Impact of Pattern of Antibiotic Resistance on Outcome of Gram ...

Egyptian Journal <strong>of</strong> Medical Microbiology, January 2006 Vol. 15, No 1
<strong>Impact</strong> <strong>of</strong> <strong>Pattern</strong> <strong>of</strong> <strong>Antibiotic</strong> <strong>Resistance</strong> on Outcome <strong>of</strong> Gram
Negative Induced Spontaneous Bacterial Peritonitis
Manal A. Bahgat¹ and Mohammed A. Bahgat²
Microbiology & Immunology¹ and Tropical Medicine² Departments,
Faculty <strong>of</strong> Medicine, Zagazig University
Spontaneous bacterial peritonitis (SBP) is a bacterial infection <strong>of</strong> ascitic fluid, which is a
frequent and severe complication <strong>of</strong> hepatic cirrhosis. A vast majority <strong>of</strong> such infections are due to
enteric Gram-negative bacteria, mainly Enterobacteriaceae. The prevalence <strong>of</strong> extended spectrum
beta-lactamases (ESBLs)-producing bacteria is clearly increasing, and in many parts <strong>of</strong> the world
10-40% <strong>of</strong> strains <strong>of</strong> Escherichia coli and Klebsiella pneumoniae express ESBLs .
This study was conducted to detect the impact <strong>of</strong> pattern <strong>of</strong> antibiotic resistant in our
community, especially ESBLs-producing E. coli and K. pneumoniae, on the outcome <strong>of</strong> SBP caused
by these bacteria. Ascitic fluid samples were collecting from 52 proved cases with cirrhotic ascites
complicated by SBP for examination and culture (bedside inoculation into blood-culture bottles was
done). Also, antibiotics sensitivity tests were performed. Potential ESBLs producers were subjected
to detection <strong>of</strong> ESBLs by three dimensional method and Oxoid combination test (CDO2).
Positive cultures were obtained in 43 (82.7 %) <strong>of</strong> examined cases, among them 40 (93 %)
had Gram negative bacteria [32 ( 80 %) E.coli and 8 ( 20 %) K. pneumoniae ] and the others (7%)
were Gram positive [S.aureus]. Patients with negative or Gram positive cultures were excluded
from this study, and these with Gram negative one were treated and followed up until discharge
or in-hospital mortality. About 27.5% <strong>of</strong> studied Gram negative isolates were ESBLs- producing
(25% <strong>of</strong> E.coli and 37.5% <strong>of</strong> K.pneumoniae). There was no statistically significant difference
between three dimensional method and Oxoid combination test (CDO2) in detection <strong>of</strong> ESBLs
(P>0.05). The percentages <strong>of</strong> resistant E.coli and K.pneumoniae respectively were (43.75 % & 25%)
to cefotaxime, (46.9% & 25%) to ceftriaxone, (50% & 50%) to ceftazidime, (53.1% & 75%) to cotrimoxazole,
( 50 % & 62.5%) to norfloxacin and (12.5% & 50%) to cipr<strong>of</strong>loxacin.
In-hospital mortality due to SBP was 60 %. After uni- and multivariate analyses, only 5
variables studied were independently associated with SBP in-hospital mortality (P12 (RR=3.2), having hepatic encephalopathy (RR=2.7), jaundice
(RR=1.5), SBP caused by ESBLs-producing strains (RR=1.5) and serum Na

Egyptian Journal <strong>of</strong> Medical Microbiology, January 2006 Vol. 15, No 1
This study was conducted to detect
the impact <strong>of</strong> patterns <strong>of</strong> antibiotic resistant in
our community, especially ESBLs-producing
E. coli and K. pneumoniae, on the outcome <strong>of</strong>
SBP caused by these bacteria. Also, we were
compared between the three dimensional
method and Oxoid combination test (CDO2)
in detection <strong>of</strong> ESBLs-producing organisms.
SUBJECTS, MATERIALS AND
METHODS
This study was done in Tropical
Medicine and Microbiology & Immunology
departments, Faculty <strong>of</strong> medicine Zagazig
University in the period from October 2004 to
May 2005. This study included 52 patients
with cirrhotic ascites and primary SBP, 40
males and 12 females, with mean age
50±9.5years, The diagnosis <strong>of</strong> cirrhotic
ascites was based on the clinical, laboratory
(serum/ascites albumin gradient ≥1.1g/dl (10) ,
and ultrasonographic examination. The
diagnosis <strong>of</strong> SBP was based on presence <strong>of</strong>
ascitic PMNL count ≥250 cells/mm3
(6) .
