Ir aq i J.Pha rm.Sc i., Vol.15 (2 )  ,2006                  Cefamandol , Ce ftaz idime /Clavulanate Comb inatio n  
  

8 

Synergistic Effect of Potassium Clavulanate in Combination 
withCefamandol and  Ceftazidime on - Lactamase, Extracted From 

Resistant E.coli 
Siham S. Shaokat*1   , Hamoudi  A. Hameed**  

*Ministry of Industry and Mineral 
**Min istry  o f Industry and Mineral 
  
 Abstract  
The  aim of this s tudy was  to evalua te  in-vitro activity of Ce fa mandol and Ceftazidime , in 
combination with pota ssium clavulanate a gains t 10 uropathogenic E.coli is olated from 
patients with c hronic c omplicated urinary trac t infe ctions (UTIs), these  is olates were 
identified by the Api ide ntific ation s ystems .The a ntimicrobial s usc eptibility tests were 
determined by Kirby-Ba uer method and the minimum inhibitory c onc entra tions of 
Cefamandol and Ce ftazidime , were de te rmined, by tube method. The se isolates  we re  res is tant 
to Ampicillin (Amp), Amoxicillin (Amo), Carbenic illin (Cb), Tic arcillin (Tic), Amoxicillin\ 
Potas sium Clavulanate {Augmentin}, (Amo\CA), Tic arcillin\ Potass ium Cla vulanate 
{Timentin} (Tic\CA), Cefamandol (Cfm) and Ce ftazidime (Cfz), also re sistant to other 
antibiotic s, s uch as  Te trac ycline, C hlora mphe nicol, Trime thoprim and (50% of the  isola tes  
were re sistant to Na lidixic acid and Rifampicin). Transfer of pla smids   by dire ct c onjugation 
experime nts were performed by mating 10 stra ins   with rec ipient strain E.c oliK12C  600 Rif 
or Na l re sistant, and ce ll free  - la ctamas es  we re  prepared and detec te d by macro-iodome tric 
method. The  ac tivitie s of eac h cell free  – lac ta ma se s e xtra ct agains t Cfm and Cfz were 
determined by disks  diffusion method (microbiologic al Masuda method) and by macro-
iodome tric  me thod. The ac tivity of - lac ta mase s was inhibited by the addition of Potas sium 
Cla vulanate. Conclus ion: 
Good e ffectiveness  of Cfm\ CA a nd Cfz\ CA was  obtained against re sistant stra ins of E.coli 
due  to complica te d urina ry tra ct infec tion (UTIs). 
K ey words: - la ctamas es , Cefama ndol, Ce ftazidime , Timentin and  Augme ntin . 

الصة  خ
والنيك ض الكالف منهما مع حام ول السيفتازيديم بخلط كل  من السيفاماند عالية كل  ) ملح البوتاسيوم (  يهدف البحث الى تقييم ف

ة   عزوـل مرضة والمشخصـة والم ون الم راثيم اشريشيا القول ة نظـام التوصـيف الوظـائفي    (ضد ج طريـق رة   ) ب ـش ن ادرار  ع ـم
مصابين  بالتهابات المجاري البولية مزمنة مرضى  ة      .ال ريـق ة بط ختلـف ة الم ـادات الحيوـي راثيم لمض ـج جاد حساسية هذه ال تم اي

وسـط السـائل         ة  ال غرى بطريـق ذلك الجـرع المثبطـة الصـ ـك ي و رـب ة   , كي وـم ا مقا ـدت بأنـه وج ـيلين , و مبس ن , لال سيسـيلي موك , اال
ن  ــلي ــــ ــلين ,الكاربينسـ ــ رســـ كا ــت  , التي ــ ــ النـي كالفيو ــيوم  ــــ وتاسـ ـيلين  \ب ــ ــــ كسيس ن (امو ـي ــ ــــ وكمنت ــيوم كالف , )ا ــــ ــت بوتاسـ ــ ــ النـي  \يو

ن ن(تيكارســلي ـايمنتي ول,) ـت ن  ,  والســيفتازيديم, الســيفاماند ورامفينيكــول, وكــذلك للمضــادات االخــرى مثــل التيتراســايكلي كل , ال
رايميثوبريم  ريفامبسين,الت كانت مقاومة  لحامض الناليديكسيك وال زالت  من الع مسون بالمئة  ران   . وخ ة االقـت تم تنفيذ تجرـب

معرفة انتق شر البسيط ل ومة للريفامسين او المبا ن الحساسة للمضادات الحيوية والمقا شريشيا القولو عزلة ا ال البالزميدات الى 
ض الناليديكسيك  مضادات , لحام ميدية لل ومة بالز مل مقا الت تح ميع العز مما دل على ان ج عها ايجابية  مي كانت النتائج ج و

