6 Overview of enterobacteria. Coproculture.

from "Diagnosticul de laborator în Microbiologie"

by Prof. Dr. Mircea Ioan POPA
translated by Dr. Andrei-Alexandru MUNTEAN

6.1 Introduction
The Enterobacteriaceae family (now known as Enterobacterales) comprises a significant number of
genera of medically significant microorganisms (28) and numerous species (over 100, if we do not consider
the classification of the genus Salmonella into more than 2000 species). Of the genera included in this
large family, we will mention the following: Escherichia, Shigella, Salmonella, Yersinia, Klebsiella, Proteus,
Citrobacter, Edwarsiella, Enterobacter, Morganella, Providencia and Serratia. In the following chapters
we will only discuss the diagnosis in infections produced by microorganisms belonging to the first 6 genera
listed. Enterobacteria are Gram-negative bacilli that cannot be differentiated through optical microscopy.
Some are motile, due to the presence of peritrichous cillia (eg Salmonella spp., Proteus spp.), nonmotile
(eg Shigella, Yersinia, Klebsiella), unsporulated. Some species are capsulated (eg Klebsiella pneumoniae).

• Culture characteristics: They are non-fastidious bacteria that can multiply on simple media, aerobes,
facultative anaerobes, that have a fermentative glucose metabolism with or without gas production,
are oxidase-negative, catalase-positive, and they reduce nitrates. They form S, R (in the case of
öldc̈ultures) or M (for the capsule species) colonies. They may ferment lactose (eg Escherichia coli,
Klebsiella spp.) or are lactose negative (eg Salmonella spp., Shigella spp., Yersinia spp.). In order
to isolate the enterobacter species we can use selective, differential and selective-differential media.
Based on the level of selectivity of the media used there:
- are low selectivity media that inhibit most Gram-positive bacteria and allow the development of
all enterobacteria (eg Mac Conkey medium with low concentration of bile salts; purple crystal
may also be added),
- media medium selectivity that inhibits the multiplication of lactose-positive enterobacteria (eg
ADCL medium with deoxycholate, citrate and lactose, the Shigella-Salmonella medium with
bile salts and brilliant green or the XLD medium with xylose, lysine and deoxycholate, preferred
in many European Union countries) and
- media with high selectivity (eg Wilson-Blair medium with high concentration of brilliant green).

• Biochemical characteristics: are essential for the identification of enterobacteria. After examining
the culture characteristics (on the selective and differential media, lactose fermentation can be
identified), by subculturing the suspicious colonies on suitable media (TSI, MIU / MILF, Simmons
etc), the different biochemical characteristics may be examined the following day (fermentation
of other sugars, metabolization of a certain substrate, use of citrate as the only carbon source,
identification of enzymes, etc.) or other phenotypic characteristics (eg motility). Currently there
are test batteries and commercial systems that allow the examination of a wide range of biochemical
characteristics (eg API galleries). We would like to mention that for each phenotypic test there are
a number of variations that are presented as a percentage in the specially dedicated tables.

• Another very useful aspect for identification is the study of antigenic characteristics using known sera
(prepared in laboratory animals), with the help of different Ag-Ac reactions (see also chapters 15-20).
All enterobacteria have Ag O (LPS). Mobile enterobacteria have Ag H. Capsulated enterobacteria
have Ag K. Salmonella Typhi also has Ag Vi, Yersinia pestis Ag F1, etc. Enterobacterial infections

15
6 Overview of enterobacteria. Coproculture.

are quite varied. They can be located at the digestive tract (dysentery and dysentery-like syndromes,
cholera-like syndromes, food-poisoning and other enteral infections, etc.), at the level of the urinary
tract, the respiratory tract or nervous system. Enterobacterial infections may also infect other
specific sites or may be generalized (eg, typhoid fever, paratyphoid fever, the plague). In most
infections caused by enterobacteria the diagnosis is bacteriological, direct. In typhoid fever the
diagnosis can be both bacteriological and immunological (serological). Due to the fact that in many
of these infections the clinical specimen is represented by the fecal matter, we will next discuss the
bacteriological examination of the fecal matter.

6.2 Coproculture
In many of the infections caused by enterobacteria, which are manifested by diarrhea syndrome, we use
coproculture. In the following we will discuss the coproculture in general, not only from the point of view
of an enterobacterial infection.

6.2.1 Acute diarrheal disease

Acute diarrheal disease (ADD) has been and remains a widespread pathological entity worldwide. For
example, in our country in recent years, the number of cases of acute diarrheal diseases was 85,055 in
2000, 81,268 in 2001 and 97,317 respectively in 2002, with an incidence of 446.5 0/0000 in 2002. the same
time period, about 100 patients died from ADD each year. Diarrhea syndrome includes elimination of
more than 3 stools in 24 hours and the stools have diminished consistency (it may be that only aqueous
stools are emitted, with loss of up to 20-30 liters / day in cholera). The fecal matter can have an altered
(pathological) appearance (aqueous, mucous, purulent, bloody etc.), modified color, a particular odor etc.
Usually, diarrheal syndromes also associates with other digestive signs and symptoms (abdominal pain,
tenes, nausea, vomiting, etc.) or general (fever, altered general condition, etc.). It is very important to
note that ADDs leads to water and electrolyte losses and the most important intervention to consider is
hydroelectrolyte rebalancing.

6.2.2 Etiological agents

Acute diarrheal disease may be of infectious and non-infectious etiology. Next we will briefly discuss
the infectious etiology in general, not just the bacterial or fungal one. It is worth mentioning that the
vast majority of infectious ADDs have a viral etiology.

