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1


Antibiotic Resistance And Failure Of Eradication
Of
Helicobacter Pylori

In Egypt

Wesam Abdel G
han
y

Hasan
e
in
, Hanan M
ohammed
Elsayed, Raghda
Abd Ellatif H
afez, Osama M
ohammed B
asha

and

El sayed Hamdy

Moham
m
ed
.




Departments of Microbiology & I
mmunology, internal medicine
,

Faculty of
Medicine, and

Botany Department,

Faculty of Science
,

Zagazig University
.


Abstract

Background
:
Helicobacter pylori
is
is

a Gram

negative bacterium that colonizes
human gastric mucosa and is one of the most common bacterial
pathogens
world
wide with a prevalence of upto 90 % in developing Countries
.
It is the
primary cause of peptic ulcer disease and an etiolog
ic agent in the development of
gastric cancer.
H. pylori
infection is curable with regimens of multiple antimicrobial
agents. However, antibiotic resistance is a leading cause of treatment failure.
The
aim
of this study is to
assess the prevalence of
H.pylori

in gastric biopsies taken from
Egyptian patients by using invas
ive methods and
study the

role of antimicrobial agents
in elimination of this bacterium
.
Methodology
:F
rom 50 patients,
3 antral ga
s
tric
biopsies were taken from the greater curvature
about 2 cm f
r
om pylorus
.
T
he first
biopsy was for direct G
ram
's

stain and culture
(using Blood agar)

to apply Traditional
Biochemical tests and antimicrobial susceptibility test

to different antibiotics
,
The
second biopsy was used for rapid urease test

(usin
g
modified Christensen
'
s urea broth
)

and

t
he third biopsy was kept in deep freezer at
-
70
0
C

in brain heart infusion broth

for PCR assay using Ure
c gene
.
Result
s

:A
mong 50 patients, 2
2(44%) were positive
by culture
, 1
7(34%) were positive by direct G
ram's stain with 77.3% sensitivity, 100%
specificity

and 90% accuracy,
while 19(38%) were positive by
rapid urease test

with
63.6% sensitivity, 82.1% specificity and 74% accuracy
,

and 2
5
(
50
%) were positive by
PCR with 95.5% sensitivity, 89.3% specificity an
d 92% ac
curacy .By antimicrobial
2


Susceptibility testing using disc diffusion method, it was shown that,
the highest
susceptibility of the isolated
H.pylori

strains was to
amoxicillin

(90.9%)

followed

by
tetracycline (81.8%)

,
Gentamicin (54.5%)

Erythromycin

(18.2%)

and
Ciprofloxacin

(9.1%)
,However

no one

(zero%) were highly sensetive

to

Metronidazole
.

Conclusion
:
Some of antibiotics widely used in Egypt are no
longer

suitable for treatment of
H
elicobacter pylori


and new antibioticts

regimen

are need
ed to eradicate this organism
.






Key words:

H.pylori

and antimicrobial agents eradication.



Introduction
:




H.
pylori

is a Gram negative spiral shaped, measuring 2 to 4
µ
m

in length and 0.5
to 1
µ
m in width,

multi
-
flagellated bacteria found almost

exclusively on human
gastric mucosa. It contains a hydrogenase which can be used to obtain energy by
oxidizing molec
ular hydrogen (H2) that is produced by intestinal bacteria
.(Olson and
Maier,
2002
)
,
The bacterium is a microaerophilic and capnophilic organism, slowly
growing with rigorous culture demands.
(Mégraud and Lehours, 2007)



Helicobacter pylori

play
s

a
vi
tal role in the pathogenesis of several gastro
duodenal pathologies makes its diagnosis necessary in

many different circumstances.
S
ince, the diagnosis of this bacterium is an essential element in the management of
many common gastrointestinal pathologies
(Fock and

Ang, 2010)
.










Several virulence factors of
H. pylori
have been identified. The most studied is
CagA

(Cytotoxin associated gene Antigen) which according to numerous studies, is
associated with peptic ulcers, precancerous

conditions and gastric cancer in the
Western world
.
(Kusters
et al.,

2006)
,
Another well
-
known virulence factor,
VacA

(vaculating cytot
oxin A)

is associated with peptic ulcer
,
adenocarcinoma and

gastric
cancer

(Wada
et al.,
2004)

.





