KLEBSIELLA GENUS

These bacteria are normal members of the microbiota in humans and other animals
and in certain conditions can become pathogens. Klebsiella species are routinely
found in the human nose, mouth, and gastrointestinal tract as normal flora; however,
they can also behave as opportunistic human pathogen These are widely spread in
the environment: nts and also live in the intestines of humans and animals and in the
respiratory tract of the humans and animals. Klebsiella organisms can lead to a wide
range of disease states, notably pneumonia, urinary tract infections, septicemia,
meningitis, diarrhea, and soft tissue infections
Morphological characters: - these bacteria are Gram-negative bacteria, short, in
pairs or in short chains - are nonmotile (means that they don't have flagella) - don't
have spores - have a prominent polysaccharide-based capsule to protect them
Culture characters: - are aerobic bacteria and facultative anaerobes - can grow on
ordinary lab culture medium and do not have special growth requirements on solid
culture medium generate type M (mucoid) colonies (round, bright big sized colonies,
white-grey colour) Biochemical characters: - catalase positive oxidase negative
ferments sugars (glucose) to produce lactic acid and gas. - ferments lactose to
produce lactic acid - do not gererate hydrogen sulphide - can use citrate as as their
sole carbon sourse Antibiotie sensitivity test these bacteria respond to
Aminoglycosides, Cephalosporins.

PROTEUS GENUS These bacteria are widely spread in the environment as

saprophytes, being found They are opportunistic pathogens, commonly responsible
for urinary and septic in decomposing animal matter, in soil, in the water, on plants,
foods infections, often nosocomial. Three species - P. vulgaris, P. mirabilis, and P.
penneri-are opportunistic P. vulgaris occurs naturally in the intestines of humans and
a wide variety of P. mirabilis causes wound and urinary tract infections and once
attached to the human pathogens. animals, and in manure, soil, and polluted waters.
urinary tract, infects the kidney more commonly than E. coli. Morphological
characters: - these bacteria are Gram-negative bacteria have many flagella used to
move (they are highly motile) - don't have spores - do not have capsula to protect
them Culture characters: are aerobic bacteria and facultative anaerobes - can grow
on ordinary lab culture medium and do not have special growth requirements - on
solid culture medium generate type S (smooth) colonies (round colonies, translucent,
soft white colour and black colour in the center) Biochemical characters: - catalase
positive - oxidase negative - ferments sugars (glucose) to produce lactic acid and
gas. - reduce nitrate to nitrite, - don't ferments lactose - don't gererate hydrogen
sulphide - can use citrate as as their sole carbon sourse - specific tests include
positive urease Antibiotic sensitivity test -these bacteria are sensitive to Ampicillin,
Aminoglycosides, Cephalosporins

YERSINIA GENUS These bacteria are widely spread in the environment and a live
in the intestines Some members of Yersinia are pathogenic in humans; in particular,
Yersinia Also important species are Yersinia enterocolitica and Yersinia Yersinia
may be associated with Crohn's disease, an inflammatory autoimmune Also Yersinia
is implicated as one of the causes of reactive arthritis worldwide of the animals
(mammalian and birds) pestis is the causative agent of the Pneumonic plague (a
severe lung infection). pseudotuberculosis condition of the gut. Morphological
characters: - these bacteria are Gram-negative bacteria - have flagella used to move
(they are motile) at 28-30 degrees Celsius - do not have flagella (they are non
motile) at 37 degrees Celsius - don't have spores -do not have capsula to protect
thenm Culture characters: - are aerobic bacteria and facultative anaerobes - can
grow on ordinary lab culture medium and do not have special growth requirements -
on solid culture medium generate type S (smooth) colonies (round, medium sized
colonies, transparent, soft white-grey colour) Biochemical characters: - catalase
positive - oxidase negative - ferments sugars (glucose) to produce lactic acid, but
without gas. - don't ferments lactose - don't gererate hydrogen sulphide - do not use
citrate as as their sole carbon sourse - specific tests include positive urease
Antibiotic sensitivity test -these bacteria are sensitive to Aminoglycosides,
Cephalosporins