Patients with secondary peritonitis or had
previous attacks <strong>of</strong> SBP were excluded from
this study. Cases with negative or Gram
positive cultures were excluded from the
study <strong>of</strong> in-hospital mortality (i.e. death
during hospitalization for an SBP episode)
predictive factors. All patients were submitted
for the followings:
1- History taking and clinical examination
with emphasis on symptoms and signs
attributed to SBP and liver cell failure.
2-Ascitic fluid examination and culture:
A-Paracentesis was performed for all
patients at admission and ascitic fluid
samples were examined microscopic-ally
(for white cell count) and chemically (for
assay <strong>of</strong> total protein, albumin, LDH and
glucose levels) to confirm the diagnosis
<strong>of</strong> SBP.
B-Also, another 10ml <strong>of</strong> ascitic fluid
samples were inoculated into blood
culture bottles (oxoid) at the patient’s
bedside, incubated at 37°C for 7 days, and
examined daily for turbidity. These
cultures were subcultured after 2 and 7
days on chocolate-enriched agar plates for
aerobic and anaerobic growth. The plates
were incubated for 1 or 2 days at 37°C,
and organisms were identified (11).
C-Colonies were subjected to identification
by standard bacteriological methods,
according to Baiely and Scott (12) and by
API E (bio-Mérieux) for GNB species
identification.
D –Gram negative bacteria (GNB) were
tested for antibiotic sensitivity by disc
diffusion method according to Bauer et al.
(13)
using the following antibiotic discs
(Oxoid): ceftriaxone (30 µg), cefotaxime
(30µg), ceftazidime (30 µg), cipr<strong>of</strong>loxacin
(5µg), norfloxacin (5µg) and Cotrimoxazole
( 25 µg).
E- Each Klebsiella pneumoniae or
Escherichia coli isolate should be
considered a potential ESBLs-producer if
the test results are as follows: ceftriaxone
< 25 mm , cefotaxime < 27 mm,
and/or ceftazidime < 22 mm. (13).
3-Potential ESBLs producers GNB were
subjected to detection <strong>of</strong> ESBLs by three
dimensional method and Oxoid
combination test (CDO2) that depends on
comparing the inhibition zone give by
ceftazidime (CAZ 30 µg) and ceftazidimeplus-clavulanate
(30 µg/ 10 µg) according
to NCCLs methodology. A difference <strong>of</strong>
≥5mm between the zone <strong>of</strong> CDO2 and CAZ
alone was taken to indicate ESBLs
production, advocated by the manufacturer
(15) .
4-Blood Samples which were obtained at
admission from all patients were examined
for the followings: complete blood cell
count, liver function tests, urea and
creatinine, and serum Na .
5- The severity <strong>of</strong> liver disease was assessed
by the Child-Pugh scoring system, in which
5–15 points are assigned and a higher score
indicates greater severity <strong>of</strong> liver failure.
Patients in Child-Pugh class A have
preserved liver function (5–6 points),
patients in class B have moderate liver
failure (7–9 points), and patients in class C
have severe liver failure (10–15 points) (11) .
6-Abdominal ultrasonographic examination
was carried out using real- time equipment
(ALOKA SSD-500) with convex linear
transducer <strong>of</strong> 3.5 MHz frequency.
7-All cases <strong>of</strong> SBP were treated empirically
with I.V cefotaxime ( Claforan, Aventis) 2
gm every 8 hrs for 7 days (6) beside other
needed therapy. The antibiotic was changed
in some cases who still alive according to
results <strong>of</strong> drug sensitivity tests. These cases
188

Egyptian Journal <strong>of</strong> Medical Microbiology, January 2006 Vol. 15, No 1
were followed up until discharge or inhospital
death.
8-All data were collected, tabulated and
statistically analyzed using the computer
program SPSS version10.
RESULTS
Causative organisms were isolated
from ascitic fluid were mainly Gram-negative
bacilli (GNB) (93%), E.coli was the most
frequently isolated organism (80%) followed
by K.pneumoniae (20%) (Table 1).
