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ISOLATION AND MOLECULAR IDENTIFICATION OF MDR
Pseudomonas aeruginosa FROM ANIMALS AND PATIENTS IN
BASRAH PROVINCE
Ghasaq K. Shamkhi, Bassam Y. Khudaier*
Microbiology Department, Veterinary Medicine College, Basrah University. Basrah, Iraq
(Received 7 March 2020, Accepted 11 May 2020)
Key words: Molecular Identification, Pseudomonas aeruginosa, Animals, Patients.
*Corresponding author: bassamy10@yahoo.com
ABSTRACT
Throughout the period from October 2018 to February 2019, 278 test samples were
collected from animals and human, (55%) animal samples which are distributed to (52.3%)
swab samples were from the environment of slaughters and (47.7%) milk samples were from
cow and buffalo which collected in sterile containers. The result showed that Pseudomonas
was found in (44%) samples on pseudomonas agar distributed in (24%) samples from
slaughters, (20%) samples from milk. (45%) human samples that are distributed to (48%)
swab samples were from diabetic foot patients and (52%) swab samples were from patients
suffering from burns in hospitals of Basrah province. The results showed that Pseudomonas
was found in (56%) samples on pseudomonas agar, (18%) samples from diabetic foot and
(38%) samples from patients suffering from burns. 46 isolates were identified using VITEK 2
Kit. 25 samples identified as Pseudomonas aeruginosa which presented (54%).
Antimicrobial susceptibility testing of 31 P. aeruginosa isolates compared to 13 different
antibiotics was done by the disk diffusion method. Completely isolates were resistant to as a
minimum of 8 antibiotics; they exhibited the form of the multiple resistance to the
antibiotics. Thirty-eight samples were tested for 16S rRNA by conventional PCR assay, 19
from animal sources and 19 from patients' sources. 18 animals and 19 patient samples were
demonstrated distinct bands with approximately 618bp corresponding for P. aeruginosa.
INTRODUCTION
During the history, an essential cause of morbidity and mortality is the infectious
diseases, by the development of the antibiotic history through the twentieth century; there
was a growing confidence that the need for infectious disease specialists would all disappear
(1). P. aeruginosa, like many other nonfermenting gram-negative rods, is a saprophytic
organism widespread in nature, particularly in moist environments (water, soil, plants, and
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sewage), and endowed with only weak pathogenic potential. However, because of its ability
to survive on inert materials and its resistance to most antiseptics and antibiotics, P.
aeruginosa has become an important and frequent nosocomial pathogen. Indeed, in hospitals,
sinks, respiratory therapy equipment, and antiseptic or detergent solutions can act as
reservoirs of P. aeruginosa (2).
P. aeruginosa is an essence of opportunistic pathogen of human. It causes an
infection in urinary tract, dermatitis, infection in soft tissue, bacteremia, respiratory infection,
infection of bone and Joint, gastro-intestinal infections, particularly it causes different types
of infection such as in people suffer from AIDS, Immunocompomised conditions, burns and
cancers and the other diseases. The bacterium causes pneumonia, chronic lung infections,
endocarditic and septicemia. From 10.1% of all nosocomial contaminations, this bacterium
was the 4th utmost frequently isolated nosocomial pathogen reason (3).
P. aeruginosa has increasing veterinary importance, it is a relevant cause of bovine
mastitis, and in diverse animal species it was make various localized infections. The nonclinical
strains of P. aeruginosa from animals and environment have acquired much less care
and rare genetic investigation is identified on bovine strains (4).Raw milk is widely used up
from olden times and the need for it is constant all over the world. Milk has a high nutrition
importance so is considered a major nutrition for human. It is naturally a respectable medium
for growing of microbes. Quality control of raw milk is an essential importance (5).
Defaulting of sanitized condition such as bulk tank unsuitable cleaning, dirty udders,
equipment of milking, milk handling technique and inappropriate storing will raise the
amount of Gram-positive and Gram-negative bacteria in the bulk tank milk (6). The bacteria
in the family Pseudomonadaceae are among the most main spoilage bacteria emerging in
chilled raw milk. They are reflected psychrotrophs, growing well at communal refrigeration
temperatures (0-15°C). The main psychrotrophic micro flora come across in raw milk are
Gram negative rods with Pseudomonas spp. (7) which forms at least 50% of all bacteria in
milk (8).