• Bacterial etiologies include:

- Salmonella spp.
- Shigella spp.
- Escherichia coli (EPEC / enteropathogenic, ETEC / enterotoxigenic, EHEC / enterohemor-
rhagic, EIEC / enteroinvasive, EAEC / enteroaggregative, DAEC / diffusely adherent)
- Klebsiella spp.
- Other enterobacteria (Yersinia enterocolitica, Citrobacter spp., Proteus spp., etc)
- Vibrio cholerae and other vibrions
- Other Gram-negative bacilli (Aeromonas spp., Alcaligenes spp., Pseudomonas spp., etc.)
- Bacillus cereus
- Campylobacter jejuni
- Clostridium perfringens
- Clostridium difficile
- Clostridium botulinum
- Staphylococcus aureus etc

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 6.2 Coproculture

• Fungal etiologies include:

- Candida albicans (in AIDS patients), etc.

• Parasitic etiologies includes:

- Giardia lamblia
- Entamoeba hystolitica and Entamoeba coli
- Trichinella spiralis
- Cryptosporidium parvum
- Strongyloides stercoralis, etc

• Viral etiology includes:

- Rotaviruses
- Norwalk viruses and similar to Norwalk viruses
- Caliciviruses
- Astroviruses
- Coronaviruses
- Enteroviruses
- Adenoviruses, etc.

6.2.3 Classification of ADD according to the pathogenic mechanism:

From the diagnostic point of view and for the treatment it may be important to elucidate the type of
mechanism involved. Thus, ADD may be produced through:

- Non-inflammatory mechanism, when microscopic in the preparation between the slide and the cover-
slip, no leukocytes are detected (eg ADD produced by Vibrio cholerae, ETEC, Clostridium perfrin-
gens, Staphylococcus aureus, Giardia lamblia, rotaviruses, Norwalk viruses, Cryptosporidium parvum
etc)

- Inflammatory mechanism, when microscopically in the preparation between the slide and the cov-
erslip, polymorphonuclear leukocytes (eg ADD produced by Shigella spp., EIEC, Salmonella enter-
itidis, Vibrio parahaemolyticum, Entamoeba hystolitica, etc.) are present.

- Invasive mechanism, when microscopically in the preparation between the slide and the coverslip,
mononuclear leukocytes (eg ADD or diarrheal syndrome produced by Salmonella Typhi, Yersinia
enterocolitica etc) are present.

As can be noted, the simple collection of the clinical specimen followed by the preparation of a wet-
mount (see Chapter 7) and its microscopic examination can give important information regarding the
probable etiology and therapeutic attitude to follow. It is obvious that in a ADD for which the etiological
agents are viral or the pathogenic mechanism includes the action of a toxin, antibiotics are contraindicated.

6.2.4 Indications of coproculture in bacteriology

- When an infection with Shigella spp., Vibrio cholerae, (pathogenic types of ) E. coli , Campylobacter
spp., Yersinia enterocolitica or Salmonella spp. is suspected, especially in patients of extreme age
(the very young and the very old) or immunosuppression

- About 1 week after the onset of typhoid fever, etc.

- When the ADD presents the risk of extension at the level of a community (nurseries, kindergartens,
camps, public feeding units, drinkable water distribution stations, etc.)

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6 Overview of enterobacteria. Coproculture.

- When ADD occur in patients who have been treated with antibiotics and diarrhea syndrome persists
after discontinuation of antibiotic therapy

- When diarrhea syndrome persists for more than a week or a relapse occurs

- For the purpose of checking the health status of persons working in public food units (according to
the norms established by the Ministry of Health)

- For research purposes, etc.

We will now discuss the diagnosis, applying the general diagnostic workflow, which we will discuss, with
its particuliarities, for the respective enterobacteria in the following chapters.

1. Collection and transport of faeces: The recommendations mentioned in chapter 6 will be respected,
in particular the fact that the collection of c.s. must be done before the administration of antimi-
crobial drugs. The following can be sampled and examined:
- Spontaneous stool represents the most commonly used clinical specimen. The defecation takes
place in a cardboard container or in a single-use plastic container (which can then be decon-
taminated and disposed of without problems). At the same time we also perform macroscopic
examination of c.s. from the point of view of smell, color or appearance. We use for picking
the included spatula of the transport medium, and we’ll choose portions of the sample from
which we are most likely to isolate the pathogen (mucopurulent fragments, portions of blood,
etc.) or different portions of a stool where we do not notice the aforementioned aspects. We
take a quantity of about 1 gram which we suspend in the transport environment (minimum 2
ml in the case of aqueous stools).
- Rectal swab is recommended in case of chronic infection with Shigella spp., when we suspect
the portage of Shigella spp. or Salmonella spp. The sterile swab is gently inserted and the
secretions of the rectal mucosa are removed by spinning to collect material from the rectal
crypts. Then we introduce the swab in the transport environment. To be able to close the
container, we cut the stem of the swab.
- Nelaton probe can be used when looking at collecting c.s. from the sigmoid colon. Gently insert
the probe to the desired level (about 10-12 cm in case the patient is a child and approximately
15-20 cm in an adult patient), adapt the syringe to the other end of the probe and aspirate
the c.s. We introduce the c.s. in the transport medium.
If the c.s. cannot be processed immediately or within 1 hour, we use a transport medium, cold
transport (+4 ◦ C) or transport in a transport medium maintained at 4 ◦ C. Most commonly we
use the Cary-Blair medium (see chapter 9). This environment ensures the survival of pathogenic
enterobacteria for 24-48 hours, while inhibiting the multiplication of the association flora. When V.
cholerae infection is suspected, alkaline peptone water serves both as a transport and enrichment
medium.

