Proceeding of The 2nd International Conference of Biotechnology and Environment (ICBE 2018)

Alexandria-Egypt

Molecular Identification and Antibiogram of Multi-Drugs Resistant

Staphylococcus aurues Isolated from Diabetic foot Infections in

Thi-Qar Province, Iraq

Muslim D. Musa1 ; Khwam R. Hussien1 ; Abbas D. Muttar2
1
Al-Nasiriyah Technical Institute, Southern Technical University,Iraq.
1
muslim1983@stu.edu.iq, 1krhussein@stu.edu.iq ,
2
Veterinary College / Thi-Qar University, Iraq
dr.abbas_2012@yahoo.com

Abstract
The most important complications of diabetes mellitus are neuropathy and diabetic
foot infection (DFI), additionally multi-drugs resistant isolates has been documented
widely thought out the world, which lead to raising the coast and hospital stay. The
study is aimed to identify the most prevalent microorganism causing DFI and
screening these isolates for important virulence factors by molecular techniques, also
reveals its antibiogram. Swabs from DFI (n=53) were cultured on Blood,
MacConkey, and Manitol salt agar plates. Bacterial species were identified by
morphology, biochemical tests and API20-Staph. Polymerase chain reaction (PCR)
was used for conformational identification of Staphylococcus aureus with specific
primer targeting 16S-rRNA, and for screening for pantonvalintine-leuckocidine (PVL)
as virulence factor. Antibiogram was accomplished according to disc diffusion
method. Out of 53 swabs, bacterial growth, Staph.aureus 38(58.4%), which was the
most prevalence species. Confirmative identification of Staph.aureus revealed that all
isolates were positive and showed bands of 16S-rRNA of Staph. aureus with expected
size 125bp. leuckocidine was detected in 27 (71%). Antibiogram test showed variable
sensitivity and resistance against antibiotics. The most effective antibiotic was
Imipenem. In conclusions, multi-drug resistance Staph.aureus prevalent in DFI most
of these isolates are the causes of it and not transit coloniser.
Key words: Staphylococcus aureus, diabetes, PCR, Antibiogram

1- Introduction

Diabetes is a chronic metabolic disease, effecting a millions of peoples around the

world and considered one of the most important public health concern (1). Among
many complications of this disease, diabetic foot ulcer (DFU) which is the most
common, late onset, costly effective and considered the major cause of disability and
hospitalization(2).. Indeed it has been reported that about 25% of diabetic patients will
develop foot ulcer during their lifetime, and approximately 40-80% of these ulcer
undergo infection (3). The infection of diabetic foot can worsen the situation from
simple injury to gangrene and/or osteomyelitis leading to lower extremity amputation.
It is believed that, the impaired micro-vascular circulation limited the access of
phagocytic cells which favouring the infection of ulcer foot in diabetic patient (4).
The microbial infection can be either mono-microbial or poly-microbial. Although,
many micro-organisms implicated in DFI including Escherichia coli, Proteus spp.,
Pseudomonas spp., and Enterococcus spp (5). The infection caused by Staphylococcus
aureus constitute about 10-40% of cases (6). Staphylococci are a frequent commensal
bacteria of human skin and mucosa, being one of the major cause of infections in
Proceeding of The 2nd International Conference of Biotechnology and Environment (ICBE 2018)
Alexandria-Egypt

humans, ranging from minor skin infections to severe infections such as septicaemia,
endocarditis and osteomyelitis (7).These bacteria possesses many virulence factors that
enable them to cause damage in soft tissue, the most important one that is usually
encountered in severe tissue necrosis is the cytotoxin panton-valentine leukocidin
(pvl), whose locus is carried on a bacteriophage, found commonly in strains isolated
from community-acquired skin and soft tissue infections(8). Additionally, the presence
of multi-drugs resistant isolates has been documented widely thought out the world,
which lead to raising the coast, hospital stay and appropriate treatment. Multi-drugs
resistant Staph.aureus isolates have increased in prevalence recently as a results of
attempts to control the disease caused by these bacteria (9). The association of multi
drug resistant (MDR) pathogens with diabetic foot ulcers increases the clinical
conditions, complicate the treatment process further and possess a great challenge to
the physicians or the surgeon in treating this condition(10), the appropriate
identification and selection of antibiotics based on the antibiogram is extremely of
significant value in management of this infection, furthermore, the specific micro-
organism causing diabetic foot infection(DFI) is differed not only from one country to
another but also from one hospital to other (11). Thus this study was aimed to identify
the most prevalent microorganism causing this condition by molecular means and its
antibiogram in Thi-Qar province, Iraq.

2- Materials and Methods

 Samples Collection
Pus and wound secretions were collected from 53 diabetic patients suffering from
diabetic foot ulcer with varying grads attended to Diabetic Center in Thi-Qar province
in the period extended from first of December 2016-to last of April 2017. Sterile
swabs were used for pus and wound secretions collection from the deeper point of the
ulcer by rotary movement. The swabs were transported in cooled container to
microbiology laboratory at Technical Institute - Community Health Dept. for
bacteriology work within two hours.