منت,أعاله الت  ميع العز ود في المحيط الصلب ان ج طريقة الي جد ب مات البيتاوو زي ألن ماندول , الكتاميز -جة  كما تم تقييم السيفا
ما مع الكالفوالنيت رعـي      , والسيفتازيديم  بخلطه وسـط ألز ي ال النتشـار ـف ة  ا طريـق وب الم ماسـودا    (بطريقة اليود  ة  الـع طريـق

كروبايولوجية النيك مع , ) الماي ض الكالفو عد إضافة حام ملة ب كا ق تثبيط    مناط زالت  ميع الع والسيفا وأعطت ج السيفتازيديم 
ة    , ماندول مزمـن ة ال ولـي ي الب ار ـج ات الم عالج ناجح اللتهاـب خلطات ك كر باستخدام هذه ال جعلنانف ـات    , مما ي ـد اجـراء الدراس ع ب

زمة   .السريرية الال
  
  
  

  
  
  
  

1corresp ond in g author  email a lbia tyss84@yah oo.coma lbia tyss84@yah oo.com  
Rec eived 11 - 4-2005 
Acc ep te d  2 5-7-2006     
 

mailto:albiatyss84@yahoo.com


Ir aq i J.Pha rm.Sc i., Vol.15 (2 )  ,2006                  Cefamandol , Ce ftaz idime /Clavulanate Comb inatio n  
  

9 

In troduction  
Clavula nic ac id  is a - lacta m; structura lly it 
differs from pe nicillins  in two re sp ects , the 
re plac ement o f Sulfur in the pe nicillin 
thiazo lidine  ring with oxyge n in the clav am 
oxazo lidine  ring and the a bs enc e of the  s id e 
chain at pos ition 6. 
Clavula nic ac id  a na turally oc curring clav am 
is olated  fro m S trep to myce s clav uligerus ha s 
poo r antiba cterial a ctiv ity but exerts  a  p otent 
andirreve rs ible inhibito ry e ffec t on - 
la ctama se s es pec ia lly pe nicillinas es  b y 
bloc king the  a ctiv e sites of thes e enzyme s and 
is  s trongly s ynergistic with mo st o f the - 
la ctamine s in vitro(1,2).Due to this comb ination, 
Amoxic illin is  p rote cted fro m d egrad ation and 
its  s pec trum is therefore e xtende d to includ e 
ba cteria  normally res is tant to  a moxycillin and 
othe r  - la ctam antibiotics  (3) . In the ca se  of 
- lac ta m re sistant ba cteria a ba cteria l e nzyme, 
- lac ta mas e, c le ave s the - lac ta m ring and 
re nd ers  the antibiotic inac tive -lac tamas es  are a 
la rge and div erse  gro up of e nzyme s in whic h 
fo ur c linic ally relev ant clas se s are known (4).  
- lac ta mas es  c ontinue to  b e the lea ding ca us e 
of res is ta nc e to  - lac ta m a ntibiotic s a mong 
Gra m-negativ e ba cteria . In rec ent ye ars there 
ha s be en an increas ed  inc id enc e and 
preva le nc e of extende d-spe ctrum -la ctama ses 
(ESBLs ), enzyme s that hydrolys e a nd  ca us e 
re sistance  to  oxyimino-cep ha lo spo rins  and 
aztreo nam. The majority o f ESBLs are de rive d 
from the wide sp re ad broad  - spe ctrum - 
la ctama se s TEM-1 a nd  SHV-1. ESBLs hav e 
be come  wide sp re ad througho ut the  wo rld and 
are now found in a  significa nt p ercenta ge  of 
E.coli a nd Kle bs ie lla pneumo niae  strains  in 
ce rtain countrie s (5,6,7,8). The re  a re  also  new 
fa milies  of ESBLs , including the 
ce fo ta ximas e(CTX-M) and  OXA- typ e 
enzymes , ce ftazidimase  , a s well as  no vel 
unre la te d - lactamase s (9,10). The stab ility of 
different c ephalos porins to the mos t important 
- lac ta mas es  wa s as se ss ed and many clinical 
stud ie s ha ve shown that up to 75% of the  - 
la ctama se s re sp ons ible fo r - la ctam resis tance 
in G-nega tive  bac te ria we re  R-pla smid 
med ia te d(11,12,).Re ce ntly, new fo urth generatio n 
ce pha lo sp orins , s uc h as  Ce fe pime, Cefpirome, 
Cefos elis ,  Ce fd itoren, Ce fo zop ra n (13,14), were 
introd uc ed into antiba cterial c he motherap y and 
their ac tiv itie s were compared  with o ther -
la ctams  s uch as  Ce ftazidime,Imipe ne m and 
Carba penem , aga inst P.ae ruginos a, 
Ente rob ac te riac eae  (E.co li, Kle bsiella 
pneumo niae ) a nd G-pos itive  bac te ria. In 
ad dition s eve ra l drug comb inations  hav e bee n 
produce d whic h c ontain both a - lac tam 
antibiotic a nd a - la ctama se  inhibitor; the 

