сучасні досягнення фармацевтичної технології та біотехнології modern ach

765 Pages·2016·10.03 MB·English

МІНІСТЕРСТВО ОХОРОНИ ЗДОРОВ’Я УКРАЇНИ
НАЦІОНАЛЬНИЙ ФАРМАЦЕВТИЧНИЙ УНІВЕРСИТЕТ

СУЧАСНІ ДОСЯГНЕННЯ ФАРМАЦЕВТИЧНОЇ
ТЕХНОЛОГІЇ ТА БІОТЕХНОЛОГІЇ

MODERN ACHIEVEMENTS OF PHARMACEUTICAL
TECHNOLOGY AND BIOTECHNOLOGY

ЗБІРНИК НАУКОВИХ ПРАЦЬ

ХАРКІВ
2016
ISSN 2519-2655
УДК 615.1
С 89

Редакційна колегія:
академiк НАН України Черних В.П., проф. Гладух Є.В.,
проф. Стрельников Л.С., проф. Половко Н.П., доц. Манський О.А.,
доц. Калюжна О.С., доц. Шпичак О.С.

С 89 Сучасні досягнення фармацевтичної технології та біотехнології :
збірник наукових праць. – X.: Вид-во НФаУ, 2016. – 764 с.
ISSN 2519-2655

Збірник містить матеріали V Науково-практичної інтернет-конференції з
міжнародною участю «Сучасні досягнення фармацевтичної технології та
біотехнології» (18 листопада 2016 р.).
Розглянуто теоретичні та практичні аспекти розробки, виробництва,
контролю якості, стандартизації та реалізації лікарських засобів на сучасному
етапі.
Для широкого кола магістрантів, аспірантів, докторантів, співробітників
фармацевтичних та біотехнологічних підприємств, фармацевтичних фірм,
викладачів вищих навчальних закладів.

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Матеріали подаються мовою оригіналу

ISSN 2519-2655 УДК 615.1
©НФаУ, 2016
2
UDK 616.9:616-036
OCCURRENCE OF tonB GENE AND ITS RESTRICTION ENDONUCLEASE
ANALYSIS IN NASOPHARYNGEAL ISOLATES OF Haemophilus influenzae
FROM LARYNGOLOGICAL PATIENTS
Andrzejczuk. S.1, Kosikowska U.1, Chwiejczak E.2, Malm A.1
1Department of Pharmaceutical Microbiology with Laboratory for Microbiological
Diagnostics, Medical University of Lublin, Chodzki Str. 1, 20-093 Lublin, Poland;
2Students Scientific Association at the Department of Pharmaceutical Microbiology
with Laboratory for Microbiological Diagnostics,
Medical University of Lublin, Poland