P. aeruginosa is a multidrug-resistant common opportunistic pathogen with much
importance in medical and veterinary side. This bacteria is a communal causal agent of local
infections in humans, and it is a source of severe infections in an immune-compromised
patients and causes mortality in patients with cystic fibrosis (CF) (9). The worldwide
occurrence of multi-drug resistant bacterial strains in hospitals and communal stays to be a
problematic of due scientific anxiety, particularly contaminations caused by Pseudomonas
species and P. aeruginosa in particular. P. aeruginosa is an opportunistic pathogen with
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intrinsic resistance to various antibiotics and disinfectants including anti-pseudomonal
Penicillins, Ceftazidime, Carbapenems, Aminoglycosides and Ciprofloxacin (10).
It has necessary to develop genotype-based characterization systems which are
capable of exactly detecting these bacteria in spite of any phenotypic alterations, DNA
marker permits rapid identification of species, among DNA markers. The Polymerase Chain
Reaction (PCR) is greatly sensitive specific and fast technique which improve the recognition
of P. aeruginosa especially with using specific primer for 16S rRNA (11).
Therefore, the aim of this study was detect the presence of P. aeruginosa in animal
and human subjects using culture, biochemical tests, VITEK and PCR. The other goal is to
test the susceptibility of the isolated strains to commonly used antibiotics.
MATERIALS AND METHODS
Collection of specimens
A complete of 278 samples were collected from October 2018 to February 2019. One
hundred fifty three (55%) from animals, of which (52.3%) swabs were from the environment of
slaughters and (47.7%) samples were from milk of cow and buffalo collected by plastic sterile
containers, from different areas in Basrah city (Abu-Sukhair, Slaughter of Basrah). One hundred
twenty five (45%) samples were collected by sterile swabs with media. (52%) swabs from
patients suffering from burns and (48%) swabs from diabetic foot wounds of both genera
from Al- Fayhaa hospital, Basrah province. All specimens were labeled and transported
within two hours by a sterile samples collection container to the laboratory then cultured in
brain heart infusion broth (BHIB).
Culturing of specimens
The specimens were incubated at 37ºC for 24 hrs. The growth then transferred to
MacConkey agar plates and incubated at 37°C for 24 hrs to distinguish between the
fermenting and non-fermenting lactose bacteria. The colonies from primary cultures were
further purified by re-culture into pseudomonas agar and incubated at 37ºC for 24 hrs.
Identification of P. aeruginosa
Preliminary identification of the pure colonies by Gram staining, biochemical tests:
catalase test, oxidase test, and kliglar iron agar (12), Then the results were further confirmed
with the VITEK 2 Kit.
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Antimicrobial susceptibility test
According to NCCLS (13), The antimicrobial sensitivity of the identified isolates was
detected by disc diffusion method of 13 different antibiotic with their indicated
concentrations: Ampicillin (30μg), Amikacin (30μg), Cefotaxime (30μg), Ciprofloxacin
(5μg), Gentamycin (10μg), Ceftazidim (30μg), Imipenem (10μg), Tetracycline (30μg),
Trimethoprim (5μg), Meropenem (10μg), Levofloxacin (15μg), Nalidixic acid (30μg),
Amoxicillin (30 μg), The Mueller-Hington (MH) agar plates were covered with P.
aeruginosa growth after standardizing the inoculum turbidity with 0.5 McFarland
standards(14). The diameters of inhibition zones were measured after 24 hrs with ruler.
Detection of P. aeruginosa by molecular technique
DNA extraction and purification
The DNA of P. aeruginosa isolates was extracted and purified according to the company
instructions (Geneaid, Lot No. FC26108-G/ Korea).
DNA amplification by PCR:
Amplification of 16S rRNA gene was conducted using specific primers table 1, achieved
on thermocycler instrument (Techne, UK). The total volume of the reaction mixture was 50
μl (Table 2) with the reaction condition (Table 3).
Agarose gel electrophoresis
Agarose gel was prepared according to Sambrook et al., (15). The agarose gel was
prepared in concentration of (1.5%).
Table 1: Oligonucleotide primer sequence and PCR product size for 16S rRNA gene
amplification in P. aeruginosa isolates.
Gene Primer Sequence
MW
(bp)
16S rRNA
F: 5'-GACGGGTGAGTAATGCCTA-3
R: 5'-CACTGGTGTTCCTTCCTATA-3'
618
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Table 2: Mixture of PCR (50 μl) for amplification of 16S rRNA gene in P. aeruginosa
isolates.
Reagents
Reaction Volume
(50 μl)
F Primer 2
R Primer 2
DNA template 10
Nuclease-free water 36
Total 50
Table 3: The conditions of PCR assay for amplification of 16s rRNA gene in P.
aeruginosa isolates.
