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SCRIPTA MEDICA (BRNO) – 75 (1):
29–60, February 2002
CONFERENCE ABSTRACT
TOMÁ·EK’S DAYS 2001 10TH CONFERENCE OF YOUNG
MICROBIOLOGISTS
Brno 6. – 8. 6. 2001
Conference held by:
Microbiological Institute of
Medical Faculty of Masaryk University in Brno
Czechoslovak Society for
Microbiology Society for Microbiology and
Epidemiology of Czech Medical
Society of Jan Evangelista Purkynű
Anna’s Faculty Hospital in Brno
ABSTRACT
SECTION 1: DIAGNOSTICS OF
MICROBES FOR MEDICAL AND VETERINARY
MICROBIOLOGY
M. Stfiítecká., J. Svoboda,
L. Mejzlíková (Institute for Microbiology of Medical
Faculty of
Masaryk University and St. Anna’s Faculty
Hospital Brno, Czech Republic): Difficulty of
identification of -haemolytic
Streptococcus.
Haemolytic streptococci are the
most common human pathogens out of genus Streptococcus.
They can initiate acute disease
of respiratory tract, skin or urogenital tract.
When determining group of
streptococcus, serological methods should be primarily used. Not
only serological methods are
used in routine practice for rapid identification of A-group, even
though theirs reliabilities
are lower. Bacitracin test make use of high sensibility of A-group
Streptococcus to bacitracin. We
have proved that some species of C and G-group are bacitracin
sensitive and on the other hand
found A-group Streptococcus bacitracin resistant.
Using latex agglutination
(Pastorex strep, Biorad) we identified in total 262 strains of -
haemolytic streptococci. Out of
this amount 26 % of A-group, 18 % of B-group, 39 % of C-group,
only 1 % of F-group
(surprisingly) and 16 % of G-group.
A-group streptococci showed 9–23 mm zone around
bacitracin (0,004 j, Itest plus s. r. o, Hradec
Králové), about 4 % were
bacitracin resistant. Only 4 % of B-group streptococci showed zone
around bacitracin at all. However
31 % of C-group streptococci shoved 7–20 mm zone around the
disc. No zones were observed with
F and G group streptococci.
It is clear, that particularly C-group streptococci might be
confused with A-group streptococci.
M. Sládeková (HPL Microbiological
Laboratory, Bratislava, Slovakia): The proof of
toxin
A producing by the strains Clostridium difficile.
Clostridium difficile (next
only C. d.) is a recognised major cause of nosocomial antibiotic-
associated diarrhoea and
pseudomembrannous colitis. Importance of disease and next therapy
charging the patient led to focus
this problem. Monitoring of the complications and proof of the
production of toxin A by strain
colonizating gastrointestinal tract of the patients has to find out the
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extent and prevalence of
certain strains C. d. circulating in European
hospitals. The analysis of
molecular and phenotypic methods, and the analysis of the
antimicrobial susceptibility of the strains
isolated from patients provide
surveillance and recommendation of the antibiotic therapy of disease
caused by toxigenic strains of
C. d. across Europe.
The authors focus on the
qualitative detection of the strains C. d. producing
toxin A in faecal
samples by dot-blot method. The aim of the work is find out
the extent of C. d. toxin A strains in
our region of Bratislava before
joining to ESGCD European Study Group on C. d. We
present the
analysis of the occurrence of the strains C. d. producing
toxin A according to the age category and
sex, based on the type of
diagnosis and hospital department with respect to the antibiotic therapy.
To assemble new knowledge of the
occurrence toxigenic strains of C. d. and detailed
analysis of
these data could help to create guidelines on prevention, diagnosis, treatment
and surveillance of
C. d. infections.
Ş. Ga‰paroviăová1,
Ş. Jurgo‰2, D.Dani‰3,
D. Huăková1, Ş. Vozárová1 (1HPL Ltd.
Microbiological
Laboratory, Bratislava, Slovakia;2Private
Gastroenterological Laboratory, Bratislava, Slovakia;
3Institute
of Pathology of Teaching Derer’s Hospital, Bratislava, Slovakia):
Asset of westernblot
method in diagnostics and
strategy of antimicrobial therapy of Helicobacter pylori.
The discussion about
etiopathogenetical importance of Helicobacter pylori (HP)
virulence
factors and their influence on determination of eradicational therapy start is
still actual. The main
dispute is over the toxic proteins CagA (cytotoxin
associated with gene A) and VacA (vacuolating
cytotoxin), pathogenetical or
protective respectively significance. These are isolated from patients
with active chronic gastritis
and duodenal ulcers. HP virulence factors prevalence in reflux
gastroesophagitis patients is
identical to duodenal ulcers patients, but different in functional
dyspepsic patients.