CAMPYLOBACTER GENUS These bacteria are widely spread inside the intestines
of animals and birds. Humans can contract the disease from eating food
contaminated with Campylobacter species. Another source of infection is contact
with infected animals, which often carry Campylobacter asymptomatically.
Campylobacter jejuni is the representative species of this genus and is recognized
as one of the main causes of bacterial foodborne disease Identification methods for
Campylobacter jejuni: 1. The Direct method By making a fixed and colored
preparation and using optical microscopy Campylobacter species generally appear
as curved or comma-shaped rods, Gram negative bacilli, able to move via unipolar
or bipolar flagella, without spores and capsula. 2. The indirect method through
cultivation and isolation They generally survive in environments with low oxygen and
can grow only on culture media with nutrients. On solid culture media these bacteria
cause two types of colonies: a) small, round, transparent, without colour, colonies,
having regular contour and generating hemolysis on blood-agar culture medium. b)
flat big, round colonies having regular contour and generate hemolysis 3.
Biochemical tests - survive in environments with low oxygen - oxidase positive -
catalase pozitive can't ferment glucose 3. Antibiotic sensitivity testing these bacteria
respond to different antibiotics only after the antibiogram has been performed

HELYCOBACTER GENUS First Helycobacter was included as a species in

Campylobacter genus but after, to scientific studies, Helycobacter formed a new
genus The representative species of this genus is called Helycobacter pylori. This
species usually is found in the stomach . Up to 85 % of people infected with ence
symptoms or complications. Acute infection may appear as an acute gastritis with
abdominal pain (stomach ache) or nausea. Where this develops into chronic
gastritis, the symptoms, if present, are often those of nonulcer dyspepsia: stomach
pains, nausea, bloating, belching, and sometimes vomiting or black stool. To avoid
the acidic environment of the interior of the stomach (lumen), H. pylori uses its
flagella to burrow into the mucus lining of the stomach to reach the epithelial cells
underneath, where it is less acidic. H. pylori is able to sense the pH gradient in the
mucus and move towards the less acidic region (chemotaxis). This also keeps the
bacteria from being swept away into the lumen with the bacteria's mucus
environment, In addition to using chemotaxis to avoid areas of low pH, H. pylori also
neutralizes the acid in its environment by producing large amounts of urease, which
breaks down the urea present in the stomach to carbon dioxide and ammonia. These
react with the strong acids in the environment to produce a neutralized area around
H. pylori. Identification methods for Helycobacter pylori 1. The Direct method By
making a fixed and colored preparation and using optical microscopy we can see
helix-shaped Gram-negative bacili about 3 um long with a diameter of about 0.5um
with high motility owing to four to six flagella, without spores and without capsula. 2.
The indirect method through cultivation and isolation They generally survive in
environments with low oxygen and can grow only on culture media with nutrients. On
solid culture media like BHI (Brain-heart infusion), Hinton or chocolate-agar these
bacteria cause small, round, transparent, colonies only after 3 to seven days after
cultivation 3. Biochemical tests - survive in environments with low oxygen - oxidase
positive catalase pozitive produce large amounts of urease important for
Helycobacter pylori that allow this bacteria to survive long periods of time inside the
stomach (months, years and in some cases all life) 4. Antibiotic sensitivity testing As
treatment she standard first-line therapy is a one-week "triple therapy consisting of
proton-pump inhibitors such as omeprazole and the antibiotics clarithromycin and
amoxicillin. Variations of the triple therapy have been developed over the years, such
as using a different proton pump inhibitor, as with pantoprazole or rabeprazole, or
replacing amoxicillin with metronidazole for people who are allergic to penicillin. In
areas with higher rates of clarithromycin resistance, other options are recommended
The failure of the initial treatment requires additional rounds of antibiotic therapy or
alternative strategies such as quadruple therapy, which adds to "triple therapy" a
bismuth colloid, such as bismuth subsalicylate

