Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5thInternational Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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COMPARISON OF AEROKEYII SCHEME AND API20E
SYSTEM FOR IDENTIFICATION OF AEROMONAS
HYDROPHILA ISOLATED FROM WELLS WATER IN THIQAR
PROVINCE-IRAQ
Muslim Dhaher Musa* , Waffa Abdulelah Ahmed* *
* Department of Nursing , Technical Institute , AL-Nasiryah , Southern Technical
University, Thi-Qar , Iraq
* * Department of Microbiology , College of Veterinary Medicine , University of
Baghdad, Baghdad , Iraq
Key words : A.hydrophila , Wells, AerokeyII.
ABSTRACT
Untreated water is well-known source of A.hydrophila which is in addition to its enteropathogenic
potential ,also display resistance to commonly used antibiotics , so this study aimed to compare Aerokey
II and API20E in identification of A.hydrophila with studying of some virulence factors and
antibiogram profile in untreated wells water in Thi-Qar province. Isolation was conducted by employing
Ampicilin Blood Agar (ABA30) and MacConkey agar medium, the suspected colonies were identified
by using biochemical scheme (AerokeyII) , and API20E . The results of this study revealed that
A.hydrophila was recovered from 8 out of 30 wells with incidence rate (27.6%), incidence variation was
noted among different regions which was statistically significant . All isolates showed β-hemolysis of
human erythrocytes , 75% have proteolytic activity and 50% of isolates were DNase positive. Results
of Antibiogram analysis revealed that all isolates exhibit resistance in percentage (100%) to five
antibiotics including ;Clindamycin , Cephalothin , Vancomycin , ticarcilin-clavulanic acid , Ceftazidime,
and the resistance to Cefoxitin was 75%, while all isolates were (100%) susceptible to Gentamicin ,
Amikacin, Chloramphenicol, Ofloxacin ,Ciprofloxacin, Naldxic acid, Imipenem , Norfloxacin, and
Deoxycyclin. The susceptibility to Ceftriaxone was 62.5% , Streptomycin 87.5% and Trimethoprime
87.5% . The study concluded that , high correlation between Aerokey II and API20E in identification of
A.hydrophila and untreated wells water are an important source of multi-drug resistant enteropathogenic
A.hydrophila which pose public health threat especially to individuals using this kind of water source .
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INTRODUCTION
Members of genus Aeromonas are roughly categorized into two groups based on
their motility and growth temperature ; motile mesophilic and non-motile
psychrophilic (1). Members of the mesophilic group have been implicated in
intestinal and extra-intestinal infections ranging from acute gastroenteritis to lifethreatening
cases such as septicemia , necrotizing fasciitis and myonecrosis (2).
Among the species belonging to this group , A. hydrophila has gained much interest
due to its frequent association with human gastroenteritis ,which is occurring mainly
in young, elderly and immunocompromised people (3).Untreated water is the most
important source of Aeromonas infection , and these bacteria are commonly found
in such water sources in developing countries(4). A.hydrophila has the ability to
express a number of extracellular enzymes and toxins including protease; lipase,
DNase, hemolysins and enterotoxins which have been considered as virulence factors
(5). In early studies, it has been shown that enteropathogenic potential of
A.hydrophila could be investigated in relation to their phenotypic markers including
; hemolysis of human red blood cells and Voges-Proskauer which are associated well
with production of enterotoxins (6).
Multi-antibiotic resistant has been frequently identified in A.hydrophila more than
other Aromonas species (7) .Much concern was paid for contribution of untreated
water sources as path for dissemination of antibiotic resistance bacteria to humans
and animals(8,9,10). Periodic monitoring of antibiogram profile of these bacteria in
different geographical areas and from different sources is required for appropriate
choice of antimicrobial agent for perfect therapy(11). According to our best
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Proceeding of 5thInternational Scientific Conference,College of Veterinary Medicine
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knowledge no previous study concerning the incidence and antibiogram profile of
A.hyrophila isolated from well water in south of Iraq especially in Thi-Qar province
, so this study aimed to isolate and identify A.hydrophila from well water in Thi-Qar
province and to determine some virulence factors that are important in
pathogenicity (phenotypically) with studying the antibiogram profile.