The <strong>Antibiotic</strong> resistance <strong>of</strong> the isolated GNB
is shown in Table 3. The percentages <strong>of</strong>
resistant E.coli and K.pneumoniae
respectively were (43.75 % & 25%) to
cefotaxime, (46.9% & 25%) to ceftriaxone,
(50% & 50%) to ceftazidime, (53.1% & 75%)
to co-trimoxazole, ( 50 % & 62.5%) to
norfloxacin and (12.5% & 50%) to
cipr<strong>of</strong>loxacin. All ESBLs- producing strains
were also resistant to norfloxacin and cotrimoxazole
and 63.7 % <strong>of</strong> them were
resistant to cipr<strong>of</strong>loxacin.
About 27.5% <strong>of</strong> studied Gram
negative isolates were ESBLs- producing
(25% <strong>of</strong> E.coli and 37.5% <strong>of</strong> K.pneumoniae)
Table 4.
When the results <strong>of</strong> three dimensional
method were compared to results <strong>of</strong> Oxoid
combination test (CDO2). There was no
statistically significant difference between the
two methods in detection <strong>of</strong> ESBLs (P>0.05)
Table 5.
In-hospital mortality due to Gram
negative induced SBP was 60 % <strong>of</strong> studied
cases. Two infected cases with resistant Gram
negative bacteria to cefotaxime (one <strong>of</strong> them
ESBLs producer) were being alive when
results <strong>of</strong> ascitic fluid culture appeared,
cefotaxime was replaced by cipr<strong>of</strong>loxacin 400
mg I.V. every 12 hrs for one week, and both
<strong>of</strong> them survived (Table 6).
Table 1: Results <strong>of</strong> ascitic fluid culture <strong>of</strong> patients with SBP (n=52).
1- Negative culture 9/52 17.3%
2- Positive culture: 43/52 82.7%
a. Gram +ve (S.aureus) 3/43 7%
B-Gram –ve: 40/43 93%
• E.coli
• K.pneumoniae
32/40 80%
8/40 20%
189

Egyptian Journal <strong>of</strong> Medical Microbiology, January 2006 Vol. 15, No 1
Table 2: Characteristics <strong>of</strong> studied SBP patients* (n=40).
• Age (years)
• Gender (M/F)
• Clinical presentation :
- Fever
- Abd. Pain
- Abd. Tenderness
- Hepatic encephalopathy
- Jaundice
- Splenomegaly
- Hematemesis
- Hepatorenal syndrome
• Investigations:
- Leucocytosis
- Leucopenia
- Thrombocytopenia
- Anaemia (HB1.3 mg/dl)
- Serum Na (

Egyptian Journal <strong>of</strong> Medical Microbiology, January 2006 Vol. 15, No 1
Table 4: The distribution <strong>of</strong> microorganisms producing ESBLs among E.coli &
K.pneumoniae species.
Microorganism № %
E.coli 8 /32 25 %
K.pneumoniae 3 /8 37.5 %
Total 11 /40 27.5%
Table 5: Comparison <strong>of</strong> ESBLs results by Oxoid combination test and three dimensional
method.
Oxoid
combination test
three
dimensional
Fisher
extract test P value
n=20 method n=20 χ²
10 (50%) 11 (55%) 0.1 > 0.05
Table 6: Predictive factors for in-hospital mortality in patients with Gram negative SBP.
Variable
Univariate analysis
Multivariate analysis
P* RR 95%CI P** RR 95%CI
Age > 50 years 0.05 - -
Child-pugh score>12

Egyptian Journal <strong>of</strong> Medical Microbiology, January 2006 Vol. 15, No 1
other tested antibiotics. Moreover, the
proportions <strong>of</strong> cefotaxime resistant strains
among isolated Gram negative bacilli were
(43.75%) for E. coli and (25%) for K.
pneumoniae and most <strong>of</strong> them had crossresistance
to other studied antibiotics
(Table3).These high proportions which reflect
the improper use <strong>of</strong> antibiotics in our
community, make the empirical use <strong>of</strong>
cefotaxime or other 3 rd generation
cephalosporin for treatment <strong>of</strong> SBP
questionable. Also increasing the resistant E.
coli and K. pneumoniae strains to norfloxacin
(50% and 62.5% respectively) and cotrimoxazole
(53.1% & 75% respectively)
reduce the effectiveness <strong>of</strong> these antibiotics in
prevention <strong>of</strong> SBP attacks.