Steps Temperature (C˚) Time (min) No. of Cycles
Initial denaturation 95 2 1
Denaturation 95 1
Annealing 55 1 30
Extension 72 1
Final extension 72 5 1
RESULTS
In the present study, out of 278 tested samples, (55%) samples from animals, of
which (52.3%) were from the environment of slaughters and (47.7%) samples were from
milk of cow and buffalo. (45%) swabs from patients, of which (48%) samples were from
diabetic foot patients and 65 (52%) samples were from patients suffering from burns in
hospitals of Basrah province (Table 4 and 5).
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Table 4: Prevalence of P. aeruginosa isolated in the animals and environment of
slaughter.
Source of samples No. of Sample No. of
P. aeruginosa (%)
Environment of slaughter 80 12 24
cow's and Buffalo milk 73 10 20
Total 153 22 44
X2=0.053
No Significant different at p > o.o5
Table 5: Prevalence of P. aeruginosa isolated from burns wounds and diabetic foot
patients.
Sample Source No. of Sample No. of
P. aeruginosa (%)
Diabetic foot swab 60 9 18
Burns swab 65 19 38
Total 125 28 56
X2=3.635
No Significant different at p > o.o5
The present study results showed that from 278 collected samples only (55 %) were
non lactose fermenters when samples plated on MacConkey agar distributed in (56.3%)
samples from slaughter, (49.3%) milk samples, (70%) samples from burns and (45%)
samples from diabetic foot patients. Samples were plated on the selective pseudomonas agar
medium to isolate Pseudomonas spp., which had the ability to grow on this medium. P.
aeruginosa produced non fluorescent bluish pigment (pyocyanin) which diffused into the
agar. Only 50 (32.5%) samples out of (55%) showed positive result on pseudomonas agar
distributed in (24%) samples from slaughters, (20%) samples from milk, (18%) samples from
diabetic foot and (38%) samples from burns. Forty-six isolates of these positive
pseudomonas were identified using VITEK 2 Kit. The result showed that (54%) samples
identified as P. aeruginosa, Table (6).
Identification of P. aeruginosa
Identification of P. aeruginosa was achieved by examining bacterial culture
(MacConky agar and Pseudomonas agar), microscopically characteristic (Gram's staining)
and their biochemical reactions (catalase, oxidase, kliglar iron tests) (Table 6).
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Table 6: Biochemical tests of P. aeruginosa isolates from different sources.
Sample type
Oxidase
(%)
Catalase
(%)
Kliglar iron
(%)
VITEK2
(%)
Cow's and
buffallo milk
12/12 (100) 12/12 (100) 3/12 (25) 8/16 (50)
Slaughters 12/12 ( 100) 12/12 (100) 4/12 (33.3) 1/4 (25)
Burns wound 18/18 (100) 18/18 (100) 9/18 (50) 10/16 (62.5)
Diabetic foot
wounds
27/27 (100) 27/27 (100) 10/27 (37) 6/10 (60)
Total 100/100 100/100 38/100 54/100
X2 = 2.086 X2 = 2.068
No significant difference at p>0.05
VITEK2 test showed the highest percentage. (62.5%) of isolates from burn samples,
(60%) of isolates from diabetic wounds followed by cow's and buffalo milk (50%), and
(25%) from slaughter.
Antibiotic susceptibility testing of P. aeruginosa isolated from animals.
This study demonstrated that the results of 13 various antibiotics by disc diffusion
method to wards to 31 P. aeruginosa isolates, using Kirby-Bauer disk diffusion method that
is determined through measuring the diameter of inhibition zones around antibiotic discs
affording to CLSI (2007).
The results showed that 100% of P. aeruginosa isolates were resistant to Ampicillin,
Amoxicillin and Cefotaxime, 97% were resistant to Ceftazidime and Nalidixic acid, 77.4%
were resistant to Meropenem, 42% were resistant to each of Ciprofloxacin and
Trimethoprim, whereas 87% of P. aeruginosa isolates were sensitive to Imipenem, 71% were
sensitive to Amikacin, 67.7% were sensitive to Gentamicin, 61% we
re sensitive to Tetracycline, and 58% were sensitive to Levofloxacin, (Figure 1)
(Table 7).
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Figure 1: Inhibition zones of some antibiotics against P. aeruginosa by disc diffusion
method.
Table 7: Antibiotic susceptibility test to 13 antibiotics by disc diffusion test against 31 P.
aeruginosa isolates.
*R: Resistant; I: Intermediate; S: Susceptible.
No. Antibiotic
Con.