MATERIALS AND METHODS
Sampling
Ten milliliters (10 ml) of water sample was collected from thirty wells distributed
in five regions (Shatrah, Nassiryah, Sukh-alshuikh, Said –Dukhail, and Batha)
throughout Thi-Qar province .
Isolation and Identification of A.hydrophila
Water sample (10 ml) was aseptically added to 90 ml of Alkaline peptone water
broth (APW) PH=8.6, and incubated at 37C ͦ for 24 hours , then a loopful from
growth film above APW broth was streaked on Ampicillin Blood Agar 30 and on
MacConkey agar , all plates were incubated at 37C ͦ for 24 hours. A.hydrophila
identification was conducted based on biochemical scheme(AerokeyII) proposed by
(12), and confirmed by API20E
Detection of virulence factors
The ability of isolates to hemolyze red blood cells was investigated on blood agar
by observing hemolytic zone around colonies after 24 hours at 37C ͦ .
Protease activity was tested skimmed –milk agar plates . A loopful of an overnight
growth from ABA30 was streaked on prepared skim milk agar plates , incubated at
37°C for 24 hours. The clear zone around colonies considered a positive result
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DNase test was performed by inoculating bacterial culture on DNase medium
(Oxiod) and incubated at 37C ͦ for 24 hours . The positive result indicated by the
presence of clear zone around the bacterial growth after adding HCL 0.1 N (13).
Antibiogram
Antibiotics sensitivity test was carried out according to Kirby-Bauer method using
Mueller-Hinton agar . Following antibiotics discs were used , Gentamicin (10μg) ,
Amikacin (30μg), Streptomycin (10μg), Cholamphincol (30μg), Ofloxacin (5μg),
Norfloxacin(10μg), Ciprofloxacin (5μg), Clindamycin(2μg), Nalidixic acid (30μg),
Cephalothin (30μg), Cefoxitin (10μg), Ceftazidime (5μg), Ceftriaxone (30
μg),Doxycyclin (30μg),Vancomycin(30μg),Aztreonam(30μg), Trimethoprim-
Sulfamethoxazole, Imipenem (10μg),and Ticarcillin-clavulanic acid (75/10 μg). The
results of Antibiogram were interpreted based on criteria published in standards of
the Clinical Laboratory Standard Institute Guidelines(CLSI,2011) (14) and the
isolates were reported as susceptible, intermediate or resistant.
Multi-drug resistance index (MDRI) was calculated according to formula (15) :
MDRI=
Where (A) the number of antibiotics to which the isolates showed resistance , (B)
total antibiotics to which the isolates were exposed .
Multi-drugs resistance index (0.2) was considered as cut-off values in that value
equal or higher than (0.2) was considered to have high risk source were antibiotic are
often used , while value less than (0.2) indicated that strains are originated from
source where antibiotics never used .
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RESULTS
A.hydrophila recovered from 8 out of 30 (26.7%) wells selected from five regions
that covering entire Thi-Qar province , incidence was differed among the five
regions enrolled in this study, that the highest incidence 4(66.7%) was recorded in
Batha region followed by 2(33.4%) in Sukh-Alshuikh and 1(16.7%) in each of
Shatrah and Nassiryah ,while no A.hydrophila could be isolated from Said Dukail
wells water table (1).
All isolates were able to grow on ABA30 with smooth white to buff color
surrounded with β-hemolysis after 24 hours of incubation at 37C ͦ , indicated that all
isolates were resistance to ampicillin and able to hemolysis human red blood cells
.On MacConkey agar plates, all isolates produce large colorless colonies indicated
that all isolates were unable to ferment lactose figures (1 and 2 ).
Regions No of
samples A.hydrophila Percentage
Shatrah 6 1 16.7%
Nassiryah 6 1 16.7%
Sukh-alshuikh 6 2 33.4%
Said –Dukhail 6 0 0%
Batha 6 4 66.7%
Total 30 8 26.7
X2
P-Value
12.589
(p<0.05)
Table 1: prevalence of A.hydrophila isolated from wells water
Figure(1): Colorless colonies of A.hydrophila
on MacConkey agar plate
Figure(2):Growth of A.hydrophila on ABA30,
with β-hemolysis
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The results of AerokeyII biochemical scheme were presented in table (2). All
isolates were positive for Indole , Citrate utilization , and fermentation of glucose and
oxidase , while all isolates were negative for string test and resist O/129 which are
the key tests for differentiating Aeromonas from Vibrio . All isolates were , esculin
hydrolysis ,
produced acid from arabinose and resistant to Cephalothin, motile ( hanging drop ) ,
and majority of isolates were Voges-Proskauer positive.