Introduction. Bacteria have many different mechanisms of iron uptake
including small molecules called siderophores secreted outside the cells and chelating
iron from surrounding or specific proteins ExbBD and TonB on outer membrane and
playing a role as a receptors binding an iron from e.g.: hemoglobin, haptoglobin,
transferrin or lactoferrin. In Gram-negative microorganisms iron is transported
through outer membrane and then via the periplasm and the inner membrane, what
requires an energy flow. TonB protein, being an energy-transducing protein for iron
uptake, is a distinct part of a complex composed of three proteins ExbB-ExbD-TonB.
TonB protein is placed in the inner membrane and extends along the periplasm. Other
proteins are located only in the inner membrane. Due to the presence of TonB protein
both membranes (outer and inner) are connected together and allow the energy flow
required to iron transport [1,2,4,5].
H. influenzae is a part of human respiratory microbiota and also an important
pathogen especially for younger children. It is believed that the ability to growth and
pathogenicity of H. influenzae is generally associated with the requirement for iron
from external sources. Literature data indicated that in H. influenzae as well as in E.
coli an uptake and transport of iron is strongly dependent on the TonB protein [2,3,5].
Furthermore, inactivation of tonB gene in H. influenzae serotype b (Hib) caused a
lack of capability to iron uptake, transport and use of this element for life processes,
including an ability to involve infections. In consequence, bacteria became avirulent
[5].
The aim of the study. The aim of this study was to analyze the prevalence of
tonB gene in nasopharyngeal H. influenzae isolates collected from children with
recurrent respiratory tract infections who underwent adenoidectomy. Additionally, a
molecular typing on the basis of a restriction endonuclease digestion of the tonB-
positive strains was performed and a structural similarity of DNA patterns was
assessed.
Methods of research. The study included 37 isolates of H. influenzae selected
from children aged 2-5 years old with recurrent respiratory tract infections who
underwent adenoidectomy, as previously described [3]. The isolates were obtained
from nasopharyngeal swabs. Additionally, the reference strain of Haemophilus
influenzae ATCC 10211 from the American Type Culture Collection (ATCC) was
used.
Identification of haemophili were performed using the API NH microtest
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(BioMérieux, France). The tonB gene (~813 bp) was identified in a classic PCR
reaction using a specific Ton1 (5’-GCAAGCACAACAAGTGCAGCTAA-3’) and
Ton2 (5’-GCCGCCTTATCTAAACTTTCATCG-3’) primers [4]. PCR amplification
25 µL reaction mixture included: REDTag® Ready Mix™ PCR Reaction Mix
(Sigma Aldrich, Germany), 20 µM of each primer, 1 µL of genomic DNA and
nuclease-free water (Sigma Aldrich, USA). Amplification was performed using the
T20 Personal thermocycler (Biometra, Germany) and carried out for 34 cycles
consisting of: denaturation at 94°C for 1 min, annealing for 1 min at 48°C and primer
extension at 72°C for 1 min. Products were detected by electrophoresis on a 1.5%
agarose gels (Sigma Aldrich, Germany) with ethidium bromide (1 mg/mL) in 0.5x
Tris-borate-EDTA buffer at 120 V for 1.5 h. Gels were observed under UV light,
photographed and documented using the Quantum Vilber-Gel-documentation tool
(Vilber Lourmat, Germany).
All DNA probes with detected tonB gene were treated with the two FastDigest
enzymes XhoI and BglII (ThermoFisher Scientific, USA). A restriction analysis was
performed according to Matar et al. [4] and 30 µL reaction mixture was prepared in
accordance with enzymes manufacturer’s instructions. The obtained restricted
fragments were separated by electrophoresis on 2% agarose gels at 120 V for 2 h. As
described above, gels were observed under UV light, photographed and documented
using abovementioned documentation system (Figure 1).