3



Antibiotic resistance is an ever increasing problem with the treatment of most
microbial infecton including
H
e
licobacter pylori

and has become a growing problem

world wide with eradication of this organism


(Buta

et al.,

2010)
.








AIM OF THE WORK


So this study was conducted to eval
u
ate

recent

changes of antibi
otic resistant
pattern

of
H
e
licobacter pylori


isolated from Egyptian patients to the most common
antibiotics used for treatment of this organism
.




Materials and methods
:

50 patients
with age ranging from 18 to 70 years
suffering fro
m abdo
minal complaints underwent

gastroendoscopic examination.
F
rom
each patient
, 3 antral gatric biopsies were taken from the greater curvature about 2 cm
f
r
om pylorus
.
(
Prior to endoscopy aconscent were taken
from each patient)
.
The
first biopsy was transported
to Microbiol
. Lab. Within 2 hrs for direct G
ram stain and
culture
,
The second biopsy was used for rapid urease test

and

t
he third biopsy was kept
in deep freezer at
-
70
0
C

in brain heart infusion broth (BHIB: Oxoid)


containing 10 %

glycerol for PCR assay
.





The culture was performed on
Blood agar (Oxoid Columbia agar base)

figure
1
.

Plates were incubated at 3
7
0
C for
3
-
5

days in a microaerophilic atmosphere (Campy
Pak; Becton Dickinson). Identification of
H. pylori

was made by Gram staining of

the
colonies, lack of aerobic growth on blood agar plates, and testing for the presence of
urease, oxidase and catalase.
(Logan and

Walker, 2001)
.

and an

inoculum was spread
all over agar surface using cotton
swab for application of antimicrobial Susceptibility
testing using; Amoxicillin, Tetracycline, Gentamicin, Erythromycin, Ciprofloxacin and
Metronidazole.

The Second biopsy was
used for Rapid urease test

where
inoculated in
10% Urea bro
th with phenol red as an indic
ator. The presence of urease was indicated
by colour change

from yellow to pink.
(Aydin

et al.,
2004)
.
The
third biopsy was used
for

extraction of DNA using DNeasy Blood and Tissue Kit(Qiagen Gmbh
,
4


Hilden,Germany).The extracted DNA was used for

PCR using

primers for the
amplification of a

highly conserved region

Ure c
gene
( glm M
gen
e
)

in which

t
he size
of the

amplified product

obtained is 294
base pair

in (photo
1
)
.

The PCR protocol
used was described by
Lu
et al
.

1999
. PCR reactions were performed in a total
reaction volume of 50 ul containing 25 ul 2x PCR Master Mix (Ferments USA)which
compose of (Tag DNA polymerase
recombinant in reacton buffer 0.05 units
\
ul,Mg
cl2,4 Mm, d NTP s

(dATP, d GTP,

dTTP

and dCTP,0.4Mm of each).To 25 ul 2x
Master Mix we added 5 pm of each of the forward primer (5 AAG CTT TTA GGG
GTG TTA GGG GTT T3)and the reverse primer(
5 AAG CTT AC
T TTC TAA CAC
TAA ACG C3).(Biron,Germany).Then 5ul of the template DNA were added

and the
volume was
com
p
leted to 50

ul

by nuclease free water.The samples were maintined at
94
0
C

for 5 minutes and then 34 cycles (94
0
C

for 1 minute ,at 56

0
C

for
1

minute and
at 72
0
C

for 2

minutes) using DNA thermal cycler(Perkin Elmer,USA).
The PCR
products were run on a 2% agarose gel electrophoresis
and the products were
visualized by ethidium bromide using

UV transillumination(Cole
-
Parmer instrument
CO, Chicag
o USA)
.