inhibito r ha s high a ffinity for  - la ctama ses  it 
irre vers ibly b inds to it, a nd the re by pres erves  
the a ctivity of the - la ctam. Curre ntly, four 
penic illin inhib itor co mbinations are in clinic al 
use : Amp ic illin Sa lb ac ta m (Una syn) , 
Amoxic illin Clav ulana te  (Augmentin), 
Tic arcillin – Cla vulanate (Timentin) and 
Pip ra cillin-Tazoba ctam (Zos yn) (15,16) .Urina ry 
trac t infe ctio ns (UTIs ) c ause a s ignifica nt 
hea lth p rob le m and  E.c oli ha s be en rep orte d to  
be the p rimary pa thoge n in ap pro ximately 8 0% 
of ca ses . E.coli, exp re ss  structures  ca lled  
adhes io ns, fimbriae  or pili that help them bind  
to  s pec ific tis sue (17).  
Aim of the Study         
The  aim of the  stud y is to  ev aluate, the  
fo llowing c omb inatio ns  , Cefama nd ol / 
Cla vula nate and Cefta zidime/Clav ulana te for 
their in vitro antimic ro bial ac tivity a ga inst 
comp lica te d urinary tra ct infec tio ns ca us ed by 
- lac ta mas e producer E.coli. 
Ma terials and Methods 
 Bacteria l s trains   
Sta nda rd  str ains with plas mid  – med iate d 
beta – lactama se s wer e us ed: 
1-E.c oli K12 (TEM-1  typ e - la ctama se  with 
is oelec tric point 5 .4) co nfer plas mid(R 1 11) 
and  E.c lo aca e P99  (11 ).  2 -E.c oli K12 (S HV-1 
type - la ctama se  P itton (type II) I.p 7.7 (11). 
3-E.c oli K12  C 6 00 Rif and E.coli K12 C 60 0 
Nal Se nsitiv e to antibiotic s (11). 4-Clinic al 
is olates  o f E.co li. 5 -Pure  enzyme  o f Me d Lab s.  
All types  of a ntib io tics  powd er we re  o btained  
and  kindly provide d by SDI. 6 - E.coli ATCC 
259 22 kind ly p ro vided  b y Med ic al c ity. 
Ide ntification of E.c oli 
Strains were is olated  o n MaCc onkey a gar and 
id entified  by Ap i 20 E S ys te m (Biomerieux 
vite k, Inc ) (18). 
Antib io tic su sce ptib ility tes t (Dis k diffus io n 
method )  
 The re sistance pa ttern fo r antibiotic s w ere  
determined  by Ba uer - Kirb y (19) d iffusion 
ass ay   on Muelle r – Hinton a gar (2 0ml / plate) 
the inoc ulum was  1 04 – 10 5 ba cteria  / ml, of 6 
hours  cultures  incubate d at 3 7C0 for 24  
hours .The a ntib io tics  use d were as  follow:  
Ampicillin30g,Amo xicillin30  µg,Augme ntin 
(Amo 20µg+CA10 g), Ca rb enicillin 10 0 g, 
Tic arcillin10 0 g, Time ntin(Tic 75µg+CA 10  
g), Ce fa mandol 30 g a nd  Ce fta zidime3 0 g, 
Rifamp ic in 30  g, Nalid ixic  ac id  30 g, 
Tetra cycline 30 g, Chloramphenic ol 30  µg 
and  Co trimoxazo le   (Trimetho prime  2 .5  g + 
Sulfa metha xa zole  2 2.5 µg) Powd ers of 
Cefama ndo l a nd Ce ftazid ime were also  
obtaine d from (Rousse l, Bee cham and 
Sep acia). 



Ir aq i J.Pha rm.Sc i., Vol.15 (2 )  ,2006                  Cefamandol , Ce ftaz idime /Clavulanate Comb inatio n  
  

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Min imum inhibitor c onc entra tion  (MICs ) 
This  test me as ures  the conce ntra tion of a n 
antibiotic nece ss ary to inhibit growth o f a 
standardized ino culum under de fine d 
co nditio n .Minimum inhibitory concentra tions 
(MICs ) were d etermined  by dilutio n of 
different c onc entration of antibiotics in 
Mue ller – Hinton broth .The tub es  were 
inoc ulated with a  6  hour incub ation cultures  , 
diluted , give n a final co nc entratio n of 
inoc ulum (1 04 –  1 05 CFU/ml ) a nd  inc uba te d at 
37C .The lo wes t co nce ntra tio n o f antibiotic 
preve nting growth and rema ining clea r (fre e 
from microbia l growth ) (MIC) w as es tima te d 
after 1 8 hours  o f inc uba tion . 
Rema ining clea r (free  fro m microb ia l growth) 
(MIC) wa s e stimated after 18  ho urs of. As 
co ntro l, fully sensitiv e E.c oli K12 stra in wa s 
te sted  und er the  s ame  co nd itions . Ta ble1 and 
Tab le  2 s ho ws normal MICs v alues  and 
diame te rs  o f zo ne of inhib ition ac cording to 
the method  reco mmended  by the Natio nal 