2. Examination of fecal matter: The fecal matter is examined macroscopically and microscopically.
From a microscopic point of view, each time wet-mount preparations will be made (between the
slide and the coverslip). The fecal matter is suspended in a drop of 1% methylene blue and the
preparation will be examined initially with the 10x objective, then with 40x objective. For example,
the presence of over over 40 PMN / high power field, accompanied by macrophages and red blood
cells, raises the suspicion of bacterial dysentery. Colored smears may be useful when suspecting the
diagnosis of enterocolitis due to Campylobacter spp. or of pseudomembranous colitis after antibiotic
therapy.

3. Cultivation of faeces: Typically, for cultivation we will use 3 different media, namely:
- a sample with selenite sodium broth and 2 Petri dishes
- one with low selectivity medium (eg Mac Conkey)

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 6.2 Coproculture

- another with medium selectivity medium (eg ADCL or XLD). These selective media are also
differential media.
We choose characteristic fragments of the c.s. (containing mucus, pus, etc.) and inoculate 2-3 loops
in selenite broth (enrichment medium especially for Salmonella spp.). We make a homogeneous
suspension in the liquid media. We take a loopful (or a drop) from the suspension and place it on
the Mac Conkey environment. The method of streaking differs from that presented in Chapter 10.
We plate the c.s. using parallel streaks on a half plate, near the edges of the plate, without touching
the edges. Without sterilizing the loop, we touch the last 3-4 streaks and disperse the c.s. in other
parallel streaks, perpendicular to the first, on half of the available environment, up to the edges of
the plate, without touching them. Touching the last 3-4 streaks again, we spread the c.s. similarly
on the plate left uninoculated. Incubate for 18-24 hours at 35-37 ◦ C.
The following day, starting from the tube with selenite broth we streaked as mentioned above a
plate with Mac Conkey and a plate with ADCL (or XLD); incubate these plates for 18-24 hours at
35-37 ◦ C. We examine the previously streaked plates to identify suspicious colonies.
On the Mac Conkey agar, lactose-positive colonies are 1-3 mm in size, red, and may have an
opalescent halo; they are typically S-type but may also be M-type. Lactose-negative colonies are
1-2 mm in size, they are not colored, they are transparent, they are usually type S.
On ADCL, most lactose-positive bacteria are inhibited; small, red, S-type colonies may appear late,
lactose-negative colonies are 1-2 mm in size, are not colored, are semi-transparent, of type S; if they
produce H2 S the center of the colony is black.
Usually we monitor the occurrence of lactose-negative colonies, and for the identification steps we
will subculture (by picking the colonies without touching the culture medium) 3 colonies if the
patient is a child and 3-5 colonies if the patient is an adult. If there are no lactose-negative colonies,
we will subculture (without touching the culture medium) 10 colonies in the child and 10 colonies
in the adult (if the identification is considered necessary, eg in epidemic ADD outbreaks).

4. Therefore, after the appearance of the isolated colonies on the selective-differential media we proceed
to identify the species involved pathogens based on morphological and stain characteristics, culture
(see chapter 11), biochemical (see chapters 11 and 12), antigenic (see chapters 11-20, in especially
chapter 17), of pathogenicity, on the basis of sensitivity to bacteriophages and possibly on the basis
of molecular characteristics. For interpretation we will use the different tables available in medical
treaties or the manufacturersŕecommendations in the case of using commercial diagnostic systems.

5. For the strains considered to be pathogenic, we will perform the susceptibility studies on antibiotics
and chemotherapeutics, usually by diffusimetric methods (standardized diffusimetric antibiogram,
comparative antibiogram, E-test).
Other particular issues will be presented in the following chapters.

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7 Escherichia. Uroculture
The genus Escherichia comprises commensal germs that are ubiquitous (in water, soil, etc.) and in
the human intestine they play a role in the synthesis of vitamins (symbiosis). Are Gram-negative bacilli,
aerobic, facultatively anaerobic, lactose positive. The type species is Escherichia coli.
Although most E. coli strains are saprophytic or conditionally pathogenic (begin involved in oppor-
tunistic infections with locations outside the digestive tract, eg peritonitis, cholecystitis, wound infections,
endometritis, pneumonia, etc.), there are certain strains that are characterized by particular pathogens.
Among these types of E. coli (pathotypes) are distinguished three major groups:

• Pathogenic types involved in acute diarrheal diseases (ADD)

- Entero-aggregative E. coli (EAggEC) involved in the appearance of a persistent aqueous ADD
with mucus, especially in infants
- Enterohemorrhagic E. coli (EHEC), for example serotype O157:H7, which develops two cyto-
toxins and is involved in the emergence of an aqueous ADD, which can become severe and may
be accompanied by a serious complication, the uremic hemolytic syndrome
- Enteroinvasive E. coli (EIEC) that develops one or more enterotoxins producing dysenteriform
syndrome
- Enteropathogenic E. coli (EPEC) that causes diarrhea in the young child, sometimes of max-
imum severity (eg malignant diarrhea in newborns)
- Enterotoxigenic E. coli (ETEC) which develops two enterotoxins (thermolable and thermostable)
and produces holeriform syndrome
- Diffusely adherent E. coli (DAEC) which produces persistent aqueous diarrhea in children aged
1-5 years

• Pathogen types (groups) involved in urinary tract infections (UTI)

• The type of pathogen involved in meningeal infection in the newborn.