LABORATORY DIAGNOSIS IN INFECTIONS CAUSED BY TREPONEMA

PALLIDUM Treponema pallidum is a bacterium which causes syphilis, disease
acquired by close sexual contact To identify these kind of bacteria we can take
samples from patients such as: blood for serological tests and - serousness from
exudative lesions Therefore, demonstration of treponemes in lesion material,
serologic reactions beside clinical manifestations are used for diagnosis. In many
cases, clinical manifestations are highly characteristic. In patients with acquired
venereal syphilis, there is an initial genital tract lesion (primary stage) followed by
disseminated lesions (secondary stage) and, in approximately one-third of untreated
individuals, cardiovascular and neurologic problems (tertiary stage). Identification
methods for Treponema pallidum: 1. The direct method Treponemes is a motile
spirochaete, helically coiled, corkscrew-shaped cells, 6 to 15 um long and 0.1 to 0.2
um wide about its longitudinal axis and bending, flexing and snapping about its full
length. (Levaditi method) microscopy, can be visualized by using dark-field
microscopy Treponema pallidum exhibits characteristic motility that consists of rapid
rotation Treponemes in tissues can be visualized by silver impregnation methods
Live treponemes, which are too slender to be seen by conventional light Other
method to identify this bacteria is by immunofluorescence 2. The indirect method
through cultivation and isolation Traditionally this organism has been considered a
strict anaerobe, but it is now known to be microaerophilic. Treponema pallidum have
not yet been cultured in vitro Definitive diagnosis of syphilis is complicated by the
inability to cultivate T pallidum in vitro in laboratory. This bacterium can be kept in the
laboratory only by inoculation in rabbits or by cultivation on embryonic eggs and
tissue culture
Serological tests Serologic tests are a mainstay of syphilis diagnosis. They are the
only means of identifying infected individuals. Two terms relevant to syphilis
serodiagnostic testing are sensitivity and specificity The perfect test, not yet
developed, would detect 100 percent of the treponemal infections and would be
nonreactive in all other diseases. Sensitivity refers to the ability to detect the tested
variable, in this case syphilis. A false-negative occurs when serum from a syphilitic
patient fails to react. when the variable is not present (ie, to Specificity refers to the
ability to recognize exclude syphilis in nonsyphilitic patients). A false-positive occurs
when serum from a nonsyphilitic patient reacts positively In general, treponemal
tests are more sensitive and more specific than the nontreponemal tests. More than
200 serologic tests have been developed over the years and these tests are divided
into two general categories: A. "nontreponemal" tests, which measure antibodies
directed against lipid antigens, principally cardiolipin, thought to be derived from host
tissues Examples for "nontreponemal" tests are a. the Venereal Disease Research
Laboratory (VDRL) and Rapid Plasma Reagin (RPR) tests which are quantitative
tests b. the Bordet-Wassermann test and Kolmer test (also quantitative tests). A
number of clinical conditions may cause false-positive nontreponemal tests. These
include leprosy, tuberculosis, malaria, infectious mononucleosis, collagen disorders,
systemic lupus erythematosus, rheumatoid arthritis, pregnancy, and drug and
alcohol abuse. The results of nontreponemal tests usually shows the extent of
infection; titers tend to be highest during secondary syphilis and subside during
subclinical infection (latency) or following antibiotic therapy B. "treponemal," which
detect antibodies directed against protein constituents of T pallidum subsp pallidum.;
examples are: - The Fluorescent T pallidum Antibody-Absorption (FTA-ABS)
Haemagglutination for T pallidum HATp) test. - TPI (Treponema pallidum
immobilization) The treponemal tests often remain reactive for life.
Both treponemal and nontreponemal serological tests have been highly standardized
by the Centers for Disease Control and Prevention (CDC). The sensitivity of the
nontreponemal and treponemal tests varies with the stage of the disease (There are
three stages for syphilis: primary, secondary and tertiary or late stage. 4. Antibiotic
sensitivity testing Penicillin treatment eradicates all stages Penicillin remains the
drug of choice for treating syphilis