Abbreviations: M – 100 bp DNA ladder; lane 2 – PCR product of H. influenzae
ATCC 10211 digested with XhoI; lane 3 – PCR product of H. influenzae ATCC
10211 uncut by BglII; lanes 5, 7, 11, 13, 15, 17, 19 – PCR products uncut by BglII;
lanes 4, 6, 10, 12, 14, 16, 18, 20 – PCR products digested with XhoI; lane 8 – PCR
product uncut by XhoI; lane 9 – PCR product digested with BglII
Figure 1. XhoI and BglII restriction endonuclease digestion results of PCR
products of tonB gene (~813 bp) in nasopharyngeal Haemophilus influenzae strains
isolated from children with recurrent respiratory infections who underwent
adenoidectomy.
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Main results. According to our results, among 37 nasopharyngeal H.
influenzae isolates from children with recurrent respiratory infections who underwent
adenoidectomy the occurrence of tonB gene as revealed by PCR product ~813 bp was
observed in 33/37 (89%) of isolates.
Analysis of restriction enzyme digestion of DNA fragments with detected tonB
gene in H. influenzae strains are summarized in Table 1. A digestion with the
endonuclease XhoI showed that PCR products obtained from 32/37 (86%) of strains
and the reference strain H. influenzae ATCC 10211 were cut by this enzyme into two
fragments of ~204 and ~609 bp. In case of a reaction with the BglII enzyme, PCR
products from 15/37 (41%) strains were cut into two fragments of ~388 and ~425 bp.
On the basis of the results of PCR products digestion involving XhoI and BglII
endonucleases, a four possible restriction patterns were defined: I (X+B+) – PCR
products cut by both XhoI and BglII, II (X+B-) – PCR products digested only with
XhoI, III (X-B-) – uncut PCR products and IV (X-B+) – PCR products digested only
with BglII. The obtained results showed that most isolates were characterized by the
II restriction pattern – 18/37 (49%), followed by the I pattern with 14/37 (38%)
representatives.
Conclusions. The high prevalence of tonB gene among nasopharyngeal H.
influenzae isolates in children with recurrent respiratory infections who underwent
adenoidectomy suggests its importance not only as a virulence factor responsible for
pathogenicity and invasive infections but also for facilitating bacteria survival in a
human body and colonization by them of e.g. nasopharynx.
Table 1. Restriction patterns of PCR products of tonB gene (~813 bp) after
digestion with XhoI and BglII in nasopharyngeal Haemophilus influenzae strains
isolated from children with recurrent respiratory infections who underwent
adenoidectomy.
XhoI BglII
Restriction
Fragment No. of products Fragment No. of products
pattern
length (bp) length (bp)
digested uncut digested uncut
I (X+B+) 14 - 14 -
II (X+B-) 18 - - 18
~204, ~609 ~388, ~425
III (X-B-) - - - -
IV (X-B+) - 1 1 -
X+ – PCR products digested with XhoI, B+ – PCR products digested with BglII,
X- – PCR products uncut by XhoI, B- – PCR products uncut by BglII
References
1. Choby JE, Skaar EP. Heme synthesis and acquisition in bacterial pathogens.
Journal of Molecular Biology. 2016;428(17):3408-3428.
2. Jarosik GP, Sanders JD, Cope LD, Muller-Eberhard U, Hansen EJ. A
functional tonB gene is required for both utilization of heme and virulence expression
by Haemophilus influenzae type b. Infection and Immunity. 1994;62(6):2470-2477.
3. Kosikowska U, Korona-Głowniak I, Niedzielski A, Malm A.
Nasopharyngeal and adenoid colonization by Haemophilus influenzae and
Haemophilus parainfluenzae in children undergoing adenoidectomy and the ability of
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bacterial isolates to biofilm production. Medicine (Baltimore). 2015;94(18):e799.
4. Matar GM, Chahwan R, Fuleihan N, Uwaydah M, Hadi U. PCR-based
detection, restriction endonuclease analysis, and transcription of tonB in Haemophilus
influenzae and Haemophilus parainfluenzae isolates obtained from children
undergoing tonsillectomy and adenoidectomy. Clinical and Diagnostic Laboratory
Immunology. 2001;8(2):221-224.
5. Morton DJ, Hempel RJ, Seale TW, Whitby PW, Stull TL. A functional
tonB gene is required for both virulence and competitive fitness in a chinchilla model
of Haemophilus influenzae otitis media. BMC Research Notes. 2012;5:327.
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UDK 616.311
THE PREVALENCE OF THE SELECTED GENES RESPONSIBLE FOR
BIOFILM FORMATION IN Candida glabrata ISOLATED FROM THE ORAL
CAVITY IN IMMUNOCOMPROMISED PATIENTS
Biernasiuk A., Sternik M., Malm A.
Department of Pharmaceutical Microbiology with Diagnostic Microbiology Unit,
Medical University, Lublin, Poland