7.1 Laboratory diagnosis of infections caused by E. coli

Regardless of the location of the infection, the microbiological laboratory diagnosis is bacteriological,
direct.
In the following, we will discuss the microbiological laboratory diagnosis of E. coli infections of the
digestive system:

1. The collection and transport of the clinical specimen must be done according to the known rules.
The clinical specimen is usually represented by the diarrhea but we could also collect vomit fluid or
a certain incriminated food. The faeces are examined macroscopically and sent to the laboratory
as mentioned in the previous chapter. Next we will discuss the diagnosis of an infection caused
by EHEC (eg O157:H7) but, we emphasize that in medical practice, starting from c.s. represented
by fecal matter, we can isolate any microorganism involved etiologically in ADD, as the case may
be. The fecal matter may have a haemorrhagic appearance and on macroscopic examination of c.s.
we can observe the presence of strands of epithelial mucosa. The transport will be carried out as
mentioned in chapter no. 28.

2. The microscopic examination of the clinical specimen includes the preparation of wet-mounts, be-
tween the slide and the coverslip; we note the presence of red blood cells and PMN leukocytes.

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 7.2 Uroculture

3. Cultivation on culture media of the clinical specimen is performed in such a way that one can
obtain isolated colonies and a pure culture, respectively, which will be identified (see chapter no.
28). In order to isolate EHEC, in addition to the media mentioned in the previous chapter, it
is recommended to use Mac Conkey media with D-sorbitol (also reffered to as SMAC), because,
unlike most E. coli strains, EHEC does not ferment sorbitol (or ferments it late). In this sense, the
suspected colonies, which we will subculture (without touching the medium) in order to identify
them, will be colorless, with dimensions of 1-3 mm, usually S-type (the sorbitol-positive colonies
will have a pink color).

4. The identification of the pathogenic microorganism involved will be based on several characteristics:
• Morphological and strain characteristics: They are Gram-negative bacilli.
• Culture characteristics: They produce the colony types mentioned above.
• Biochemical characteristics (for EHEC will be examined at the reference center level; processing
of cultures in which EHEC is present requires additional safety measures):
- They Ferment glucose (with gas production), are lactose-positive, do not ferment (or are
late fermenters of) sorbitol, do not produce H2 S.
- Can produce indole, are urease-positive, mobile (but there are also immobile strains).
- Conventional multi-test media (TSI, MIU, MILF) or multi-test galleries API 10 S, API 20
E, API Rapid 20 E or other variants can be used

Table 1: Common biochemical characteristics for E. coli

Test Result
Glucose fermentation Positive
Lactose fermentation Positive
H2 S production Negative
Gas production Positive (+)
Mobility Variable
Indole Positive
Urease Positive
Simmon’s citrate Negative
Sorbitol Negative (or late)

• Other tests that can be studied to identify the bacteria:

- Testing of antigenic characteristics: Somatic Ag can be identified by agglutination or latex
agglutination reactions (monovalent anti-O157 and anti-H7 serums can be used as well);
toxins can be identified by ELISA techniques, latex agglutination, reverse passive latex
agglutination (VET-RPLA), or by seroneutralization of the cytotoxic effect on Vero cells.
- Pathogenicity testing: EHEC produces lethal hemorrhagic enterocolitis in pigs; the cyto-
pathic effect on Vero cells mat also be studied
- Molecular biology assays (PCR, detection of pathogenicity genes starting from c.s. or from
colonies isolated on Mac Conkey with D-sorbitol).

5. Antibiogram is usually recommended only for monitoring the occurrence of the phenomenon of
resistance to antibiotics and chemotherapeutics; it is realized by diffusimetric methods.

7.2 Uroculture
7.2.1 Introduction
The urinary tract is normally uncontaminated, however the distal urethra may have a very diverse
microbial flora, begin contaminated with microorganisms from the genital area or colon. Among these

21
7 Escherichia. Uroculture

microorganisms we can isolate from the distal urethra coagulase-negative staphylococci, E. coli, Klebsiella
spp., Proteus spp., diphtheromorphic bacilli, enterococci, streptococci, saprophytic neisseria, Mycoplasma
spp., Ureplasma spp. yeasts (eg Candida spp.), Clostridium spp., different Gram positive or Gram negative
anaerobes, lactobacilli, non-tuberculous mycobacteria, S. aureus, etc. could be identified. Microbial
colonization of the distal urethra explains why in the vast majority of cases we will perform a quantitative
uroculture.
Although regardless of the care with which we will collect the c.s. (urine) it will be contaminated, by
the quantitative assessment of the number of colony-forming units (CFUs) in the freshly collected urine,
ä midstream, clean catch specimen,̈ we can specify whether or not the patient has a urinary infection.
Several studies have shown that when the presence of 105 bacteria / ml is demonstrated, it indicates a
urinary tract infection (UTI) in about 80% of cases, while a value of 104 bacteria / ml indicates UTI in
less than 30% of cases. Please note that asymptomatic bacteriuria should not be treated.
Due to these results, in medical practice it is considered that we are in the situation of a UTI when the
number of bacteria / ml urine is ≥ 105 / ml.
However, we would like to emphasize that the interpretation of the result does not have to be made
mechanically and the final decision regarding the identification of the bacterium, the susceptibility test-
ing to antibiotics and the drafting of the analysis bulletin respectively should be take into account the
macroscopic aspect of the urine (eg cloudy, purulent), the result of the microscopic examination of the
urinary sediment (eg the presence of PMN), the number of colony-forming units / ml of urine and clinical
elements (eg dysuria, frequent urination, fever). In other words, the collaboration between the clinic and
the laboratory is essential. For example, in the case of suggestive elements, even if the number of bacteria
is 104 / ml, we can make the decision to repeat the uroculture and isolation of the same bacterium may
indicate the presence of UTI. On the other hand, the isolation of more than 104 colony forming units / ml
in the case of staphylococci or yeast is considered significant, as is the presence of Listeria monocytogenes
in the urine of pregnant women or the presence of Salmonella Typhi are significant regardless of the value
of CFU / mL.
To emphasize once again the importance of the urine collection and transport, we should mention that
urine is a very good culture medium for most bacteria that can contaminate the c.s. and a urine sample
that initially has (at the time of sampling) 103 bacteria / mL, will contain 105 bacteria / mL after 2 hours
at room temperature.
UTI classification can be done according to several criteria. Lower UTIs include cystitis and urethritis,
prostatitis and epididymitis (according to some authors). Upper UTIs include acute pyelonephritis, papil-
lary necrosis (which may be an infectious complication of acute pyelonephritis or may occur in the absence
of infection - drug side-effects, ischemia, other diseases), renal abscess, or chronic pyelonephritis (accord-
ing to some authors). Invasion of the urinary tract can occur by ascending, lymphatic or hematogenous
routes.
There are a number of factors that favor the appearance of UTIs, of which we mention: obstructions
that lead to urinary stasis (adenoma or prostate carcinoma, other tumors, urethral or bladder strictures,
urethral compression during pregnancy, foreign bodies, urinary calculi, urinary catheterization, etc.),
vesicoureteral reflux, other functional factors.