Committe e fo r Mic ro biology Labo ra tory 
Stand ards (FRANCE) (20). 
Tra nsfer  of g ene tic  info rma tion by dire ct 
con jug atio n method .  
Conjugal tra ns fe r o f 3 GC resis ta nt ESBL 
pro ducing s trains  wa s do ne at 35°C- °C 37°C 
in liquid med ium {Brain hea rt infusion (B.H)}  
or in so lid me dia { Trypticas e S oya  agar 
(T.S.A) o r Mue ller – Hinto n (M.H)} us ing   E. 
coli K12 C 6 00 Rif and E.co li K12 C 600  Na l as  
re cipient.Equal v olumes  (1 mL) o f culture of 
the d onor and the  rec ip ie nt s train  (108-109 
CFU/ml) grown with a gita tion in tryptic  s oya  
bro th were mixed  a nd incuba te d  s ta tic ally for 
18 hours  at 35° C. Trans co njuga nts we re  
selec ted on M.H aga r conta ining Nalid ixic  
acid( 150  g /ml) o r Rifa mpicin(30 0 g /ml) to  
inhibit the growth of donor and   
Amoxic illin ,Tica rc illin,a nd e ftazidime to  
inhibit the gro wth o f recipient s train (11). 

  
Table  (1) S ta nda rd o f MICs  a nd dia me te rs  () o f zo ne  o f inhibition o f ce phalosporins 

ce pha losporins   
 
 
 

Abbre v ia tions 

Critic al 
c oncentratio ns 

In g/ml 

o f * 
Zone  of 

Inh ibit ion 
 

Pote ncy of 
disk / g/ml 

First ge ne ratio nc c C d D 
Cefalothin Ctn 8 32 1 8 1 2 3 0 
Cefaloridin Cfr  8 32 1 8 1 2 3 0 
Cefalexin cfx 8 32 1 8 1 2 3 0 

Se cond ge ne ratio n  
Ce fa mand ol Cfm 8 32 2 2 1 5 3 0 
Cefuroxim Cx m 8 32 2 2 1 5 3 0 
Ce fo xitin  Cxt 8 32 2 2 1 5 3 0 

Third ge ne ra tion  
Cefotaxime Ctx 4 32 2 1 1 5 3 0 
Ceftriaxone Cro 4 32 2 1 1 5 3 0 
Ce fo tia m Ctm 4 32 2 2 1 5 3 0 

Cefmenoxi me Cmx  4 32 2 1 1 5 3 0 
Ce ftazid ime  Cfz 4 32 2 1 1 5 2 5 
Ce ftizoxime  Zox  4 32 2 1 1 5 3 0 

Cefope ra zo ne Cfp 4 32 2 1 1 5 3 0 
Cefod ia zine Hr221 4 32 2 2 1 5 3 0 
Moxalac ta m Mox 4 32 2 3 1 7 3 0 

MIC c : Se ns itive  s trains , MIC>C: Res istant strains , C< MIC C In te rme diate,  D: Se nsitive s trains , 
<d Resistant s trains  d < D   =d ia meter (in 11).  
 
 
 
 
 
 
 
 
 
 
 



Ir aq i J.Pha rm.Sc i., Vol.15 (2 )  ,2006                  Cefamandol , Ce ftaz idime /Clavulanate Comb inatio n  
  

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Table  (2) Standard va lues  o f MICs  and  d ia me ters  () o f z one  o f inhibition of Pe nicillins . 
 

Pe nicillins   
 
 
 

Abbre viatio ns 

Critica l 
co ncentra tion

s 
In g /ml 

of * 
Zone  of 

Inh ibit io n 
 

Pote nc y 
o f disk / 
g/ml 

Gro up A c C d D 
AMPICILLIN Amp  4 16 1 7 11 10 

AMOXYCILLIN Amo  4 16 2 1 14 25 
AUGMENTIN Amc  4 16 2 1 14 Amo 20 + 

CA 10 
TIMENTIN Tim 1 28 128 1 3 13 Tic  7 5+ 

CA 10 
CARBOXYPENICILL  

CARBENICILLIN Cb 1 28 128 1 5 15 1 00 
TICARCILLIN Tic 1 28 128 1 3 13 75 

AMIDINOPENICILLIN       
MECILLINAM Me c 1 8 2 3 17 25 

UREIDOPENICILLIN       
MEZLOC ILLIN Me z 8 32 2 1 16 75 
AZLOCILLIN Azl 1 6 128 1 9 10 75 
P IPRACILLIN P ip 1 6 128 2 0 13 1 00 