HAEMOPHILLUS INFLUENZAE Haemophillus influenzae are opportunistic

pathogens that usually live in their host without causing disease, but cause problems
only when other factors such as viral infection create an opportunity for them to
penetrate human organism. Haemophillus influenzae first described in 1892 during
an influenza pandemic and first considered that produce influenza. After laboratory
res proven that influenza is produced by influenza virus and not by haemophilus
influenzae, this bacteria producing just flu-like symptoms. earch, it has been H
influenzae causes: - bacteremia, - pneumonia, - bronchopneumonia, -
otorhinolaryngology infections such as epiglottitis, - eyes infections, ear infections, -
meningitis., - osteomyelitis, - arthritis and - neonatal infection. To identify these
bacteriase can take samples from humans such as: - The nasal sample - The naso-
pharynx sample The pharynx sample - The sputum samples Different secretion
(eyes secretions, ear secretions) Blood Cerebro-spinal fluid The transport must be
carried out within a maximum of 2 hours. The samples should be stored less than 24
hours at room temperature.
Identification methods for Haemophillus influenzae are: 1. The Direct methood By
making a fixed and colored preparation and using optical microscopy we can see
Gram-negative bacilli, sometimes coccobacilli without spores, non-motile, with or
without capsula (some strains have capsula, some not) 2. The indirect method
through cultivation and isolation These bacteria can not grow on ordinary lab culture
medium and have special a. Factor X (Protoporfirin) growth requirements such as
two important growth factors: is a thermostabile factor that have an important role in
some enzyme synthesis - b. Factor V (nicotinamide adenine dinucleotide) - is a
thermolabile factor that have an important role in oxido reduction processes These
bacteria can grow on different culture medium such as: Blood agar (Hinfluenzae will
grow in the hemolytic zone of Staphylococcus aureus on blood agar plates; the
hemolysis of cells by S. aureus releases factor V which is needed for its growth. H.
influenzae will not grow outside the hemolytic zone of S. aureus due to the lack of
nutrients such as factor V in these areas - Chocolate agar - Miller-Hinton medium
with antibiotics (Vancomycin, Polymyxin and - BHI medium (Brain-Heart-Infusion
medium) On these culture media the colonies of H. influenzae appear as convex,
smooth, 3. Biochemical tests - oxidase positive Bacitracin) pale, grey and
transparent ones. catalase positive 4. Antibiotic sensitivity testing These bacteria
respond to the following antibiotics: Ampicillin Sulbactam, Cephalosporins of the
second and third generation Fluoroquinolones.

PSEUDOMONAS GENUS The representative species for this genus is

Pseudomonas aeruginosa (also called P. aeruginosa is found in hospital
environments, beeing the second-most Pseudomonas aeruginosa is an opportunistic
human pathogen and generate Pyocianic). common infection in hospitalized patients
(generating nosocomial infections) various infections, especially at children and
patients with low immunity, cancer, burned, diabetic, politraumatized persons. Can
generate various infections like: - wound infections - eyes infections - ear infections
infections of the respiratory tract - endocarditis vaginal infections - infections of the
digestive tract food poisoning To identify these kind of bacteria we can take samples
from humans such as: - pus blood cerebro-spinal fluid - sputum - eyes secretions ear
secretions - vaginal secretions faeces The transport of the samples must be carried
out within a maximum of 2 hours. The samples should be stored less than 24 hours
at room temperature
Identification methods for Pseudomonas aeruginosa: 1. The Direct method By
making a fixed and colored preparation and using optical microscopy we can see
straight thin, Gram-negative bacilli, sometimes having long filamentary forms, without
spores, having flagellum or flagella (one or more) providing motility, without capsula
2. The indirect method through cultivation and isolation These bacteria can grow on
ordinary lab culture medium generating colonies type S, R, or M with a characteristic
smell of lime flowers, beta hemolytic (on blood agar) culture medium On liquid
culture medium cause uniform disorder of the liquid medium and cause a film on the
surface of the liquid medium and beneath the culture medium have a green-blue
colour - due to some pigments such as pyoverdine (a fluorescent yellow- green
siderophore) or additional types of siderophore, such as pyocyanin (a blue colour
siderophore) and thioquinolobactin. 3. Biochemical tests strictly aerobic specie:s
oxidase positive - catalase positive glucose is oxidised in oxidation/fermentation test
- citrate positive - indole negative 3. Antibiotic sensitivity testing Most Pseudomonas
species. are naturally resistant to Penicillin and the majority of related Beta-lactam
antibiotics, but a number are sensitive to Piperacillin, Imipenem, Meropenem,
Ticarcillin, or Ciprofloxacin. Aminoglycosides such as Tobramycin, Gentamicin,
Amikacin are other choices for therapy.