Test Reaction No postive (%)
Growth on ABA30 + β-hemolysis 8/8 (100%)
Indol + 8/8 (100%)
Citrate utilization + 8/8(100%)
Glucose fermentation with gas + Gas production 8/8(100%)
Oxidase + 8/8(100%)
String - 8/8(100%)
Resistance to O/129 + Resistance 8/8(100%)
Motility + 8/8(100%)
Esculin hydrolysis + 8/8(100)
Voges-Proskauer + 6/8 (75%)
Acid from arabinos + 8/8(100%)
Resistance to Cephalothin 30μg + Resistant 8/8(100%)
Table (2) Results of biochemical tests for identification of A. hydrophila
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A.hydrophila were also subjected to API20E (analytical profile index , bioMérieux
) and results of current study indicated that biochemical tests in Aerokey II are
highly discriminative and are excellent in identification of A.hydrophila to species
level as all positive isolates were also identified by API20E as A.hydrophila
Detection of virulence factors revealed that all isolates in the current study were
Virulence factor
Region
No of
isolates
Protease DNase Hemolysis
Shatrah 1 1(100%) 1(100%) 1(100%)
Nassiryah 1 1(100%) 1(100%) 1(100%)
Sukh-alshuikh 2 1(50%) 0(0%) 2(100%)
Said –Dukhail 0 --- ---- ----
Batha 4 3(75%) 2(50%) 4(100%)
Total 8 6(75%) 4(50%) 8(100%)
Figure (3) shown API20E (analytical profile index) result of A.hydrophila
identification according of API20E data
Table (3): Virulence factors of A.hydrophila from wells water in different regions
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able to express virulence factors , that all isolates were β-hemolytic on blood agar
regardless of region of isolation , and 75% of these isolates were positive for
protease test and 50% were positive in DNase test ,figures (4 and 5). A.hydrophila
isolates from Shatrah and Nassiryah wells water were expressing the three virulence
factors ,while the isolates from Sukh-alshuikh showed no DNase activity and only
one isolate was expressing protease activity , while three isolates from Batha were
protease positive and two isolates were DNase positive , table (3).
Results of antibiogram profile analysis presented in table (4) revealed that ,
Aminoglycosides ( Gentamicin, Amikacin) ,Quinolone and Flouroquinolones,
Carbapenem, Doxycyclin and Chloramphenicol were absolutely active against
A.hydrophila as all isolates were (100%) susceptible in this study , also Streptomycin
and Trimethoprim-Sulfamethoxazol have excellent activity ( ≥ 80% of isolates were
susceptible ) while Certiaxon had moderate susceptibility (60% of isolates were
susceptible ) and low susceptablitiy (≤30% of isolates were susceptible ) was
recorded for Cefoxitin .
Figure (4): DNase activity of A. hydrophila Figure (5) protease activity of
A.hydrophila on Skimed-milk agar
Table (5): Antibiogram Profile of A.hydrophila Isolated From Wells Water
Table (4): Antibiogram profile of A.hydrophila
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On the other hand absolute resistance (100%) of A.hydrophila was observed
against five antibiotics : Ticacillin-clavulnic , Ceftazidime, Vancomycin,
Cephalothin, and Clindamycin, while the resistance to Cefoxitin was 75% . In this
study two isolates were (25%) resistant to at least five antibiotics with multi-drug
resistance index MDRI(0.26) and six isolates were (75%) resistant to six antibiotics
with MDRI(0.31).