7.2.2 Etiological agents

The microorganisms most commonly involved in the production of a urinary tract infection are, in
descending order: E. coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Klebsiella
aerogenes (formerly Enterobacter aerogenes), Pseudomonas aeruginosa, Proteus mirabilis, Proteus vul-
garis, Citrobacter freundii, Citrobacter diversus, Enterococcus faecalis, coagulase-negative staphylococci,
Adenoviruses which can cause hemorrhagic cystitis in children. Less frequently, in descending order, the
etiology of UTIs can be represented by Staphylococcus aureus, Acinetobacter calcoaceticus, beta-hemolytic
streptococci belonging to groups A, B, C or G, Morganella morgannii, Providencia rettgeri, P. stuartii, Ser-
ratia liquefaciens, Serratia marcescens, Candida albicans, Salmonella spp., Corynebacterium urealyticum.
Regarding E. coli, the most common microorganism involved in UTI, there are certain uropathogenic
groups, e.g. O1, O2, O7 etc. Certainly, there are differences in the etiology of UTI by age group, sex and
hospitalization status (inpatients vs outpatients).

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 7.2 Uroculture

7.2.3 Performing the uroculture

1. Collecting and transporting the clinical specimen Urine, blood (eg in acute pyelonephritis) or even
CSF can be sampled in the UTI. Next we will discuss urine collection and transport. Apart from
the general recommendations mentioned above, we must emphasize some particular aspects:
- If the urine cannot be sampled first thing in the morning, it should be sampled 3-4 hours after
the latest urination
- Collection is done after washing the external genitalia and of the perineum (soap and water
may be used); there are specific recommendations for the female and the male and the young
child respectively
- It is preferable for the collection to take place in an adequate space, near the laboratory, and
the medical staff should carefully explain the procedure, and may even assist the collection
- Urine collection at home is contraindicated (there may be collection errors and prolonged
transport time will lead to the multiplication of bacteria, as mentioned above); after collection,
if the urine is not processed immediately (or within 30 minutes after sampling), the c.s. should
be refrigerated and transport should be carried out at +4 ◦ C.
- The indication is to collect a midstream, clean catch urine sample. After washing the external
genitalia and perineum, the first 100 ml of urine is voided in the toilet (this stream of urine will
removing part of the germs from the distal urethra). Only afterwards, 20-50 ml (preferably 50
ml) of urine is collected in the sterile container. Afterwhich the patient may resume urination
in the toilet.
- There are other non-invasive or invasive collection techniques (suprapubian puncture and as-
piration, urethral catheterization, but each have their respective risks).

2. Macroscopic and microscopic examination of the urine

We initially perform the macroscopic examination; the c.s. can be cloudy, opalescent, it can have
a certain odor, etc. For microscopic examination of the urine we homogenize the urine by ẗurning
overẗhe 2-3 times. We place a drop of urine on the slide, cover with a coverslip and examine with
the aid of an oil immersion microscope (or phase contrast microscope). If we identify the presence
of at least one bacterium in each field, in each of the 5 consecutive fields, we can, with a convenient
degree of certainty, consider that we have bacteriuria ≥ 105 bacteria / ml. Alternatively, you can
use Gram stained smears. It is necessary to simultaneously evaluate the presence of pyuria (over 10
leukocytes / mm3 of urine). A number of other tests for the assessment of pyuria and bacteriuria,
such as the Griess test (detection of nitrites in urine), detection of leukocyte esterase, bioluminescent
tests, etc., have been proposed. Pyuria and bacteriuria should be interpreted in the clinical context.
The examination of the urinary sediment allows the visualization of epithelial cells (malpighian,
bladder, urethral etc.), blood cells (leukocytes, erythrocytes), urinary casts (hyaline, leukocyte, ery-
throcyte, epithelial, granular, etc.), urinary crystals, yeasts (possibly budding, with blastoconidia),
Trichomonas vaginalis etc; the examination of the urinary sediment is very useful and should be
performed every time.