MONOBACTAM       
AZTHEREONAM Atm 4 32 2 3 17 30 

 
MIC c : Se nsitive  s trains, MIC>C: Res istant strains , C< MICC Inter media te , 
 D: Se ns itive  s trains , <d Res is tant strains d < D   =diameter ( in 11).  
Extraction of – lactamase .  
Cell free be ta  –lactamase s were p re pared  from 
strains known to  be  good  prod uce rs  o f the 
de sire d enzyme s, (– la ctama se s, typ e TEM-1 
E.coli R111  a nd SHV-1  E.c oli4 53 , R-plas mid 
med ia te d e nzymes ) and  –lac ta mas e from 
E.cloac ae  P 99( ce pha lo spo rina se ) as  re ference.  
Crude  e nzymes  we re  a ls o prepa re d from te st 
is olates  of E.c oli. Ba cteria l c ultures were 
grown ae ro bically a t 370 C in Bra in Hea rt 
Infusion b roth (Difco ) ove r night. A 20 0 ml 
flas k of the  s ame broth was  the n ino cula te d 
with 2 ml of the culture, incubated  a t 3 7°Cfor 4 
ho urs, the  ce lls  were ha rv este d b y 
ce ntrifuga tion, wa shed twice  with buffer 
phosp hate  p H 7  a nd dis rupted with ultras ound 
(s onip re p 150 HSE) a t 2 0KHZ. To remo ve ce ll 
de bris , the c rude extrac ts were ce ntrifuge d; the 
superna ta nts were collecte d in sma ll s te rile 
vials unde r as eptic co nditio ns (11).    
Detection of – lactamas e by Macr o -
io dometric method. 
1% of a garos e and0 .5 % of starch were 
diss olved  in12 0ml o f b uffer phosp ha te  and 
boiled, 18  mg o f p enicillin G po wder and0.8ml 
of iodine s olution were a dd ed at 4 0°C, the 
med ium w as s haken a nd distrib uted  in aliquo ts 
of 2 0ml in/ Pe tri- dishe s, 5 well were ma de in 
ea ch plate , the enzymes we re  a pplie d in e ac h 
well and the  zone s o f dec olorization were 
obs erve d 1-18hours a t 4°C (21) . 
 

Asses sme nt o f stability of – la ctams  to cell-
free – lactamase s( 22,23,24). 
The  ac tivity of ea ch ce ll free  –  lacta mase  
extract against eac h – lacta m a ntib io tic was  
determine d by the mic ro biologica l method  
(Mas ud a G., et a l. 197 6,mod ified by La bia.R. 
Barthelemy.M. 197 9). The surfac e of a  Muller 
Hinto n agar wa s se ede d with a susp ens ion of 
– la ctam sensitiv e ind ic ator E.co li ATCC 
259 22. Four disc s co ntaining – la ctams  under 
te st we re  p la ce d nea r filter pa pe r disc s; e ach  
imp re gna ted with 30l of the enzymatic  
extract. The  p la te s were incuba te d at 3 7°C for 
18hours, the  – lacta mas e ac tivity was  
obs erved  like half mo on zone of inhibitio n. 
Unc hange able inhibition zo nes  de monstra te  
stability o f the antib io tic to the enzyme.  
Inh ibition by Cefamandol or  Ceftazidime 
/Clavulana te Modifie d io dometric method (21). 
Modifie d iod ometric  me thod (Labia , R . , 
Barthelemy . M.19 79),w as used  witho ut 
incorpo ra tion of penic illin G in the  me dium, 
five s wells  we re  ma de in the p la te in which 
10l of e nzyme extrac t , 30  l of po ta ss ium 
clavula na te  and  30l o f Ce fa mandol or 
Cefta zidime were a dd ed. The re sults we re  
noted  a fter 4-18  ho urs a t 4°C, ab sence  of 
dec olorization zone  indicated  pos itive rea ctio n. 



Ir aq i J.Pha rm.Sc i., Vol.15 (2 )  ,2006                  Cefamandol , Ce ftaz idime /Clavulanate Comb inatio n  
  

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Masu da micr obiolog ic al me th od 
Ten c linica l is olates  we re  sc re ene d fo r – 
la ctama se  inhib itor us ing10l p otass ium 
clav ulana te in c omb inatio n with 3 0 l of 
Ceftazid ime  o r Cefama ndo l. Sensitiv ity disc s 
co ntaining Ce ftazid ime o r Cefamand ol and a 
filte r disc  incorpo ra te d with 30l e nzyme and 
10l pota ss ium clav ulanate were p la ce d o n 
agar p la te  o n which a  ba cterial s us pension of 
se ns itiv e E.c oli (s ta nd ard) was  sp re ad the 
inoc ulum was  1 04 – 10 5 CFU / ml, o f 6 ho urs 
cultures  a t 3 7C0 for 2 4 hours  a cc ord ing to the 
metho d re commend ed by the Natio nal 
co mmittee  for microbiologic al Lab oratory 
standards  (25) .  
 