On the other hand, it is a worldwide
tendency that ESBLs-producing strains are
increasing in proportion
(7,17) ,and their
presence in our community was reported
(18) ,so it is important to pay attention to this
growing problem and to investigate deeply
their extension and its resistance pattern.
There was no statistically significant
difference between three dimensional method
and Oxoid combination test (CDO2) in
detection <strong>of</strong> ESBLs (P>0.05). Oxoid
combination test (CDO2) was proved by Ho,
et al and Fam and El-Damarawy to be a
sensitive, convenient and inexpensive method
which can be incorporated into routine
clinical laboratory service (19,20) .
The in-hospital mortality <strong>of</strong> our SBP
cases was 60% which is similar to that reported
by Henz et al (1995) (21) and more than the
results <strong>of</strong> other authors (30-50%) (22) .This high
mortality reflects the severity <strong>of</strong> our cases (65
% had Child-Plugh score >12). The multivariate
analysis demonstrated that from a total <strong>of</strong> 24
variables analyzed ( most <strong>of</strong> them in table 2),
only Child-Pugh score >12 , having hepatic
encephalopathy, jaundice, SBP caused by
ESBL-producing strains and serum Na

Egyptian Journal <strong>of</strong> Medical Microbiology, January 2006 Vol. 15, No 1
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7- Park,YH; Lee, HC; Song,HG et al (2003):
Recent increase in antibiotic-resistant
microorganisms in patients with
spontaneous bacterial peritonitis
adversely affects the clinical outcome in
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18:927-33.
8- Kang,CI; Kim, SH; Park, WB et al (2004):
Clinical outcome <strong>of</strong> bacteremic
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extended spectrum beta-lactamaseproducing
Escherichia coli and Klebsiella
pneumoniae. Korean. J. Intern. Med. ;
19:160-4.
9- Rupp ME, Fey PD.(2003): Extended
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producing Enterobacteriaceae: considerations
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Dupeyron C. (2002): Nosocomial
Spontaneous Bacterial Peritonitis and
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11- Bailey, V R. and Scott, E G.(1990):
Diagnostic Microbiology. 8 th ed. The C V.
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12- Bauer, A W; Kirby, W M; Sherris; J C.
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13- National Committee for Clinical
Laboratory Standards. (1999).
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susceptibility testing. NCCLS approved
standard M100-S9. National Committee
for Clinical Laboratory Standards, Wayne,
PA.
14- Carter MW, Oakton KS, Marner M, et
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(1999):Spontaneous bacterial peritonitis
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18- Sadeq, RA ; El-Hosiny, M ;Mowafy, H.
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comparative study. Z.U.M.J.;7:2303-14.
19- Ho PL, Chow KH, Yuen KY, et al.
(1998).Comparison <strong>of</strong> a novel, inhibitorpotentiated
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Antimicrob. Chemother; 42 (1):49-54.
20- Fam NS and El-Damarawy M (2002).
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11(3):569-580.
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(1995): Spontaneous bacterial peritonitis:
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Schweiz. Med. Wochenschr;125:2379-86
[English abstract].
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7 [English abstract].
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Egyptian Journal <strong>of</strong> Medical Microbiology, January 2006 Vol. 15, No 1
اثر نمط المقاومة للمضادات الحيوية على عاقبة مرضى التهاب الصفاق البكتيرى
التلقائى الناتج عن بكتريا سلبية الجرام .
منال بهجت ، محمد بهجت
قسمى الميكروبيولوجى والامراض المتوطنة – طب الزقازيق
يعتبر التهاب الصفاق البكتيري التلقائي-‏ وهو عبارة عن عدوى بكتيرية للسائل الاستسقائى بالبطن-‏
يعتبر من
المضاعفات المتكررة والخطيرة لتشمع الكبد،وهو ناتج في معظم الحالات عن عدوى ببكتيريا معوية سلبية الجرام،‏
خصوصا ً الأمعائيات
.
وقد ازداد فى الفترة الأخيرة انتشار البكتريا المفرزه لإنزيمات البيتالاكتاميز واسعة المجال ،
حيث يصل مدى انتشارها في عدد من الأماكن إلى
و(‏ الكلبسيله الرئوية).‏
٪٤٠-١٠
من السلالات المعزولة ل(الايشيريشيا القولونية)‏
وقد صممت هذه الدراسة لتحديد اثر نمط المقاومة للمضادات الحيوية خصوصا ً للبكتريا سلبية الجرام المفرزه
لإنزيمات البيتالاكتاميز واسعة المجال،‏
على عاقبة مرض التهاب الصفاق البكتيري التلقائي الناتج عن هذا النوع من
البكتريا ، حيث تم جمع عينات من السائل الاستسقائى من ٥٢ مريضا بتشمع الكبد المصحوب بالتهاب الصفاق البكتيري
التلقائي لفحصها وزراعتها
الحساسية للمضادات الحيوية
.