Statistical


methods
:

The statistical analysis was performed using SPSS vers
i
on 16
.P values less than 0.05 were considered to indicate significance




Result
:
Among
50

patients,
22

(
4
4%) had positive
Culture

results,
17
(
3
4%) had
positive
direct G
ram's stain
findings,
19(38%)

had

positive
R
apid urease

test results,
and
2
5

(
50
%) had positive
PCR

results.
In accordance with the culture as a gold
standard
; the sensitivity, specificity, positive predictive value, Negative predictive
value and accuracy for Direct Gram's stain were
77.3%, 100%, 100%, 84.8% and
92%.respectively,
the sensitivity, specificity,
positive predictive value, Negative
predictive value and accuracy for

rapid urease test were 63.6%, 82.1%,
73.7
%,
74.2
%
and
74
%.respectively

and the

sensitivity, specificity, positive predictive value,
Negative predictive value and accuracy for

PCR were

9
5.5%, 89.3%, 87.5%, 96.2%
and 92
%.respectively as shown in table (1)which indicates that,
The PCR method is
5


the most sensitive method (95.5%) followed by direct Gram stain (77.3%). Direct
Gram stain method is the most specific method (100
%) followed by PCR method
(89.3
%
). while the rapid urease test is the least specific (82.1%)
.A
s regarding to
antimicrobial
s
usceptibility testing by disk diffusion method, it was shown that,
the
highest susceptibility of the isolated
H.pylori

strains was to
amoxicillin (90.9%)

followed by
tetracycline (81.8%)

and
Gentamicin (54.5%)
. On the other hand, all
the isolated
H.pylori

strains were resistant to
metronidazole (91.0%)
.
as shown in
table (2) from which it was concluded that the

th
e antimicrobial agent
Amoxicillin

and
Tetracycline

are the most drugs of choice for
H.pylori

treatment.
The result is
statis
tically significant (P<

0.0
01
)
.





Figure (
1
)
:

Agarose gel showing


H.pylori

DNA band at 294 bp.

Lane(1):



Molecular size standard( 1
-
kb DNA lad
d
er,Gibco BRL).

Lane(
3,
5,

6 &
7): Positive gastric biopsy specimens for
H.pylori
.

Lane(2

& 4
):

Negative

cases
.

Lane(
8
):

Negative

control.






6


Table

(
1
)
: Validity of different laboratory methods used as confirmed by
Culture for identification of
H.pylori



Accuracy



(%)


PV


+ve
-
ve


Specificity



(%)

Sensitivity



(%)


Culture method

(reference test)


Positive Negative

n= 22 n= 28

Screening test


90.0


100 84.8


(100.0)


(77.3)



17 (a) 0 (b)


5 (c) 28 (d)

Direct Gram stain

Positive

Negative


74.0


73.7 74.2


(82.1)


(63.6)


14 (a) 5 (b)


8 (c) 23 (d)

Rapid urease Test

Positive


Negative


92.0


87.5 96.2



(89.3)



(95.5)


21 (a) 4 (b)


1 (c) 24 (d)

PCR

Positive

Negative



Table (2
):
antimicrobial susceptibility of the isolated
H.pylori

strains.






Resistant strains


No
.

(%)

Intermediate strains


No
.

(%)

Sensitive strains


No.

(%)

Antimicrobial



agents


0

0.0


0

0.0


1

4.5


8

36.3


9

40.9


20

90.9


2

9.1


4

18.2


9

40.9


10

45.4


11

50.0


2

9.1


20



90.9


18



81.8


12



54.5


4



18.2


2



9.1


0



0.0

Amoxicillin

Tetracycline

Gentamicin

Erythromycin

Ciprofloxacin

Metronidazole

?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
7


Discussion
:

I
n the routine clinical diagnostics, rapid urease test,
histopath
ological examination, culture, d
irect Gram's staining and PCR assay are
valuable methods for detection of
H.pylori

in gastric biopsies.
(Taj
et al.,

2003)
.In our study, Culture technique was used as a gold standard
for diagnosis
of
H.pylori

infection where 22(44%) among 5
0 patients was
positive using
c
ulture
. Since,
culture proved a very successful method for detecting
H. pylori
in
gastric biopsy specimens

and this coincide with that reported by
Ozcakir
et

al.,(
2008)

who

reported that among 148 patients with dyspeptic complaints,
70
(47.2%) were positive by using c
ulture
.
The rapid urease test is a simple,
inexpensive method
for

detecting
H. pylori
in gastric tissues
, in our study,
19(38%) among 50 patients was positive using
rapid urease test and this agreed
with that reported by
Oyed
eji
et al.,(
2002)

who
reported the positive rate of 435
stomach mucosal biopsies taken from 145 consecutive patients
,
was 61 (42.1%)
using RUT.
and disagreed with that reported by
Sengupta
et al.,
(2002),
found
that of 25 patients, 24 were positive(96%) by
using RUT
.this difference may be
attributed to
that false
-
positives can occur owing to the presence of other urease
-
positive bacteria in the gastric tissues or reflux of alkaline bile into the stomach
(
el
-
Zimaity, 2000
)
.
the Direct Gram's stain showed
17
(34%) was positive and
this is in close agreement with that of
Murata
et al.,
(2002)

who

found that direct
gram
's

stain was positive 609(44.6%) in specimens taken from tunica mucos
vestibulum ventricul and tunica mcosa corpus ventriculi.