 
 

Results a nd Discuss ion   
Dis k aga r diffus io n te st (S usc eptibility te st) 
 Ac co rding to the  results  of Susce ptibility 
te st   .The  res is tanc e patterns  of E.c oli RIII 
(TEM-1 be ta-la ctama se) and E.c oli K12 (SHV-
1) type    - lac ta mas e Pitton (type  II ) I.p 7 .7 
were c ompa re d w ith te n s trains  the y we re  
re sistant to Ampicillin , Amoxic illin , 
Carbenic illin P ip ra cillin , Augmentin , 
Time ntin ,  Cefama ndo l and  Ce ftazid ime . 
The y we re  a ls o re sistant to  o ther a ntib io tics  
suc h as Tetracycline ,Chlo ra mphenico l and  
Trimethoprim and (50%) we re  re sistant to  
Rifamp ic in a nd  Nalidixic acid . The  res ults  
indicated  d is se minatio n of resista nce  a mong 
clinic al iso la tes of E.coli in Ira q table 3. 

Table  (3) Se nsitiv ity Te sts of S trains  De te rmine d by Disk  Diffusion Te st 

 
Abb revia tion s : Amp :Amp ic illin ; Am o:Amoxic illin ;Amc:Amo xic la ve ; Cb : Ca rb en icillin ;T ic: Ic arcillin  ;T im : 
T imen tin  ;Cfm: C efama ndol ; Cfz:Ce ftazidime   ;  T c :T etracyclin ;Cm:Chlo ra mphen ic ol ;T m:T rime th op rim ;Rif: 
Rifa mpic in  ; Nal :Nalid ixic a cid ;(see table1  ) 
 
 

 
 

Detection of   - lac tamases 
This  tes t is bas ed  o n the re ac tion o f the (oic ) 
ac id  of penic illin with io dine .  - lac ta mas e 
hydrolyze  p enic illin to pe nicilloic  a cid , whic h 
in turn reac t with iod ine , the  p re sence  of  - 
la ctama se  in a  tes t s ys te m wa s s ho wn b y 

dec olorizatio n of starch –  iod ine co mplex . The  
re sults of de te ctio n o f  - lac ta mas es   by 
io dome tric metho d were pos itive  for 10 s trains  
comp ared with standard ne ga tive  E.c oli   K12 C 
600  Rif and  po sitive  –  lacta mas es R11 1 
(TEM-1), p re se nted in Fig 1. 

 
 
 
 
 
 
 
 

 
 
 
 
 
 
 
 

No of 
is olate s   

DIAMETERS OF ZONE OF INHIBITION/MM 

 Amp  Amo  Amc  Cb  T ic  T im Cfm C fz  T  Cm T m Rif Na l  
1 0 0 4 0 0 5 .5 2 3 0 0 0 16 0 
2 0 0 4 .5 0 0 7 3 3 0 0 0 0 18 
3 0 0 3 .5 0 0 8 2 5 0 0 0 18 0 
4 0 0 4 0 0 8 .5 0 4 0 0 0 10 0 

5 0 0 5 0 0 9 6 15 0 0 0 15 0 
6 0 0 5 .5 0 0 9 10 15 0 0 0 0 0 
7 0 0 6 0 0 7 11 11 0 0 0 0 18 
8 0 0 6 0 0 6 .5 5 10 0 0 0 0 19 
9 0 0 6 0 0 9 .5 5 11 0 0 0 0 19 
10 0 0 7 .5 0 0 9 7.5 13 0 0 0 10 18 

E.c oil( 24) 

SHV-1 
0 0 1 9 0 0 2 3 19 24 0 0 22 0 20 

E .coil 
TEM-1 

0 0 2 1 0 0 2 4 20 26 0 0 21 0 20 

E .c loacae  
P99 

0 0 4 15 13 5 6 4 - - 0 0 0 



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Min imum inh ib itor y co ncentra tions  
    The  inhib ition of beta-lacta mas e pro ductio n 
by pota ssium clav ulana te  ha s bee n 
de mons tra te d with many s trains  o f bac te ria, 
this effe ct p otentiates  the  a ction of many b eta 
la ctams , suc h a s Amp ic illin, Amoxicillin, 
Carbe nicillin a nd Tica rcillin. Ma ny clinical 
re ports of co mbination of Amo xicillin with 
Clavula nic ac id (Augme ntin) have  bee n 
encoura ging, in urina ry trac t infe ctions  d ue  to 
– lacta mas e-p ro duc ing orga nisms  typ e TEM 
and S HV, whilst Amo xicillin a lo ne  ha d no 
effe ct, the  add ition of Cla vula nic ac id  (as  s alt) 
drama tica lly change the ha lf mo on inhibitio n 
zo ne to co mplete inhibitio n zone (3,4,11). 
          Our inve stigations  indica ted res is tant 
pheno type  of Augmentin and Time ntin; the 
diame te rs  o f zone of inhibitio n range d from 
(3 .5 mm-7 .5mm)fo r Amc and ( 5.5mm-9.5mm) 
fo r Tim, while The  s tand ard d ia meters  zo ne s 
of inhib ition we re  for Amc(14-21mm) and for 
Tim is(13 mm). The critica l normal MICs  of 
Augmentin and Time ntin were (4-16  g /ml), 
(1 28 g /ml) res pec tive ly. 
          The  minimum inhibitory concentra tions 
were stud ie d fo r ten c linica l isolates  of E.co li 
in c omp aris on with s ta nd ard res istant stra ins, 
TEM-1 - lac ta mas e co ded  for plasmid  R1 11, 
E.coli K12 S HV-1  - lacta mase  Pitton (type  II) 
co ded  for p la smid 45 3 I.p 7.7, E.cloac ae  P9 9 
ce pha lo sp orina se  I.p 8 .3  (France) a nd  E.co li 
ATCC 25 922  (Med ic al city hos pita l) se nsitiv e 
strains as  references , the  MICs of Cfm, Cfz, 
were very high, the ra nge  o f MICs  for Cfm 
was  5 12 - 20 48 g/ml fo r Cfz 64 - 32  g/ml. 
The se  res ults are indica te d in Table 4. 
 