3. Urine cultivation Usually, for cultivation we can use 3-4 different media. For reasons of economy we
could seed half a plate with Mac Conkey medium (without crystal violet), half a plate with blood
agar and one plate sector with tellurite agar and eosin-methylene blue (EMB) agar respectively. if
the microscopic examination of the urine is positive. If the microscopic examination is negative, we
streak only half a Petri plate with Mac Conkey medium.
A variant of streaking for Mac Conkey and blood agar media is with the use of a 1 µl calibrated
loop with. We initially perform a streak in the center of the half plate and then spread the c.s. in
parallel streaks, perpendicular to the first streak. Finally without sterilizing the loop, we spread
the c.s in parallel streaks at an angle of 45◦ C to the previous streak. After an incubation period of
18-24 hours at 35-37 ◦ C, we count the number of colonies that have developed on the agar plate,
and multiply by 1000 to estimate the number of bacteria / ml of urine.

23
7 Escherichia. Uroculture

Urine may also be cultivated after dilution (eg 1/100) is frequently used, with the aid of a calibrated
mechanical/manual pipette. We inoculate 0.1 ml of 1/100 diluted urine onto a plate with Mac
Conkey medium, incubate under the known conditions and after the appearance of colonies calculate
the number of bacteria / ml by multiplying the number of CFUs to the inverse of the dilution and
inverse the inoculated volume.
For our example, let’s consider we obtained 100 colonies on the Mac Conkey agar. 100 (colonies
grown on the agar plate) } 100 (dilution vactor) } 10 (volume) = 105 CFUs/mL

4. Interpretation of quantitative uroculture results

- Isolation of a bacterium with a concentration of over 105 / ml of urine confirms a UTI in men
and women with characteristic symptoms; [If the patient is asymptomatic, the UTI certification
is given by isolating the same bacteria in the second urine sample
- Isolation of two different bacteria, each with a concentration of over 105 / ml of urine, requires
repeat collection and cultivation; if the result is similar, a mixed UTI is possible (otherwise it
is a very likely a contamination)
- Isolation of three different bacteria, even if the concentrations for each one exceed 105 / ml of
urine, requires repeat analysis; most likely it is a contamination or that the c.s. Was collected
too long ago and improperly stored (eg kept at room temperature and not refrigerated)
- Isolation of a bacterium with a concentration of 104 / ml of urine leads to the recommendation
to repeat the analysis (there are some exceptions, where the result is considered positive, eg for
yeasts with a concentration of more than 104 / ml of urine, coagulase-negative staphylococci
in concentration of over 5x 104 / ml of urine, etc)
- Lack of bacterial multiplication or development of CFU in 103 / ml urine concentration is a
negative uroculture
- We want to emphasize again that the microbiological result should be analyzed in the clinical
context and correlated with the microscopy result and in the case of certain bacteria (eg
Salmonella Typhi) the result is positive regardless of the concentration of CFUs / mL of urine.

5. In the case of a positive uroculture, the isolated bacterium is identified (on the basis of morpholog-
ical and staining characteristics, culture, biochemical, antigenic and other characteristics) and the
susceptibility to antibiotics and chemotherapeutics is tested, usually by diffusimetric method (see
chapter 14).

24
8 Klebsiella
from "Diagnosticul de laborator în Microbiologie"
by Prof. Dr. Mircea Ioan POPA
translated by Dr. Andrei-Alexandru MUNTEAN

8.1 Introduction
Klebsiella microorganisms are immobile, unsporulated enterobacteria, characterized by intense metabolic
activity on carbohydrates that they hydrolyze with acid and, sometimes, gas production. Glucose is de-
graded by gas production. There are several species for example Klebsiella pneumoniae, K. ozaenae, K.
rhinoscleromatis and K. oxytoca. Klebsiella pneumoniae is the main species of the genus and includes
Gram-negative, capsulated bacilli (the capsule can be 2-3 times larger than the diameter of the bacterium).
Initially considered as an etiologic agent of pneumonia, only 1-3% of pneumonias have been shown to be
produced by Klebsiella pneumoniae. Yet, the pneumonia is severe, necrotic and hemorrhagic, especially in
patients with a altered immune systems. K. pneumoniae is a conditioed pathogen, which can be involved
in a wide array of diseases such as infectious food poisoning, upper respiratory tract infections, urinary
tract infections etc. Klebsiella pneumoniae is increasingly isolated in nosocomial (hospital/healthcare)
infections, see also Chapter 23.

8.2 The microbiological Laboratory diagnosis of Klebsiella pneumoniae

Although not the most representative clinical entity, we will continue to discuss the diagnosis of pneu-
monia produced by Klebsiella pneumoniae. The diagnosis relies on clinical, paraclinical and laboratory
elements; the bacteriological diagnosis begin useful in establishing the etiology and the appropriate an-
tibiotic treatment.
The microbiological laboratory diagnosis is bacteriological, direct, including the following steps.

1. The collection and transport of the clinical sample must be carried out in accordance with a series
of general rules (as close as possible to the onset of the disease, before the patient has received
antibiotics, as quickly and correctly from the point of view of the techniques used, complying with all
the rules of asepsia and antisepsis etc). In infections caused by Klebsiella pneumoniae, fecal matter,
urine, blood, pus, etc. can be collected. Next we will discuss the case where c.s. is represented
by sputum (see also chapter 6). Macroscopic examination of the sputum yields a dark-red, mucoid
appearance, similar to "currant jelly".