(mcg)
No. of isolates
R (%) I (%) S (%)
1 Amoxicillin (AX) (30) 31 (100) - -
2 Amikacin (AK) (30) 7 (22.5) 2 (6.5) 22 (71)
3 Ampicillin (AM) (30) 31 (100) - -
4 Ceftazidime (CAZ) (30) 30 (97) - 1 (3)
5 Cefotaxime (CTX) (30) 31 (100) - -
6 Ciprofloxacin (CIP) (5) 13 (42) 4 (13) 14 (45)
7 Gentamicin (CN) (10) 9 (29) 1 (3.2) 21 (67.7)
8 Imipenem (IPM) (10) 3 (9.7) 1 (3.2) 27 (87)
9 Levofloxacin (LEV) (15) 6 (19.4) 7 (22.6) 18 (58)
10 Meropenem (MEM) (10) 24 (77.4) - 7 (22.6)
11 Nalidixic acid (NA) (30) 30 (97) 1 (3) -
12 Tetracycline (TE) (30) 8 (25.8) 4 (12.9) 19 (61)
13 Trimethoprim (TMP) (5) 13 (42) 8 (25.8) 10 (32.3)
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Molecular identification by PCR assay:
Detection of 16S rRNA gene in animals.
Nineteen P. aeruginosa isolated from animals were subjected to PCR assay to detect
16s rRNA gene, Eighteen isolates exhibited clear bands of approximately 618bp which
corresponding for detection of P. aeruginosa strains.
Detection of 16S rRNA gene in patients.
Nineteen P. aeruginosa isolates from patients were subjected to PCR assay to detect
16S rRNA gene. The results showed that 100% isolates exhibited clear bands of
approximately 618bp which corresponding for identification of P. aeruginosa strains (Figure.
2).
Figure 2: Agarose gel electrophoresis of PCR-amplified 16S rRNA gene of P. aeruginosa
isolates. Lane M: DNA marker; Lane 1-6: 16S rRNA gene (618bp); and Lane C:
Negative control.
DISCUSSION
In the present study, Brain heart infusion broth was applied as supplement medium
whereas MacConkey agar and pseudomonas medium were applied to detect and isolate P.
aeruginosa. (55%) P. aeruginosa isolates were obtained from 278 from animal samples, of
which (52.3%) swabs were from the environment of slaughters and (47.7%) samples were
from milk of cow and buffalo collected in sterile containers. (45%) samples from patients, of
which (52%) swabs were from patients suffering from burns and (48%) swabs were from
diabetic foot patients in hospitals of Basrah province. Moreover, out of 18 P. aeruginosa
cases, 8.6 % were under Wagner,s Grade III ulcer. The percentage of P. aeruginosa in the
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present study were the topmost infections in comparison with the rate in the study of
Hutschinson and McGuckin (16). The uppermost ratio of P. aeruginosa was obtained from
burn samples (27.7%) This agreed with the study of R'auf (17) who documented the
uppermost ratio of P. aeruginosa through burn infection then followed by wound samples.
This is for the reason that it was the 3rd most-communal pathogen related with infections
which acquired in hospitals (18). Regarding the isolation of Pseudomonas from burns, the
results come in an agreement with the study of Abbas and his colleagues (19) who revealed
that Pseudomonas spp. gave a positive growth in 71% of burns samples.
The main organisms which cause milk spoilage were Pseudomonas species through
the production of lipolytic and proteolytic enzymes (20). No difference in smell or
appearance between milk contaminated with disease-causing bacteria from non-contaminated
milk (21). So, overall of 73(47.7%) samples of raw milk were analyzed for the detection of
Pseudomonas spp. The number of Pseudomonas spp. was 12 isolates. 10(13.6%) isolates
were recognized as P. aeruginosa by biochemical identification. The result were more than
to those described by El- Zubeir and El-Owni (22) in Sudan who isolated P. aeruginosa in a
percentage of 6.6% from raw milk samples. However, the results showed less prevalence rate
of pseudomonas compared to that was documented by Jyoti and his assistance (23) in India
who showed that P. aeruginosa was isolated in a percentage of 11.11% of the raw milk
samples and higher than those found by Hussein (24) in Iraq who isolated P. aeruginosa in a
percentage of 3.7% from raw milk samples. The variance among the current and the earlier
studies may be attributed to the technique of sampling, treatment of samples and the kinds of
media. Bacterial identification was directed based on morphological and biochemical tests
(25).