Table  (4) Minimum Inhibito ry  
Conce ntra tions OF Four Ant ibiotic s 
To wards Te n Uropathoge nic  E.c oli 
Comparing  w ith Standard Strains  

*Isoe lectric  point , ** e phalosporinase 
 
 
Inh ibition of – lacta mase s  
          Figure 2 sho ws  c ompa ris ons  b etween 
antibiotic -e nzyme  interactions, by the  highly 
sensitiv e double disk tec hniq ue which 
demo ns trated  hydrolys is  of Cefta zidime and  
Cefama ndo l by – lacta mas e-p ro duc ing E.co li. 
The  e nzyme s ob ta ined fro m 10 s trains  
hyd ro lyzed , Ce fa mandol and  Ce fta zidime, b ut 
were highly s ta ble to  a ll – lactamase s teste d 
when c omb ined with potas sium c la vulanate . 
  Enzymes extracte d fro m E.coli standards  
harbo ring plas mid R1 11 TEM-1 o r S HV-1 
harbo ring pla smid R 4 53 -la ctama ses  we re  
inhibite d with p otass ium c la vula nate when 
comb ined with Amoxic illin or Ticarcillin( Fig 
3 A, B)  howev er -lac ta mas e of E.cloa cae  
was no t affected  b y Augmentin and  inhib ited  
by Tica rc illin and hydrolyze d a ll 
cep halos po rins  re pres ented  in Fig 4.  In 
contras t -la ctama se  unde r te st we re  highly 
re sistant to  Amo \ CA, Tic\CA, Fig 5 : sho w 
inhibition o f enzymes  b y io dometric method. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

No . of Is olate     

E.c oli Ce fa mandol Ce ftaz idime  

1,2 ,3 512 `64 

4,5 ,6 1 02 4 32 

7,8,9,10 2 04 8 32 

E.c oli(4 53)(24) 
SHV-1 (7.7 )* 

16 0 .0 5 

E.co li(R1 11) 
TEM-1 (5 .4 )* 

32 0 .0 5 

E.c lo acae (P 99 )**(8 .3 )*   

Figure  (1):  Io dome tric  Me thod fo r 
De te ctio n o f  -Lac ta mase  (Enz ymatic 
Re action at (4 C )No.1 , No.2 , No.3:  -
Lac tamase  from E.coli Clinical Is olates  , 
No .4 : TEM -1 -Lactamase  as 
Standard , No .5 :  - La ctamase  ne gative   

E.c oil ATCC 2 5922 . 



Ir aq i J.Pha rm.Sc i., Vol.15 (2 )  ,2006                  Cefamandol , Ce ftaz idime /Clavulanate Comb inatio n  
  

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SHV -1  (P453) 

 

 

 

 

 

 

 

 

 

 

 

 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
 
 
 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 

 
 
 
 
 
 
Direc t co njuga tion  method 
       It wa s fo und tha t c opies  o f the ge nes  for 
ampicillin, tica rc illin, te trac yc line , and  
chloramp he nicol resis ta nce  co uld be  
transferre d by direct conjuga tion me thod  fro m 
donor c ell to  rec ip ie nt c ell. The  res ults  we re  
pre se nted  a s fo llow: 
 

Figure (2 ):Antib io tic – e nz yme  inte rac tions, by 
the  highly  se ns itive  double  dis ks technique  (25) 
de monstrate d the  inhibition of  - la ctamases 
by  c lavula nic acid. A a nd B: 1  an d 2 -dis ks of 
Ce ftaz idime  (Cfz ) 30µg, and Ce famandol 
(Cfm)  30µg , 3 and 4 disk s at a  distance of 2  cm  
fro m Cfz  and Cfm impre g nate d with (10 µl) of  
e nzy me (- la ctamase )5 a nd 6  disks 
impre gnated with 1 0µl e nz yme  + 10µl  

Clav ulanic  a cid (CA). 
 