2. The microscopic examination of the clinical sample includes the preparation of at least two smears
of the clinical sample collected and transported properly, which will be stained with methylene blue
(MB) and Gram’s stain respectively. The smears are examined with the aid of the immersion optical
microscope and the presence of cells at the alveolar level (eg alveolar macrophages), the presence of
inflammatory cells (eg leukocytes) and the presence of Gram-negative, relatively large, encapsulated
bacilli (surrounded by a large capsule) are noted. The microscopic examination must demonstrate
the quality of the clinical sample and carry out a presumptive identification of the microorganism
involved (see also chapter 52). In methylene blue staining, the capsule appears as a halo around
the bacteria. Using anti-capsular antibodies, rapid identification including directly on the clinical
sample can be achieved by the capsule swelling reaction (see Chapter 12, point 12. 14).

3. Cultivation on culture media of the clinical sample is performed in such a way that isolated colonies
and a pure culture can be obtained, which will be identified (see also chapters 9 and 10). Klebsiella

25
8 Klebsiella

pneumoniae can develop on ordinary culture media in 18-24 hours, at 35-37 ◦ C. It is preferred to use
poorly selective culture media (eg MacConkey). Klebsiella pneumoniae does not develop on highly
selective media and is partially inhibited on moderately selective ones. On solid media Klebsiella
pneumoniae forms large lactose-positive, M-type colonies that are elevated, creamy, often referred
to as a "drop of honey", which give the appearance of "dripping" on the surface of the culture
medium. They tend to be confluent and it is difficult to identify individual colonies. Inoculation
can also be performed in sensitive laboratory animals (eg white mice), see also Chapter 11.

4. The identification of the pathogenic microorganism involved will be based on several characteristics:
• Morphological and staining characteristics: They are Gram-negative bacilli, with large dimen-
sions, with a large, voluminous capsule, arranged either isolated, in pairs or in short chains.
Multiple passages on culture media may lead to the loss of their capsule.
• Culture characteristics: They produce large, type M, lactose positive colonies (2-3 mm at
24 hours, over 4 mm at 48 hours), elevated, creamy, like a "drop of honey", which give the
appearance of "dripping" on the surface of the culture medium, with a tendency towards
confluence.
• Biochemical characteristics:
- Klebsiellas are a lactose positive microorganism - this can be seen, for example, at the
isolation step on the MacConkey media.
- Other biochemical characteristics are studied after the subculture of some colonies on multi-
test media (TSI, MIU), on Simmons citrate media or in commercial multi-test systems (API
20E gallery).
- Klebsiella pneumoniae is glucose positive (with gas production), lactose positive, does not
produce H2 S, non-motile, urease positive, indole negative, able to grow using citrate as the
only carbon source.
- They produces acetoin, a characteristic evidenced through the Voges-Proskauer reaction.

Table 1: Common biochemical characteristics for Klebsiella pneumoniae.

Test Result
Glucose fermentation Positive
Lactose fermentation Positive
H2 S production Negative
Gas production Positive (+++)
Mobility Negative
Indole Negative
Urease Positive
Simmon’s citrate Positive
Acetoin production Positive

• Antigenic characteristics: Serums with known antibodies are used to identify Klebsiella strains
by agglutination, coagglutination or latex agglutination techniques. There are over 80 types of
K antigen in Klebsiella pneumoniae. These reactions can be performed in reference laboratories.
• Pathogenicity characteristics: White mouse inoculation can be used (see Chapter 11).
• Other characteristics / tests used in identification that can be used (in reference centers):
- Bacteriophage sensitivity testing (some of the schemes have been established in the Cantacuzino
Institute)
- Grouping by antibiotic and chemotherapeutic resistance
- Molecular biology characteristics (identifying the plasmid spectrum, identifying the plasmids
involved in virulence, etc.).

26
 8.2 The microbiological Laboratory diagnosis of Klebsiella pneumoniae

5. The antibiogram (used to check the susceptibility to antibiotics and chemotherapeutics in order
to establish the treatment) is mandatory. and is usually done by diffusimetric methods. The
determination of the MIC and the MBC may be necessary (see Chapter 14).

27
9 Proteus
9.1 Introduction
The genus Proteus is composed of pleomorphic germs (hence the name of the genus), highly mo-
bile, Gram-negative, aerobic facultatively anaerobic, which do not ferment lactose, produce urease, pro-
duce phenyl-alanine-deaminase, are unsporulated, non-encapsulated. There are 2 main species, Proteus
mirabilis and Proteus vulgaris.
Usually microorganisms of the genus Proteus are pathogenic involved in diseases that occur outside the
digestive tract (they are part of the gut microbial flora). There are, however, infectious food poisoning with
Proteus spp. While most known for urinary tract infections (UTIs), Proteus spp. may produce other
infections (skin, genital, other locations, with possible evolution towards bacteremia in patients with
deficient immune status). It is oftentimes associated with nosocomial (hospital / healthcare) infections.

9.2 The microbiological laboratory diagnosis of Proteus spp.

In the following, we will discuss the diagnosis of a UTI.
The diagnosis is bacteriological, direct, including the following steps.
1. The collection and transport of the clinical sample must be carried out in accordance with a series
of general rules (as close as possible to the onset of the disease, before the patient has received
antibiotics, as quickly and correctly from the point of view of the techniques used, complying with
all the rules of asepsis and antisepsis etc). In infections caused by Proteus spp., urine, pus, various
purulent exudates, fecal matter, vomit fluid, food, blood, etc. can be collected. Next we will discuss
the case where the c.s. is urine (see also chapter 29). Macroscopically, the urine may be turbid,
purulent. Due to the production of indole, it sometimes causes purple urine bag syndrome. Urine
transport should be completed quickly, within a maximum of 30 minutes, and if this recommendation
cannot be met, the c.s. must be stored and transported at + 4 ◦ C.