Discussion
Antibiotics
Potency
1 2 3 4 5 6 7 8
Diameter of zone
Susceptibilit
y (%)
R I S
Gentamycin 10 S S S S S S S S 12 13-14 15 100%
Cholamphincol 30 S S S S S S S S 12 13-17 18 100%
Ofloxacin 5 S S S S S S S S 12 13-15 16 100%
Amikacin 30 S S S S S S S S 14 15-16 17 100%
Clindamycin 2 R R R R R R R R 14 15-20 21 0%
Naldxic acid 30 S S S S S S S S 13 14-17 18 100%
Streptomycin 10 S S I S S S S S 12 13-16 17 87.5%
Cephalothin 30 R R R R R R R R 14 15-17 18 0%
Norfloxacin 10 S S S S S S S S 14 15-18 19 100%
Vancomycin 30 R R R R R R R R - - 17 0%
Doxycyclin 30 S S S S S S S S 10 11-13 14 100%
Aztreonam 30 I I I I I R I I 15 16-21 22 --
Cefteriaxon 30 S I S I S S S I 13 14-20 21 62.5%
Trimethoprimsulfamethaxazon
S I S S S S S S 10 11-15 16 87.5%
Imipenem 10 S S S S S S S S 13 14-15 16 100%
Ticacillinclavulanic
75
/1
0
R R R R R R R R 14 25-19 20 0%
Ciprofloxacin 5 S S S S S S S S 15 16-20 21 100%
Ceftazidime 5 R R R R R R R R 14 15-17 18 0%
Cefoxitin 10 R S R R R S R S 14 15-17 18 25%
MDRI 0.31 0.26 0.31 0,31 0.31 0.31 0.31 0.26
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Exposure of individuals to water contaminated with Aeromomas sp has been
reported to be an important cause for human diarrheal disease , as these bacteria able
to produce enterotoxins , cytotoxins, and hemolysins (2) .
The results of this study in concerning incidence of A.hydrophila in wells water
was (26.7%) convergent to those reported by previous studies including ; ( 59% ) in
Libya reported by (16) and (48%) reported by (17) and 50% reported by (18) in Iraq
.
The low incidence rate of A.hydrophila reported in current study could be
attributed to isolation procedure as they used lower Ampicillin concentration
15mg/L incorporated in blood agar (30 mg/L in recent study) ,or due to limited
number of wells enrolled in this study or due to investigation the prevalence of
genus Aeromonas without performing species identification (17) however, the results
of this study were in concordance with the results obtained by (19) and (20) in
brazil as they found A.hydrophila incidence in well water was 22.5% and 21.9%
respectively, and agreed with (21) who found the incidence of A.hydrophila in well
water (27%) and 28% reported by (22) . In this study there was variation in
prevalence of A.hydrophila from wells water among five regions enrolled. variation
in recovery of Aeromonas from different geographical locations was documented by
(23).
Identification of Aeromonas to species level based on phenotype is necessary
since there are differences in clinical importance , antibiotics susceptibility and
epidemiology of different species of Aeromonas (24) . In this study A.hydrophila has
been identified to species level by using a set of highly discriminatory biochemical
scheme (AerokeyII), that have been proposed by (12),by this scheme he was able to
identify 97% of Aeromonas to species level and 100% of reference strains . The
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results of this study found that Aerokey II is an excellent method for identification
of A.hydrophila , this result was compatible with, (25) and (24) who found that API
system alone cannot identify Aeromonas to species level unless this system used in
combination with Aerokey II .
Some researchers have stated that the hemolytic activity , or hemolytic activity
with voges-Proskauer positive , act as a good indicator of ability of these bacteria to
produce enterotoxins ( 6,26) . According to this criteria , majority of isolates in this
study were enterotoxigenic , as 100% of isolates were β-hemolytic and 75% of
isolates were V-P positive , this result was agreed with (26,27,28) .
In this study all isolates (100%) were multi-drug resistance with MDRI ranging
from 0.26-0.31 , Multi-drug resistance phenotype among Aeromonas spp has been
reported from different regions throughout the world , particularly in A.hydrophila
(10). The results of this study were agreed with (29,30,31). The resistance of all
isolates to Ticarcillin/clavulanic acid and cephalothin was not unexpected because
A.hydrophila classically resistant to Beta –lactam antibiotics, this resistance can be
explained in a part that Aeromonas spp. isolates are resistant to many β-lactams as a
result of multiple inducible, chromosomally-encoded β-lactamases which are under a
single mechanism of coordinate expression (31), and among Aeromonas spp, only
A.hydrophila express four classes of -lactamases A;B ,C, and D class(32) , the
resistance of A.hydrophila to many β-lactams antibiotics including ampicillin is a
significant public health concern , as this property may act as predisposing factor for
gastroenteritis , since ampicillin inhibiting competing microflora in intestine, that is
benefit for A.hydrophila to proliferate and producing toxins (33).
