus, Richmond, Va.

175

 Arranged by Sarah Mohssen

Section I– Microbiology By Nada Sajet

Table 2 Agents of Infective Endocarditis

Agents of Infective Endocarditis:

Aggregatibacter aprophilus

Viridans streptococci*

Nutritionally deficient streptococci (Abiotrophia spp. and

Granulicatella spp.)

Enterococci*

Streptococcus bovis

Staphylococcus aureus*

Staphylococci (coagulase-negative)

Enterobacteriaceae

Pseudomonas spp. (usually in drug users)

Haemophilus spp.

Unusual gram-negative bacilli (e.g., Actinobacillus,

Cardiobacterium, Eikenella, Coxiella burnetii)

Yeast

Other (including polymicrobial infectious endocarditis)

*Most common organisms associated with native valve endocarditis in non-drug-using

adults.

Table -3 Common Agents of IV Catheter–Associated Bacteremia:

Staphylococcus epidermidis

Other coagulase-negative staphylococci

Staphylococcus aureus

Enterobacteriaceae

Pseudomonas aeruginosa

Figure 2 Short-term, triple-lumen central venous catheter. The ends from which the catheter is accessed

are usually referred to as the hubs. After the catheter is inserted, the tip resides within the bloodstream.

176

 Arranged by Sarah Mohssen

Section I– Microbiology By Nada Sajet

Candida spp.

Corynebacterium spp.

Other gram-negative rods

2)Extravascular Infections:

Except for intravascular infections, bacteria usually enter the circulation through the lymphatic system. Most

cases of clinically significant bacteremia are a result of extravascular infection. When organisms multiply at a

local site of infection such as the lung, they are drained by the lymphatics and reach the bloodstream. In most

individuals, organisms in the bloodstream are effectively and rapidly removed by the reticuloendothelial system

in the liver, spleen, and bone marrow and by circulating phagocytic cells. Depending on the extent of

immunologic control of the infection, the organism may be circulated more widely, thereby causing a

bacteremia or fungemia..

The most common portals of entry for bacteremia are the genitourinary tract (25%), respiratory tract (20%),

abscesses (10%), surgical wound infections (5%), biliary tract (5%), miscellaneous sites (10%), and uncertain

sites (25%). For the most part, the probability of bacteremia occurring from an extravascular site depends on

the site of infection, its severity, and the organism. For example, any organism producing meningitis is likely to

produce bacteremia at the same time.

 Of importance, certain organisms causing extravascular infections commonly invade the bloodstream; In

addition to these organisms, a large number of other bacteria and fungi that cause extravascular infections are

also capable of invading the bloodstream. Whether these organisms invade the bloodstream depends on the

host’s ability to

control the infection and the organism’s pathogenic potential. Some of the organisms associated with potential

bloodstream infections from a localized site include members of the family Enterobacteriaceae, Streptococcus

pneumoniae, Staphylococcus aureus, Neisseria gonorrhoeae,

anaerobic cocci, Bacteroides, Clostridium, beta-hemolytic streptococci, and Pseudomonas. These are only

some of the organisms frequently isolated from blood. Almost every known bacterial species and many fungal

species have been implicated in extravascular bloodstream infection.

Clinical manifestations:

 bacteremia may indicate the presence of a focus of disease, such as intravascular infection, pneumonia, or

liver abscess, or it may represent transient release of bacteria into the bloodstream. Septicemia or sepsis

indicates a condition in which bacteria or their products (toxins) are causing harm to the host. Unfortunately,

clinicians often use the terms bacteremia

and septicemia interchangeably. Signs and symptoms of septicemia may include fever or hypothermia (low

body temperature), chills, hyperventilation (abnormally increased breathing leading to excess loss of carbon

dioxide from the body) and subsequent respiratory alkalosis (a condition caused by the loss of acid leading to

an increase in pH), skin lesions, change in mental status, and diarrhea. More serious manifestations include

hypotension or shock, DIC, and major organ system failure.

The syndrome known as septic shock, characterized by fever, acute respiratory distress, shock, renal failure,

intravascular coagulation, and tissue destruction, can be initiated by either exotoxins or endotoxins.

177

 Arranged by Sarah Mohssen

Section I– Microbiology By Nada Sajet

 Septic shock is mediated by the production of cytokines from activated mononuclear cells, such as tumor

necrosis factor and interleukins Shock is the gravest complication of septicemia. In septic shock, the presence

of bacterial products and the host’s response act to shut down major host physiologic systems.

Clinical manifestations include a drop in blood pressure, increase in heart rate, functional impairment in vital

organs (brain, kidney, liver, and lungs), acid base alterations, and bleeding problems. Gram-negative bacteria

contain a substance in their cell walls, called endotoxin, which has a strong effect on several physiologic

functions. This substance, a lipopolysaccharide (LPS) comprising part of the cell wall structure , may be

released during the normal growth cycles of bacteria or after the destruction of bacteria by host defenses.

Endotoxin (or the core of the LPS, lipid A) has been shown to mediate numerous systemic reactions, including

a febrile response, and the activation of complement and certain blood-clotting factors. Although gram-positive

bacteria do not contain the lipid A endotoxin, many produce exotoxins, and the effects of their presence in the

bloodstream may be equally devastating to the patient.

Disseminated intravascular coagulation (DIC) is a complication of sepsis. DIC is characterized by numerous

small blood vessels becoming clogged with blood clots and bleeding as a result of the depletion of coagulation

factors. DIC can occur with septicemia involving any circulating pathogen, including parasites, viruses, and

fungi, although it is most often a consequence of gram-negative bacterial sepsis.