2. The microscopic examination of the clinical sample includes the preparation of wet-mount (an
examination between the slide and coverslip) from the sediment resulting after the centrifugation of
the urine. Microscopic examination of the urine is very important because it is easy to perform, is
cheap and allows observation of elements that guide the diagnosis. Microscopic examination of urine
could lead to significant savings (time, reagents and materials, culture media); see also chapter 29.
Proteus spp. presents a large number of peritrich cilia that confer it a characteristic mobility (there
are, however, also strains that do not have cilia). Gram’s stain from homogenized urine can also
be performed. Proteus spp. have a marked polymorphism on the smear and can take the shape of
bacilli, coccobacilli, filamentous forms up to 30 µm. They are uncapsulated and unsporulated. The
presence of at least 1 leukocyte / microscopic field on examination with the objective of immersion
suggests pyuria, which, in a suggestive clinical context, helps define UTI. If from the homogenized
urine we draw with the calibrated loop of 0.01 ml (10 µL) urine and in the result smear we identify
at least 1-2 bacteria / immersion field, this result suggests the presence of 105 bacteria (colony
forming units) / ml and urinary tract infection respectively.

3. Cultivation on culture media of the clinical sample should be performed in such a way that isolated
colonies and a pure culture can be obtained, which will be identified (see also chapters 9 and
10). They grow on simple media including peptone water media with a characteristic "rotting"
smell. It withstands large variations in temperature and pH. It is necessary to perform quantitative
uroculture and to demonstrate the presence of over 105 bacteria / ml of urine. Isolated colonies
cannot be obtained on the usual simple media, on agar-blood, on AABTL, etc., as the invasion

28
 9.2 The microbiological laboratory diagnosis of Proteus spp.

phenomenon leads to confluent growth (see also chapter 11). This aspect suggests the presence of
Proteus spp. in the clinical sample. The "phenomenon" can be inhibited if the c.s. is inoculation is
performed on selective-differential media (eg MacConkey, ADCL, CLED). In these media Proteus
spp. produces negative lactose colonies, type S.

4. The identification of the pathogenic microorganism involved will be based on several characteristics:
• Morphological and staining characteristics: They are Gram-negative bacilli, with an accen-
tuated polymorphism (bacilli, coccobacilli, filamentous forms up to 30 µm in lenght), non-
encapsulated and unsporulated.
• Culture characteristics:
- The invasion phenomenon: represents a preliminary identification characteristic for Proteus
spp., a highly mobile bacterium; if we sow a strain that belongs to the genus on the
periphery of a Petri dish with a simple medium (2% agar), the bacteria will invade the
whole surface of the plate, developing "in concentric waves".
- In some selective media, the invasion is inhibited and isolated colonies can be obtained
(through the addition of bile acids or salts, sodium thiosulphate, etc. to the culture
medium); these colonies are lactose negative and S-type. The CLED medium is oftentimes
used for this purpose.
- The phenomenon of the "demarcation line" represents a useful character in epidemiological
studies, if the strains involved belong to the genus Proteus; if on a plate with solid medium
we cultivate 2 different stems, in 2 opposite points, the stems will develop invading to a
point near the meeting line, where a "demarcation line" will be created between the 2
stems (distance of 1 -2 millimeters); if the strains are not different, "growth in waves" will
take place without "demarcation" with the development of a "continuous" culture
- The "climbing" phenomenon represents a variant of isolation in pure culture of a Proteus
strain; cultivation of a clinical sample in the condensate fluid of a sloping agar tube incu-
bated upright will lead (if in the c.s. there is a strain of Proteus spp.) to obtain within
24 hours a pure culture of Proteus, which will develop "in concentric waves" to the upper
part of the culture media.
• Biochemical characteristics:
- Proteus spp. includes lactose-negative microorganisms.
- Other biochemical characteristics are studied after the repetition of some colonies on multi
test media (TSI, MIU), on Simmons medium or in commercial multi-test systems; Proteus
spp. is glucose positive (sometimes with gas production), lactose negative, produces H2 S,
mobile, urease positive, can grow using citrate as the sole carbon source.
- Proteus spp. produces phenyl-alanine-desaminase.
- Proteus mirabilis is indole negative and ornithine decarboxylase positive while Proteus
vulgaris is indole positive and ornithine decarboxylase negative.
• Antigenic characteristics: Serums with known antibodies (anti-O and anti-H) are used to
identify Proteus strains by agglutination techniques (at the reference laboratories).

5. Other characteristics / tests used in identification (in reference centers):

- Testing bacteriophage susceptibility
- Grouping on the basis of antibiotic and chemotherapeutic resistance.

6. The antibiogram (checking the sensitivity to antibiotics and chemotherapeutics in order to establish
the treatment) is mandatory and is usually done by diffusimetric methods. The determination of
the MIC and the MBC may be necessary (see Chapter 14).

29
9 Proteus

Table 1: Common biochemical characteristics for Proteus spp.

Test Result
Glucose fermentation Positive
Lactose fermentation Negative
H2 S production Positive
Gas production Positive (+)
Mobility Positive (+++)
Indole Proteus mirabilis. Negative
Proteus vulgaris. Positive
Urease Positive
Simmon’s citrate Positive
Ornithine decarboxylase Proteus mirabilis. Positive
Proteus vulgaris. Negative
Phenyl-alanine-desaminase Positive

30