Figure (3) A : S usce ptibility disk   diff us io n 
me tho d s hows the  activ ity of Aug me ntin 
(Ame  ) and Time ntin (T ic ) Ag ainst 
plas mid – me dia te d   - Lac ta mase 
produce d by E.co li SHV – 1 Pitton type   

II( P45 3)(11) 

 

Figure  (3 ) B : Ac tion o f inhibitor c la vulan ic  
ac id (CA ) on the  ac tivity o f s tandard -
lactama se  e xtrac te d from E.coli SHV-1 Pitton 
type  1 : Ampicillin , 2:  - Lactama se  3 : - 

Lac ta mase+c la vulanic  acid. 
 

Figure  (4) :Hydrolys is of Ce phalosprins  by 
Ce phalo spo rina se  ( β –  Lactama se  ) , extracte d 
from E.cloa cae P99  ( According to  S irot ,  

D,1 983  ) 
 

Fig ure  (5):  Inhib it ion of   - Lactamase  o f  
Clinical Is olates  (E.co li )By  Mo difie d  
Iodo me tric Me thod Compa ring With 
S tanda rd Stra in o f E.co li ATCC 25 922    -  

Lactamase  Non Produce r 



Ir aq i J.Pha rm.Sc i., Vol.15 (2 )  ,2006                  Cefamandol , Ce ftaz idime /Clavulanate Comb inatio n  
  

15 

 

 
 
 
 
 
 
 
 
 
Ten s trains  tra ns fe rred  resista nce  to 
Tica rc illin ,Ceftazidime , and o ther a ntib io tics 
after mating for 1 8ho urs, tra nsc onjugants 
de rived from the se  s tra ins prod uced β-
lactama ses, the se  results  suggested  that a ll 
strains bear plas mids and p roduce extended 
spec trum β - lac ta mas escapab le  o f hydrolyzing 
and inactivating a wid e variety of β-lactams 
includ ing third generatio n 
ce phalosp orinsp enic illins and Aztreo nam , 
se ns itive to  imipe ne m  , these result was 
simila r to the s tudies  of Ro drigues  C , e t a l  , 
Cha udhary U ,a nd Kuro kawa ,-H et a l (26,27,28 )  .  
Conclus ions  
The  clinica l isola tes in this stud y we re  very 
resista nt to  Aug mentin , Ce fa mand ol and  
Ceftazid ime  c omp aring with s ta nd ard  TEM – 1 
and  SHV – 1 (pasmidic pe nc illinases ) , E. 
clocae  P99 is ve ry resista nt to Cefalothin , 
Cefama nd ol , Cefotaxime , Ce fta zidime 
standa rd strain which produce  - -lacta mase 
(ESBLs  ) e nzymes  tha t hydrolyze  a nd ca use 
resista nce  to oxyimino –  ce phalosporins and 
aztreo name . The ma jo rity of ES BLs are 
de rived from the widesprea d broad – 
spec trum-lac ta mases  TEM -1  and SHV-1 . 
ESBLs have be come  widesp read   thro ughout 
the world  a nd are now found in a  s ignific ant 
pe rcenta ge o f E.coli and Klebsiella 
pneumo niae  s tra ins in certain co untries (6,7,9,10) . 
The  increasing e mergence  cep ha lospo rins 
resista nt E.c oli has leaded  to co nc ern ab out the 
us e of v ario us  c omb inatio n therap y . A go od in 
– vitro respons e was  observed  in our clinical 
uropathogenic  E.co li when Cfm and  Cfz , 
were mixed with different conce ntra tion o f 
potass ium Clav ulanate as inhibitor the y w ere 
effe ctive and safe fo r the trea tme nt o f UTIs 
ca used b y -lactama ses (Ceftazidima se  ) 
producing comp lica te d strains  (22,23,24,28) .  
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     E.c oli   wild type       AmpR      TicR   CfzR    CmR      Tc R     NalR RifS  o r Nal S    Ri fR  
    E.c oli) K12 C 600  (sta nda rd ) Amp

s    Tics       Cms    Tcs    Na lR RifS   o r Nal S    Ri fR 
   E.coli   K12 C 600 (Tra nsconjuga nt) Amp

R   TicR  CfzR      NalR     or    RifR      
   E.coli   K12 C 600 (Tra nsconjuga nt) Amp

R   TicR                 NalR     or    RifR      
  E.co li   K12 C 60 0(Transco njuga nt Amp

R   TicR    CmR                    NalR     or    RifR           
  E.co li   K12 C 60 0(Transco njuga nt Amp

R     CmR                   NalR     or    RifR   
  E.co li   K12 C 60 0(Transco njuga nt Amp

R    Tic R CmR   CfzR            NalR     or    RifR        
  E.co li   K12 C 60 0(Transco njuga nt Amp

R    Tic R TcR   CfzR          NalR     or    RifR      



Ir aq i J.Pha rm.Sc i., Vol.15 (2 )  ,2006                  Cefamandol , Ce ftaz idime /Clavulanate Comb inatio n  
  

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