Our study indicated Aminoglycosides ( Gentamicin, Amikacin) ,Quinolone and
Flouroquinolones, Carbapenem, Doxycyclin and Chloramphenicol were absolutely
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active against A.hydrophila, this finding was in agreement with previous studies
(11,29, 30, 31). In this study we have isolated A.hydrophila from wells water , that
have the ability to express virulence factors and majority of these isolates were
enteropathogenic and expressed resistance to most commonly used antibiotics
including the third generation of cephalosporins , this may constitute an important
health risk to population that used untreated water sources for daily usage .
فی (API20E) و أشرطة الفحص السریع (AerokeyII) مقارنة المخطط التشخیصی
تشخیص الایروموناس ھایدروفیلا المعزولة من میاه الآبار فی محافظة ذی قار – العراق
مسلم ظاھر موسى* ، وفاء عبد الالھ احمد **
*قسم التمریض ، المعھد التقنی /الناصریة ، الجامعة التقنیة الجنوبیة ، العراق
** فرع الأحیاء المجھریة ، کلیة الطب البیطری ، جامعة بغداد ، العراق
الخلاصة
تعتبر المیاه الغیر معاملة مصدر مھم لبکتریا الایروموناس و التی تعتبر بکتریا معویة ،ولھا القدرة على
مقاومة العدید من المضادات الحیویة شائعة الاستخدام ، لذا ھدفت ھذه الدراسة إلى مقارنة المخطط التشخیصی
فی تشخیص ھذه البکتریا و دراسة بعض عوامل (API20E ) و أشرطة الفحص السریع (Aerokey II)
الضراوة و نمط مقاومة المضادات الحیویة لھذه البکتریا فی میاه الآبار فی محافظة ذی قار . استخدم وسط اکار
الدم المزود بالامبسلین بترکیز 30 ملغرام/لتر و وسط المکونکی لعزل البکتریا و استخدم المخطط
فی التشخیص . وجدت الایروموناس ( API20E) و أشرطة الفحص السریع (ArokeyII) التشخیصی
ھایردوفیلا فی 8 أبار من أصل 30 بئر بواقع ( 27.6 %) کذلک أظھرت ھذه الدراسة اختلاف فی نسبة العزل
بین المناطق و بفرق معنوی عند مستوى المعنویة ( 0.05 ). و فیما یخص عوامل الضراوة فقد بینت ھذه
الدراسة أن کل عزلات الایروموناس ھایروفیلا ( 100 %) کانت قادرة على تحلیل الدم باستخدام وسط اکار الدم
اظھر فحص المقاومة للمضادات . DNase و 75 % منتجة لانزیم البروتیز و 50 % قادرة على انتاج انزیم
Clindamycin , : الحیویة أن کل العزلات ( 100 %) کانت مقاومة لخمسة انواع من المضادات الحیاتیة وھی
وکانت نسبة المقاومة Cephalothin , Vancomycin , ticarcilin-clavulanic acid , Ceftazidime
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Gentamicin , %75 . بینما کانت جمیع العزلات( 100 %) حساسة للمضادات الحیاتیة Cefoxitin لل
Amikacin, Chloramphenicol, Ofloxacin , Ciprofloxacin, Naldxic acid, Imipenem ,
Streptomycin 62.5% ) و ) Ceftriaxone و کانت الحساسیة لل Norfloxacin, and Deoxycyclin
من خلال نتائج ھذه الدراسة نلاحظ وجود توافق بین التشخیص بکلا . Trimethoprime 87.5% و % 87.5
الطریقتین و کذلک تعتبر الآبار مصدر مھم لبکتریا الایروموناس ھایدروفیلا التی لھا القدرة على إظھار مقاومة
متعددة للمضادات الحیاتیة و القادرة على أنتاج السموم المعویة و التی تشکل خطر على الصحة العامة
خصوصا بالنسبة للأشخاص الذین یعتمدون على ھذا النوع من مصادر المیاه .
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