LABORATORY DIAGNOSIS IN INFECTIONS CAUSED BY MYCOBACTERIUM

TUBERCULOSIS DIAGNOSIS OF PULMONARY TUBERCULOSIS Pulmonary
Tuberculosis is the most frequent clinical form of Tuberculosis and is caused by
Mycobacterium tuberculosis (called also Koch bacillus) and rarely by Bovis bacillus
The suspicion of clinical and radiological tuberculosis is confirmed with certainty only
by bacteriological examination We can take sputum from humans and in people who
can not be expectorates is examined the gastric lavage fluid Stimulation of bronchial
secretion is achieved with warm salt aerosols Identification methods for
Mycobacterium tuberculosis 1. The direct method identifies thin bacilli of variable
lengths with rounded ends and granular appearance - they are isolated, in groups, or
in piles By making a fixed and colored preparation and using Ziehl-Neelsen stain
they appear in bright red color on a dark blue background Mycobacterium
tuberculosis are also called alcohol-acid resistant bacilli (BAAR) If using fixed and
colored preparation we see BAAR we can diagnose pulmonary tuberculosis even if
these BAAR are few ones but we can't we can not determine the species (M.
tuberculosis or M. bovis) If using fixed and colored preparation we don't see BAAR
we can not exclude the diagnosis of pulmonary tuberculosis because under a certain
amount of sputum bacilli (40.000 bacilli /ml), the chance of highlighting on the fixed
and colored preparation is almost O.
2. The indirect method through cultivation and isolation a. M. tuberculosis bacilli
develop very hard (16-17 weeks - the limits are 2-4 weeks) and only on a specially
culture medium called Lowenstein (which is a solid culture medium placed in a test
tube). The colonies on this culture medium are type R (rough) colonies, yellow-white
colour, with cauliflower appearance and easily detached from the surface of the
culture medium b. M. bovis develop even more difficult then M. tuberculosis, in 4-8
weeks and the colonies aretype S, yellow colour and hardly removable from the
culture medium 3. Biochemical tests -the most important biochemical test used to
identify M. tuberculosis from others Mycobacterium species is Niacina test (which is
positive for M. tuberculosis and negative for other species of Mycobacterium). Other
biochemical tests are: producing thermoresistant catalase for other species) to TCH
and M. bovis is sensitive) - reducing nitrates to nitrites (which is positive for M.
tuberculosis and negative - sensitivity to thiophene carboxylic acid hydrazide (M.
tuberculosis is resistent 4. Antibiotic sensitivity testing These bacteria are resistent to
antibiotics therefore for the treatment a combination of antimicrobial substances is
used for a period of at least 6 months. This combination (four out of five) include the
following: Hydrazide (also called Izoniazide) - Streptomycin Pyrazinamide Rifampicin
Ethambutol

LABORATORY DIAGNOSIS IN INFECTIONS CAUSED BY CANDIDA ALBICANS

Candida albicans is an opportunistic pathogenic yeast that is a common member of
the human gut flora. It does not proliferate outside the human body. It is usually a
commensal organism, but it can become pathogenic in immunocompromised
individuals under a variety of conditions (such as long-term antibiotic treatment or
immune system degradation) candidiasis, which results from an overgrowth of the
fungus such as: It is one of the few species of the genus Candida that causes the
human infection We can take samples from patients depending on the location of the
infection -in cutaneous localization we harvest hair, nails - in digestive localisation we
harvest feces in bronchopulmonary localisation we harvest sputum - in cerebral
localisation we harvest cerebro-spinal fluid - in renal localisation we harvest urine - in
septicemia we harvest blood Identification methods for Candida albicans: 1. The
direct method - identifies three types of cells characteristics for Candida species:
Blastospores-big spherical cells (3-5 micrometers)Gram positive Pseudomicelii- own
cells placed head to toe in the form of chains Chlamydospores - spherical cells
bigger than blastospores cells (with double contour) and characteristics for Candida
albicans 2. The indirect method through cultivation and isolation - the colonies of
Candida albicans can grow very well on Sabouraud culture On these types of culture
media the colonies of Candida albicans are type S Candida albicans is identified on
cromogenic culture media based on the color of 3. The zimograma studies the
fermentation of sugars 4. Auxonograma studies sugar assimilation media or
cromogenic culture media. (smooth) colonies, white-yellow colour, big colonies,
creamy ones, matte surface and with the smell of yeast the colonies
5. Fast filament test is used to identify Candida albicans when filamentous
prolongations occur very rapidly in about 3 hours if the culture to be investigated
resembles in rabbit serum. 6. Chlamydospores test used also to identify Candida
albicans species. C albicans generates chlamydospores if the culture to be
investigated is cultivated on special culture media such as Tween media. 7.
Antifungal sensitivity test These yeasts are sensitive to: Clotrimazole Fluconazole
Ketoconazole Miconazole Nystatin Amphotericin B