Epidemiological studies show variable incidence frequency of CagA considering
the gender, age and geographical
distribution.
The incidence of discussed proteins was observed by indirect
determination of antibodies of IgG
class by western blot method (Euroimmun GmbH,
Germany). The samples of 30 patients were examined
by western blot method. 10
patients were HP type 1 positive and 20 patients were HP type 2 positive.
The presence of CagA was evident
in 17 samples. CagA together with VacA were present in 10 samples.
No differences in subjective
conditions and clinical status of HP type 1 and HP type 2 patients were
observed. Western blot method
evidence was compared to direct evidence of HP (urease test and
microscopically confirmation by
staining with Giemsa) in samples extracted from stomach mucous.
There was 97 % (29 of 30 samples
examined) identity of the results acquired by mentioned methods. HP
was not proved in one biopsied
sample, but nonrepresentative biopsy can not be excluded in this case.
Based on previous experiences,
western blot method usage difficulty level is comparable to the
other method ELISA,
considering the sample preparation. But it has higher sensitivity and
specificity. Due to use of
western blot method the presence overview of toxic strains of HP in
examined samples could be
determined. It offers to gastroenterologist a possibility of more exact
decision about the antimicrobial
therapy in dyspepsia problems patients.
M. Kobidová2,
K. Schwarzová1, I. âiĎnár1 (1Institute
of Preventive and Clinical Medicine,
Bratislava, Slovakia;
2Faculty of Science, Bratislava,
Slovakia): Borrelia burgdorferi
s. l. isolates
from the vector and host comparison by immunochemical
methods.
Borrelia garinii K48 tick isolate and the CSF isolate from a
patient with neuritis Borrelia afzelii
LeMo were used for comparison
of the phenotypical properties. Double diffusion methods in
agaroses by Ouchterlony and
immunoelectrophoresis with modifications were applied in the study.
Both isolates showed a different
growth rate. The patient’s isolate grew slowly and the growth
curve phases have had a 2 days
delay shift. With the increasing number of in vitro passages this
differences were reduced.
By the precipitation of antigens
with hyper immune rabbit antisera a partial homology of the
isolates was confirmed, however
simultaneously there was a relative high degree of heterogeneity
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of Osp proteins and endotoxine
like component. Further, the immunochemical analysis showed the
presence of common antigens of
LeMo isolate with B. garinii K48, but also
with other B.
burgdorferi s.
l. genotypes.
K. Tomanová, J. Smola (Institute of
Microbiology and Immunology, University of Veterinary
Medicine and Pharmacy, Brno, Czech
Republic): Lawsonia intracellularis:
Its Determination in
Horses In Czech Republic.
Lawsonia intracellularis,
previously known as ileal intracellular symbiont, is an obligatory
intracellular micro organism,
which was observed and described mainly in pigs as a contributor of
swine proliferative enteritis
complex (ileitis). The causative agent is detected by PCR method from
intestinal epithelium or in the
faeces of infected pigs. A specific and sensitive serological test was
developed for fast diagnosis of
swine ileitis, which enables to detect antibodies in the chronic forms
of this disease and
simultaneously in unapparent carriers.
For a long time, the occurrence
of L. intracellularis in samples of section materials
taken from
horses was uncertain. While four cases were recorded in the USA and one positive
PCR result was
registered in our laboratory from 7 analyzed intestinal
samples of horses. Last year for the first time,
the occurrence of this
disease with its clinical manifestations in horses was described only
in Canada.
Clinical materials, which were collected from horses (English pureblood
breeds) with
anamnesis of chronic diarrhoea in relatedness of L.
intracellularis, were investigated in our
laboratory without depending up
on the previous results. We used PCR for direct identification of
bacteria from the faeces and
rectal swabs. And we used commercial kit of immunofluorescence for
indirect detection of antibodies.
15.7 % of
positive PCR results of L. intracellularis were
registered from 51 analyzed samples
taken from horses. 76.6 % of specific
antibodies were detected from 64 analyzed blood sera.
Identification of
L. intracellularis from intestine and faeces of
horses (English pureblood and half-
pureblood breeds) has priority not only in Czech
Republic but also in Europe.