the PCR technique is an
accurate for diagnosis of
H.pylori

as among 50 patients, 24(48%) was positive
with PCR assay, this is in close coincinment with that repored by
Stella
et

al
.,(2011)
who

found that

3/7(42.8%)

patients were positive by using PCR with

glmM primers, they concluded that PCR test using glmM gene appears to be the
most reliable test for
H.pylori

diagnosis,

and
Ah Ra Cho
et al
.,(2010)

who

found that the positivity rates of infection from 90 patients was 42.2% using
PCR assay.
in relation
to validity of these tests
, it was shown that, the
The Direct
8


Gram’s staining has 77.3% s
ensitivi
ty, 100% s
pecificity, 100% PPV, 84.8%
NPV and 90% accuracy.
and this coincide with that of
Sadeghifard
et al.,
(
2006)
who

reported that the sensitivit
y,
specificity and accuracy of direct Gram stain
when C
ulture was taken as a gold standard are 89.7 %, 96.9% and 86.3%
respectively.
while Rapid urease test has
63.6% sensitivity, 82.1% s
pecificity,
73.7% PPV, 74.0% NPVand 74% accuracy when Culture was taken
as a gold
standard .
and this is similar to that of
Taj
et al.,
(2003)

who
reported the
sensitivity and specificity of urease test was 67% and 85% respectively
with
c
ulture as a reference.bu
t

disagreed with that reported by
Kalem
et al.,

(2010)

who found t
hat the sensitivity and specif
icity of RUT with a reference c
ulture
were 97.5% and 20.7% respectively.
PCR assay has
, the sensitivity,
specificity,PPV, NPV and accuracy when culture was taken as a gold standard
were 95.5%, 89.3%, 87.5%, 96.2% and 92.0%

respectively.
and this is strongly
agreed to that reported by
Stella
et al.,
(2011)

who
found that the PCR assay
using glmM primers has 100% sensitivity, 74.1%
-
84.1% specificity, 68.2%
-
75%
PPV and 100% NPV with culture and RUT as a gold standards.
in the

present
study ,the isolated
H.pylori

stra
ins were highly

sensitive to
amoxicillin
(90.9%),Tetracycline (81.8%)

and showed

intermediate

susceptibility to
Gentamicin ( 54.5%)

while it showed
91.0%

resistance to
Metronidazole

and
40.9%
resistance to
Ciprofloxacin
.

This result is very important as
metronidazole is acorner stone of
many triple
-
therapy formulation for the
eradication of
H
elicobacter pylori

and this agree
d

with several studies which
reve
a
led that resistance to metronidazole approach 90%

in m
a
ny dev
e
l
o
ping
countries and even in Western Europe it ranges from 5 to 50
%
.
In Egypt, a

universal high level primary metronidazole resistance in children

compared to
lower resistance to other selected antibotics was reported by
Sherif
et al
.,

(
2004
)
.

And our results also

agreed with
Gholam
et al.,
(2007)

who
found that 24
patients were resistant to metronidazole (84.1%), 4.1% to erythromycin and they
are highly sensitive to Tetracycline
,Amoxicillin

and furazolidene.

and
Treiber
et

9


al
.,(2002)

who
found that
H.pylori

resistance rate to Metronidazole is 29.1
-

41%.
but disagreed with that report
e
d by
Smith
et

al
.,(2001)

who documented
that
H.pylori

strains were 100% resistance to Amoxicilin.


Conclusion
: polymerase chain reaction is the most accurate for diagnosis of
H.pylori

in gastric biopsy specimens
,

Some of antibiotics widely used in
Egypt are no longer suitable for treatment of
Helicobacter pylori


and new
antibioticts regimen are n
eeded to eradicate this organism
, since,

Amoxicillin
and Tetracycline


were


taken

as agood antibiotics for treatment
.




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