The present study results revealed high resistance of P. aeruginosa isolates against
Ampicillin, Amoxicillin, Cefotaxime, Ceftazidime, Ciprofloxacin, Meropenem, Nalidixic
acid and Trimethoprim, while they were sensitive to Imipenem, Amikacin, Gentamicin,
Tetracycline, and Levofloxacin. These results are in accordance with those of other studies
conducted by Fallah and his assistance (26) in Tehran, Abbas and his colleagues (19) in Iraq,
but it contradict with the study of Odumosu and his assistance (27) in Nigeria. On the other
hand, this study showed the percentage of antibiotic resistance among P. aeruginosa isolated
from burn patients and altered clinical samples, is also low. It would hence appear that these
antibiotics are currently the only antibiotics that are prescribed for burn patients (28). Since
P. aeruginosa is generally a multidrug resistant organism, this agrees with the results that
obtained by NCCLS, (13) who found that 66% of pseudomonas strains are resistant to
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Cefotaxime However, Pseudomonas were high sensitive for Imipenem. Carbapenems are
efficient antibiotics against extended-spectrum β-lactamase–producing P. aeruginosa.
However, there are many potential health risks associated with carbapenem-resistant P.
aeruginosa (29).
The major issue confronting the treatment of P. aeruginosa diseases is the reputation
of the pathogen to have a wide array of resistance determinants. P. aeruginosa can create
resistance to antibiotics either through the expression and/or work of chromosomally
encoded mechanisms as a result of mutation or the procurement of resistance genes on
portable genetic components (plasmids) (30). The present study results showed increases in
the rate of the first and second generation of β-lactam resistance, where these antibiotics now
not active in the treatment of Pseudomonas spp. infections.
The present study demonstrated that 37 out of 38 P. aeruginosa isolated from animals
and patients showed accurate bands of approximately 618bp corresponding for identification
of P. aeruginosa strains. These results are in deal with other studies conducted by (31) in
France who found that the 16S rRNA documents analysis revealed
that P. aeruginosa sequences were existing in all environments but were most redundant in
samples from human and animals. Finally, PCR technique is a perfect molecular technique
used for identification in microbiology.
متعددة المقاومة للمضادات Pseudomonas aeruginosa عزل والتشخیص الجزیئی لبکتیریا
الحیویة المعزولة من الحیوانات والمرضى فی محافظة البصرة
غسق خالد شمخی ، بسام یاسین خضیر
فرع الاحیاء المجھریة ، کلیة الطب البیطری ، جامعة البصرة ، البصرة ، العراق
الخلاصة
خلال الفترة من تشرین الاول ٢٠١٨ الى شباط ٢٠١٩ ، جمعت ٢٧٨ عینة من الحیوانات والمرضى، (
٥٥ %) عینة من الحیوانات والتی توزعت الى ( ٥٢,٣ %) مسحة من محیط المجزرة و( ٤٧,٧ %) عینة حلیب من الابقار
موجودة فی ( ٤٤ %) بعد Pseudomonas والجاموس جمعت بواسطة الحافظات المعقمة . اوضحت النتائج بأن بکتریا
(% موزعة على ( ٢٤ %) عینة من المجزرة و ( ٢٠ %) عینة حلیب . ( ٤٥ Pseudomonas agar زرعھا على وسط
عینة کانت من المرضى مقسمة بین ( ٤٨ %) مسحة کانت من مرضى القدم السکری و ( ٥٢ %) مسحة کانت من مرضى
موجودة فی ( ٥٦ %) عینة موزعة Pseudomonas الحروق فی مستشفیات محافظة البصرة، اظھرت النتائج أن بکتریا
Pseudomonas فی ( ١٨ %) عینة من مرضى القدم السکری و( ٣٨ %) عینة من مرضى الحروق بعد زرعھا على وسط
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تم .P. aeruginosa ٥٤ %) عزلة ) حیث ظھر ٢٥ VITEK 2 kit شخصت ٤٦ عینة موجبة باستخدام .agar
اختبار ٣١ عزلة تجاه ١٣ نوع من المضادات الحیویة بطریقة انتشار القرص، جمیع العزلات ابدت مقاومة لثمانیة منھا،
١٦ بواسطة S rRNA ای انھا اظھرت نمط المقاومة المتعددة للمضادات الحیویة. تم اختبار ٣٨ عینة للکشف عن وجود
موزعة بین ١٩ عینة من مصدر حیوانی و ١٩ عینة من الانسان ،حیث اظھرت النتائج PCR تقنیة تفاعل البلمرة المتسلسل
١٩ موجبة / ١٩ عینة موجبة من المصدر الحیوانی و ١٩ / حزم واضحة على ٦١٨ قاعدة نیتروجینیة موزعة بین ١٨
لعینات الانسان.
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