Achieved results by detecting the causative agent suggest
that beside domestic pigs, horses may
be one of those very significant hosts of
these bacteria. New opportunities of microbiological
diagnostics of L.
intracellularis create the possibilities of further investigations
of etiologically
significant micro organisms in the field of gastrointestinal
diseases of horses.
P. Carasová, V. Celer (Department of
Microbiology and Immunology, University of Veterinary
Medicine and Pharmacy, Brno,
Czech Republic): Porcine circovirus type 2 –
possibilities of
laboratory diagnostics.
Porcine circovirus type 2
(PCV-2) is the cause of “postweaning multisystemic wasting
syndrome” and “porcine dermatitis
and nephropathy syndrome” in pigs. Laboratory diagnostics of
this virus is performed almost
exclusivelly by PCR and the virus itself has not been diagnosed in
the Czech Republic so far. We
have designed primers allowing specific detection of PCV-2 genome
in clinical samples and we have
succeeded in detecting the viral genome in lymphoid tissue of pigs
with suspect of disease. The
identity of resulting PCR fragments was verified by sequencing.
G. Vozárová, M. Lisalová, P.
Milo‰oviă, J. Hanzen (HPL, Ltd. Bacteriological
Laboratory,
Bratislava, Slovakia): Using of
selective media for isolation of Yersinia
enterocolitica.
Yersinia enterocolitica is an
important human pathogen who causes gastroenteritis,
lymphadenitis, arthritis and
other diseases.
Yersinia enterocolitica produces
pinpoint colonies after 24 hours of incubation on the solid agar
media. These colonies may be
overlooked on commonly used nonselective cultivation media for
examination of clinical
specimens containing a multiplicity of bacterial species. The aim of
our
research was to use selective media that may enhance detection of pathogenic
strains of Yersinia
enterocolitica
from clinical specimens in our region.
From the September 2000 we
use a selective diagnostic agar medium Yersinia Selective
Medium Oxoid. On this medium
Yersinia enterocolitica produces the typical red
colonies with a
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transparent border. For
differentiation from other mannitol-positive microorganisms that may give
a colonial morphology
resembling Yersinia enterocolitica we identify
all suspect isolates by
biochemical tests. In all strains that were
determined as Yersinia enterocolitica we
performed
serotyping by slide agglutination with rabbit antisomatic O antisera.
The results of isolations on
selective medium Yersinia Selective Medium Oxoid prove that its
using allow the detection of
Yersinia enterocolitica who is the important enteric
pathogen especially
in children and young children.
SECTION 2: MORPHOLOGICAL
AND PHYSIOLOGICAL
PROPERTIES OF
MICROORGANISMS
I. Sládková, O.
Zahradníăek (Microbiological Institute of Medical
Faculty of Masaryk
University and St. Anna‘s Faculty Hospital in Brno,
Czech Republic): Comparison of delta-
haemolytical activity of
coagulase-negative staphylococci of blood cultures and from the
respiratory system.
Coagulase-negative staphylococci
are an important group of microbes, that is able to act
sometimes as normal flora (in
skin, including outer part of nasal cavity, external ear etc.), on the other
hand, they are known as heavy
pathogens in blood system, escpecially as hospital infections – often
in form of a “catheter sepsis”.
It is questionable, whether the strains found in outer respiratory ways
(i. e. common flora) differ from
the strains from blood cultures (i. e. probably valuable pathogens)
in production of any virulence
factors. One of such factors is delta-haemolysin (or, more precisely,
delta-like haemolysin). We
compared the delta-haemolytical activity of 60 strains of coagulase-
negative staphylococci from
bloodstream and 60 strains of coagulase-negative staphylococci from
nasal cavity. We found that
majority of respiratory strains showed zero or weak haemolytical
activity; among the strains form
blood cultures, on the other hand, the ratio of strains with a strong
delta-haemolytical activity was
much higher. Nevertheless, the results are not very clear. There are
many confusing factors, at least
following ones should be taken into account: 1) the quality of the
used agar medium is not
constant, despite all the standardisation; 2) the group of “coagulase-
negative staphylococci” is not
homogenous – in the blood culture strains many species were found,
the ratio of S.
haemolyticus and S. hominis was considerably high; in
respiratory strains,
S. epidermidis was the only really frequent species; 3) only
part of the blood culture strains are real
parhogens - it is very
difficult to differentiate between real causative agents of septicaemia
and
contaminants from skin.
O. Zahradníăek (Microbiological
Institute of Medical Faculty of Masaryk University and
St. Anna‘s Faculty
Hospital in Brno, Czech Republic): Interaction of
deltahaemolytical activity
of staphylococci with haemolytical activity of Staphylococcus
hyicus and Staphylococcus
chromogenes.