بطريقتي الأبعاد الثلاثة واختبار اكسويد التوافقى.‏
‏(باستخدام تقنيه الزرع بجانب المريض في قناني مزارع الدم)‏
، كما تم إجراء اختبارات
وبالنسبة لتحديد السلالات المفرزه لإنزيمات البيتالاكتاميز واسعة المجال فقد تم ذلك
وقد تم الحصول على ٤٣ مزرعة أي جابية (٨٢,٧٪)
حالة ايشيريشيا القولونيه
إيجابية الجرام
،
٤٠ مزرعة منها نتج عنها بكتريا سلبية الجرام
٣٢))
و (٨٠٪)
٨ حالات(‏‎٢٠‎ (٪
كلبسيله الرئوية))‏
،
)
بينما أظهرت الحالات الثلاثة الباقية بكتيريا
المكوره العنقودية الذهبية).‏ وقد تم استبعاد الحالات ذات المزارع السلبية أو التى أظهرت بكتريا إيجابية
الجرام من هذه الدراسة،‏ بينما تم علاج ومتابعة الحالات ذات المزارع سلبيه الجرام حتى الشفاء أو الوفاة بالمستشفى
وقد أوضحت هذه الدراسة ان
.
٢٧,٥
المجال (%25 من حالات الايشيريشيا القولونيه و
٪ من البكتريا سلبيه الجرام المعزولة تفرز إنزيمات البيتالاكتاميز واسعة
٣٧,٥
إحصائية بين الطريقتين المستخدمتين فى تمييز البكتريا المفرزة لهذه الإنزيمات
.
٪ من حالات الكلبسيله الرئويه)،‏ ولم يظهر أى فرق ذو دلاله
إما بالنسبة إلى سلالات الايشيريشيا القولونيه والكلبسيله الرئوية المقاومة للمضادات الحيوية فقد كانت كالأتى
-:
٤٣,٧٥) ٪ و (٪ ٢٥
لسيفوتاكسم و
٪٤٦,٩) و (٪٢٥
لسيفترياكسون و
٪٥٠) و (٪٥٠
لسيفتازيديم و
(٥٣,١٪ و‎٧٥‎
(٪
لكوترايموكسازول و
٪٥٠) و‎٦٢,٥‎ (٪
لنورفلكساسين و
٪١٢,٥) و (٪ ٥٠
لسبيروفلكساسين على التوالي.‏
وقد كانت نسبه الوفيات داخل المستشفى الناتجة عن التهاب الصفاق البكتيري التلقائي ٦٠٪ من الحالات المدروسة ، وبعد
إجراء التحليل الاحصائى للمتغيرات المفردة والمتعددة ، تبين ان
: الوفيات وهى
٥ متغيرات منهم كانت مستقلة و لها دور في هذه
مجموع نقاط تشيلد ‏–بف اكثر من ١٢ أو وجود اعتلال مخي كبدى أو يرقان أو العدوى بسلالات مفرزة
لانزيمات البيتالاكتاميز واسعة المجال أو انخفاض مستوى الصوديوم بالدم.‏
ومما سبق نستنتج ان حالات التهاب الصفاق البكتيري التلقائي الناتج عن بكتيريا سلبية الجرام ومفرزة
لإنزيمات البيتالاكتاميز واسعة المجال والتي تقاوم كل عقاقير الجيل الثالث للكيفالوسبورم المستخدمة لعلاج هذه المرض
منتشرة في منطقتنا و تسبب العديد من الوفيات ، لذا يجب التنبه لها ووضعها في الاعتبار
.
كما ننصح بإجراء اختبارات
متكررة لمتابعة نمط المقاومة للمضادات الحيوية لهذه السلالات والبدء في البحث عن علاج بديل فعال لعلاج هذه العدوى.‏
كما وجد ان اختبار اكسويد التوافقي بالغ الدقة ومعقول الثمن وملائم للكشف عن السلالات المفرزة لإنزيمات
البيتالاكتاميز واسعة المجال.‏
194