Introduction. The yeasts belonging to Candida spp., including C. albicans and
other species, known as non-albicans Candida spp., are commensal organisms that
can be isolated from the oral cavity, upper respiratory tract as well as from vagina in
healthy people. These yeasts are classified also as opportunistic pathogens, which can
cause candidiasis – endogenous infections from superficial to seriously deep-seated
mycoses under predisposing conditions [1,2]. However, following the widespread
and increased use of immunosuppressive therapy together with broad-spectrum
antimycotic therapy, the frequency of mucosal and systemic infections caused by C.
glabrata has increased significantly [1,2,3]. This yeast is often the second or third
most common cause of candidiasis after C. albicans. Infections caused by C. glabrata
are difficult to treat and are often resistant to many azole antifungal agents, especially
fluconazole [2,3]. Currently, there are few recognized virulence factor of C. glabrata.
One of them is the biofilm formation. These yeasts can develop form this structure on
several surfaces such as oral, urinary and vaginal epithelia or central venous
catheters, dental prostheses and other indwelling devices. The biofilms are extremely
resistant to antifungal drugs and are a source of reinfections [2]. Biofilm formation by
yeasts is complex process regulate by several genes. Among them, EPA6 and AWP2
encodes an adhesins or cell wall proteins of C. glabrata. In turn, ACT1 gene is
responsible for encoding the structural protein actin or YAK1 encodes a protein kinase
[1].
The aim of the study. The aim of the study was to estimate the prevalence of
the selected genes responsible for biofilm formation: ACT1, AWP2, EPA6 and YAK1
in C. glabrata strains isolated from the oral cavity in patients with immune disorders.
Methods of research. Totally, 50 strains of C. glabrata isolated from patients
with immunosuppression, e.g. with cancer, chronic hepatitis C, diabetes mellitus and
elderly people aged of 65 years old or older were included in the study. The Ethical
Committee of the Medical University of Lublin approved the study protocol (No. KE-
0254/75/2011). Moreover, the reference strain of C. glabrata ATTC 90030 belonging
to the American Type Culture Collection was used as a control microorganisms. To
assess the incidence of the genes involved in biofilm formation by C. glabrata, the
isolates were grown overnight in Sabouraud Dextrose bulion (SDB) medium. The
DNA from the strains was prepared using Genomic Mini AX YEAST (SPIN) (A&A
Biotechnology, Poland), according to the manufacturer’s procedure. PCR method
was performed with ACT1, AWP2, EPA6 and YAK1 primers (Genomed S.A. Poland)
(see Tab. 1).
PCR reactions were carried out in the thermocycler and amplification
conditions were as following: initial denaturation at 95 ºC for 5 min and 48 cycles of
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denaturation at 95 ºC for 10 s, followed by primer annealing at 56 ºC for 30 s and
elongation at 72 ºC for 30 s. Final extended elongation at 72 ºC have lasted 5 min.
The polymerase chain reaction was performed in 0.5 ml microcentrifuge tubes in a
15-µl final reaction mixture containing 50-200 ng of C. glabrata DNA as template,
7.5 µl of REDtag Ready MixTM PCR Mix (Sigma-Aldrich, USA), 5.9 µl of distilled
water and 0.3 µl of each 20 µM primer. For each experiment, the size of DNA
fragments amplified by PCR were determined by direct comparison with the DNA
marker – 100 bp Ladder Plus (Fermentas, Lithuania). Control samples without
template DNA were included in each run and reproducibility was checked for each
reaction. The PCR products were separated using electrophoresis in agarose gels
(1.5%) at 120 mV for approximately 60 min at room temperature in TBE buffer (Tris
Borate Electrophoretic Buffer, 89 mMTris/HCl, 89 mM boric acid, 2.5 mM EDTA,
0.01% ethidium bromide pH 8.0) (Sigma-Aldrich, USA). Reaction products were
detected and visualized with UV light.
Table 1. Primers used for detection by PCR of the selected genes responsible
for biofilm formation in C. glabrata.
Gene Sequence primers
ACT1f 5′- ACCAACTGGGATGACATGGA-3′
ACT1r 5′- TCATTGGAGCCTCGGTCAAC-3′
AWP2f 5’-GCGGTTGCAGATTGGTAATGG-3’
AWP2r 5’-TAGGATAAGAACAGACGACGGGTG-3’
EPA6f 5′- GGATGGACGGACCCTACTGA-3′
EPA6r 5′- GCTGCATCCCAACATGGATTC-3′
YAK1f 5′- GCATGGTTCAGCGTCCAATC-3′
YAK1r 5′- CGGTACGTTCGGGGTCATAG-3′