Unlike Staphylococcus
aureus with its several types of haemolysins, majority of
non-aureus
(mostly coagulase-negative) staphylococci show at most one type of
haemolytical activity. This
activity is partially analogical to the activity of
delta-haemolysin of Staphylococcus aureus.
Nevertheless, there are some
differences between delta-haemolytical activity of Staphylococcus
aureus and haemolytical
activity of at least some of coagulase-negative staphylococci. Some
phenotypical ones are shown in
this work. The intaraction between several staphylococcal strains,
producing “delta” activity
(both S. aureus and non-aureus
staphylococci) and strains of
Staphylococcus hyicus and
Staphylococcus chromogenes was tested.
S. hyicus and S.
chromogenes
are well known for their production of another type haemolysin. The presence of
this haemolysin
inhibits all types of delta activity, but the extent of this
inhibition is different in S. aureus (inhibition
is weak) and non-aureus
staphylococci (a strong, complete inhibition).
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M. Heroldová., J. Vytlaăil,
P. Ondrovăík. (Microbiological Institute of Medical
Faculty of
Masaryk University and St. Anna’s Faculty Hospital, Brno,
Czech Republic): Relation between
production of slime and elastase
coagulase-negative staphylococci and their ability to adhere
on endoprothese surfaces.
Coagulase-negative staphylococci
(CoNS) start to play more important role as causative agent
of serious complications in
orthopaedic operations. Danger of infection is getting bigger with using
of plastic and metal catheters,
prosthetic cardiac valves and endoprothesis. CoNS are characteristic
by their ability of adherence
and making of biofilms on surface of the medical appliances.
Production of polysaccharide
slime and elastase seems to be backup factor of adherence and
following invasion agents to
organism.
The aim of our study was to find out how the production of these exosubstances
is connected
with measure of adherence CoNS to single types of materials
used for production of endoprothesis.
If there is an important difference in
production of slime and elastase between strains of CoNS
isolated directly from
orthopaedic infection and strains from other clinics.
We studied 30 strains CoNS
isolated from orthopaedic materials and 30 strains isolated from
other departments. We tried to
find out production of elastase by sprinkle method with elastin and
Congo red. Slime production
was tested on CRA agar. We did not find out any differences in
adherence, and production of
slime and elastase between both CoNS groups. Our results suggest
that there are differences in
adherence to individual type of endoprothesis materials.
D. Kotrba, M. Siglová, J. Masák,
A. âejková, V. JirkŰ, L Rybariková, P. Hron (Department of
Fermentation Chemistry and
Bioingeneering, ICT, Prague, Czech Republic): Determination
of
biofilm growth and factors influencing adhesion of selected microbes (Candida
maltosa).
Biofilm formation is common phenomenon in many environmental
conditions. Set of metodic
approaches, which allow us to determine amount of biofilm
biomass is required for characterization
of conditions both enhancing and
decreasing biofilm formation. Therefore we developed procedure
allowing observation of adhesion
on particle carriers. We employed chemical treated glass spherical
particles. Procedure included
incubation of culture in presence of carrier, during procedure
quantitative and qualitative
development of adhesion and stage of formed biofilm, were observed,
optionally morphological
alterations were watched too during adhesion process. Fluorescence
microscopy gives appropriate
informative view for qualitative determination of biofilm formation.
If images are stored, it can be
achieved easy comparison and qualitative evaluation of adhesion on
various types of carriers or in
various intervals. Fluorescence microscopy brings several advantages
in observing adhesion process.
It enables locate even individual microbes, while preparation of
samples is relatively simple.
Fluorescence microscopy is applicable even for opaque samples.
Thanks to fluorescent dye SYTO 13
specifity it is possible to make visible only viable microbes.
Determination of microbial
proteins amount by modified Bradford’s method was used for
accurate quantification of
microbial biomass bounded on carrier’s surfaces. Sonication, which is
very efficient method was applied
to detach microbes attached on carrier surfaces. Concurrently is
sonication simple and fast
method. Interval 10 minutes appeared long enough to detach bounded
cells from carrier surface by
ultrasound with frequention 35 kHz.
Determination of microbial proteins is very sensitive
method, which make possible to realize
even small amount of microbes. Its micro-volume design
in connection with Bioscreen C device
allowed us to process relatively vast variety
of samples in quite short time. For quantitavely
determination of larger part of
biomass, what is the case of Fusarium proliferatum,
it is possible to
use simple determination of dry biomass.
Important characteristic
influencing adhesive behaviour of microbes is hydrofobicity both of
cell outer layers and carrier
surface. Hydrofobicity measurement of microbes was carried out by
BATH technique, which is
unsophisticated but accurate. For carrier surface hydrophobicity it is
feasible to select contact angle
measurement by the sessile drop technique.
Results obtained by approach
described above provided us with overview about ability of
microbes to colonize various
types of surfaces under various conditions. Based on the performed
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experiment we could estimate
fundamental carrier characteristics, which are required for successful
biofilm formation.
F. RŰĎiăka (Microbiological
Institute of Medical Faculty of Masaryk University and St. Anna’s
Faculty Hospital, Brno, Czech
Republic): Ability of adherence of Candida on epithelia of
healthy
persons and persons with diabetes.
The ability of genus
Candida to adhere into epithelial cells is considered
an important factor of
virulence. It makes possible the colonization of
mucosal surfaces by this microorganism. Its
adherence into the epithelial
cells of the healthy people and into the epithelial cells of the people
with diabetes mellitus was
observed at 96 strains of Candida albicans and
135 strains of other
species within this genus.
The good ability to adhere into
the epithelial cells of the healthy people was proven in 70.8 %
strains C.
albicans. In C. tropicalis, C. parapsilosis and C.
glabrata strains. the proven ratio of this
ability was smaller. The
adherence of other tested species was only rare.
In the comparison of the average
number of the yeast cells which adhered into the epithelial
cells of the healthy people
and the epithelial cells of the people with diabetes we did not
find
a statistically significant difference in most of the observed species. Only in
C. parapsilosis and C.
lusitaniae the
number of the yeast cells which adhered into the epithelial cells of the people
with
diabetes was higher.
P. Matűjková, J. ·marda (Department
of Biology, Faculty of Medicine, Masaryk University,
Brno, Czech Republic):
Effects of colicins E1 and K on spheroplasts.
We investigated inhibition
effects of colicins E1 and K on glycine and lysozyme spheroplasts
from a sensitive strain
E. coli K12 Row, from mutant strains in proteins
BtuB, TolC, OmpA, OmpF
and tolerant strains tolA, tolB, tolQ. The colicins E1 and
K have the same mechanism of the lethal
effect, they are ionofores.
However, they differ by outer membrane receptors and by one of the
translocation system proteins.
The ability to form spheroplasts
was reduced in the following order: Row, btuB, ompA, ompF,
tolC, tolB, tolA, tolQ. Glycine
spheroplasts preparation in the latter two mutants was not successful
at all.
The effect of colicins on glycine
spheroplasts was evaluated according to the degrees of their
ability to regenerate rods on
a thin agar layer in the presence or absence of colicin. We followed the
degree of lysozyme
spheroplasts lysis expressed as a decrease of light absorbancy of
their
suspension in time.
Mutants resistant to colicin E1 – strains btuB and tolC
(total insensitivity of rods) –converted
into glycine spheroplasts showed
total insensitivity to it. However, lysozyme spheroplasts showed
rapid lysis in its presence.
Mutants in porins OmpA and OmpF
(decreased sensitivity of rods to colicin K) if converted into
glycine spheroplasts appeared
to be almost insensitive to this colicin. Lysozyme spheroplasts
showed rapid lysis just as the
resistant mutants.
Tolerant mutant tolB was fully sensitive to colicin E1 and
showed a decreased sensitivity to
colicin K. Glycine spheroplasts had the same
parameters. Lysozyme spheroplasts were also fully
sensitive to colicin E1. Colicin
K enhanced the lysis, but just in a insignificant degree. Tolerant
mutants tolA and tolQ were fully
insensitive to both colicins and retained their insensitivity even if
converted into lysozyme
spheroplasts (glycine spheroplasts could not be considered).
Glycine spheroplasts of all
of the investigated strains retained properties similar to those of
intact cells in the presence of
both colicins. Lysozyme spheroplasts of the mutants in proteins of the
outer membrane gained full
sensitivity, which could be explained by damage of the outer membrane
carrying receptors by the
preparation technique in the presence of EDTA. Hypertonic sucrose
medium could also cause
plasmolysis and insufficient contact of the outer and cytoplasmic
membrane. Lysozyme spheroplasts
of the tolerant strains retained their sensitivity or insensitivity
typical for the intact cells. So
no desicive damage of colicin translocation system takes place during
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(Abstract online - PDF)