Figure 1.The prevalence of types of combination genes in C. glabrata isolated
from the oral cavity. Explanations: I – the presence of all studied genes: ACT1,
AWP2, EPA6, YAK1; II – ACT1, AWP2, EPA6; III – AWP2, EPA6; IV – EPA6,
YAK1; V – ACT1, AWP2, YAK1; VI – AWP2, YAK1; VII – YAK1.
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Main results. Using the PCR reaction, products of the studied genes involved
in biofilm formation in C. glabrata were detected with the prevalence depending on
the strain. Seven gene combinations were defined. Type I, which included all of the
tested genes was found in the majority – 76 % of isolates. Other types were detected
to occur with lower frequency (2 – 8 %) (see Fig. 1). In the case of the C. glabrata
ATTC 90030, the presence of type I was found.
The most frequently detected genes, were AWP2 and EPA6, which occurred
with similar incidence – 94 % isolates. The remaining genes, namely ACT1 and
YAK1, were found also to occur with high frequency – 86 %.
Conclusions. The high prevalence of genes involved in biofilm formation in C.
glabrata isolated from the oral cavity in immunocompromised patients suggests that
these genes may be regarded as an important factor responsible for colonization of
human body by C. glabrata as well as its pathogenicity.
References
1. Chew S.Y., Cheah Y.K., Seow H., Sandai D., Than L.T.L.: In vitro
modulation of probiotic bacteria on the biofilm of Candida glabrata. Anaerobe 2015,
34: 132-138.
2. Riera M., Mogensen E., d’Enfert Ch., Janbon G.: New regulators of biofilm
development in Candida glabrata. Res. Microbiol. 2012, 163 (4): 297-307.
3. Rodrigues C.F., Silva S., Henriques M.: Candida glabrata: a review of its
features and resistance. Eur. J. Clin. Microbial. Infect. Dis. 2014, 33 (5): 673-688.

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УДК: 615.1/2: 33 (075.8)
ABC-ANALYSIS OF RHEUMATOID ARTHRITIS
PHARMACOTHERAPY
Gerasymova O.O., Tzhatirani Riyameke
National University of Pharmacy, Kharkov, Ukraine

Introduction. Rheumatoid arthritis is the serious autoimmune disease. It is
caused by high rates disability and mortality of patients, reducing their quality of life
and significant financial costs for treatment [2,3]. Conducting rational
pharmacotherapy of the disease and optimization of its costs are relevant and
determine the conduct of the clinical and economic evaluation of rheumatoid arthritis
pharmacotherapy.
The purpose of this study is to evaluate the structure of medicinal
preparations costs for the treatment of patients with rheumatoid arthritis in one of
health care institutions in Kharkov.
Methods of research. Supplementary clinical and economic method: ABC-
analysis [1]. Research lasted for 2015.
The main results. According to the 84 disease histories of patients with
rheumatoid arthritis aged from 39 to 62 years (30 men and 54 women) 37 trade
names (TN) of medicines (20 international non-patent names (INN)) from 13
pharmacological groups have been determined. Correlation of Ukrainian and foreign
drugs is 1:2.7.
The division TN of medications into the ABC-groups was the following, group
A – 9 medications with 79.46 % of costs from the total costs sum on all researched
medications; group B – 11 medications (15.16 % of costs), group C – 17 medications
(5.38 % of costs). The leader of ABC-rating according to TN became a
immunosuppressant “Methotrexate” (“Ebewe Pharma”, tabl. 2.5 mg № 50) which is
22.92 % of total sum costs.
The most expensive pharmacological groups – immunosuppressants (38.74 %
of costs, 2 INN, 2 TN) and nonsteroidal anti-inflammatory drugs (24.51% of costs, 2
INN, 6 TN).
Conclusion. The results of this carried out ABC-analysis can be the basis to
improve the pharmacotherapy of rheumatoid arthritis in the mentioned health care
institution.
References
1. Оцінка клінічної та економічної доцільності використання лікарських
засобів у лікувально-профілактичному закладі (супровід формулярної системи):
метод. рек. / А. М. Морозов, Л. В. Яковлєва, Н. В. Бездітко та ін. – Х.: Стиль-
Издат, 2013. – 36 с.
2. Florian MP Meier, Marc Frerix, Walter Hermann & Ulf Müller-Ladner.
Current immunotherapy in rheumatoid arthritis // Immunotherapy.  2013.  № 5(9).
 Р. 955-974.
3. Klimes J., Vocelka M., Sedovа L. et al. Medical and Productivity Costs of
Rheumatoid Arthritis in The Czech Republic: Cost-of-Illness Study Based on Disease
Severity // Value in Health. Regional Issues  2014.  № 4C.  Р.75 – 81.
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