 Isolation and Identification of Bacterial species

All swabs were immediately upon arrive streaked on Sheep blood agar 5%, and
incubated at 37 Co for 24 hours. All colonies were sub-cultured on following media;
Manitol Salt Agar, MacConkey agar and Eosin Methylin Blue agar and incubated for
24 hours at 37Co. Identification of bacterial species were done according to colonial
appearance and biochemical tests (API20E and API20 Staph, bioMérieux ).

 Molecular Identification of Satphylococcuss aureus

The whole genomic DNA was extracted from all isolates following the instructions of
Presto™ Mini gDNA Bacteria Kit (Genaid-Thiland). Staph aureus species was
identified by Polymerase Chain Reaction (PCR) targeting 16S-rRAN with specific
primer (forward F 5’-CGAAAGCGTGGGGATCAAAC-3’/ reverse R5’-
CCCAGGCGGAGTGCT TAATG -3’) designated by Eman et al.,(12). PCR reaction
was conducted in 50 µl PCR tube, containing; 5µl PCR premix (Accupower,
Bioneer), 5 µl template DNA, 1 µl of each primer (10 picomole) the remaining
volume was completed with De-ionized water. The PCR reaction tubes were placed in
the theromocycler (Block model Max Blc-4/ Esco / Singapore) which was previously
programmed to fulfil the following conditions described by Al-Shammari, (13), with
some modification as follow; initial denaturation at 95 Co for 5 minutes followed by
Proceeding of The 2nd International Conference of Biotechnology and Environment (ICBE 2018)
Alexandria-Egypt

30 cycles of denaturation at 95 Co for 15second, annealing at 59 Co for 30 second and

extension at 72 Co for 60 second and final extension was 72 Co for 5 minutes. The
amplification products were electrophoresed in agarose gel (1.5%) stained with
ethidium bromide under 80 V for 45 minutes, and the specific bands were detected
according to molecular marker (2000 bp) by UV- transillunimator.

 Screening for Panton-Valantine Leuckocidin gene (pvl)

All staphylococcus aureus isolates were subjected to polymerase chain reaction for
detection panton-valantine lueckocidin gene (pvl). Based on specific primer
designated by Lina et al .,(1999) (14) [luk-PV-1 ATCATTAGGTAAAA TGTCTG
GACATGATCCA- and luk-PV-2, GCATCAASTGTATTGGATAGCAAAAGC]
which give product size 433 bp, the amplification reaction was consist of 5µl template
DNA, 1 µl forword and reverse primer(10 picomol), 5 µl(Accupower, Bioneer), and
the rest of the volume was completed with deionized water. The amplification
reaction was as follow: initial denaturation at 94 Co for 4 minutes followed by 35
cycles of denaturation at 94 Co for 45 second, annealing at 55 Co for 45 second and
extension at 72 Co for 1 minute, then one cycle of final extension at 72 Co for 2
minutes. The expected bands were visualized by UV. Trans- illuminator according to
ladder.

 Antibiogram analysis
Antimicrobial susceptibility test was performed for all isolates according to the
criteria of the Clinical and Laboratory Standards Institute (CLSI,2012)(15). The
following antibiotic discs were used; amikacin(30), gentamycin(10µg), clindamycin(2
µg), imipenem(10 µg) vancommycin(30 µg), cefotaxim(5 µg), cefoxtin(10 µg),
azteornam(30 µg), Bacterial suspension was prepared for each isolate, and adjusted to
the 0.5 McFarland Standards, and swabbed onto Mueller-Hinton agar surface. Using
sterile forceps, the antibiotic-containing discs were placed aseptically on the
inoculated plates. The plates were incubated in an inverted position, at 37°C for 24
hours and thereafter examined for clear zones of inhibition. Inhibition zones were
measured using a transparent ruler, and recorded in millimetres (mm). A standardized
table was used to determine if the isolate was “Resistant”, “Intermediate” or
“Sensitive

3- Results and Discussion

In this study, out of 53 swabs samples, bacterial growth could be observed in 43

(81.13%) while only 10 sample gave no growth. The anaerobic culture was not
performed in this study, indeed the role of anaerobic bacteria is not clearly understood
and some studies have proposed the negligible effect in diabetic foot ulcer infection
(16)
Presumptive identification and speciation based on colonial morphology and
biochemical tests (API20E and staph20) aggregated the bacterial isolates (65 isolates)
into four species, these are Staphylococcus aureus 38(58.4%), which was the most
prevalence species followed by p. merabilis 12(18.4%), E. coli 8(12.3%) and
klebsiella pneumonia 7(10.7%). Figure 1 represent the recovery percentage of
different bacterial species isolated from DFI. The predominance of S.aureus in DFI
which recorded in this study are in agreement with previous studies (17, 18, 3).
Proceeding of The 2nd International Conference of Biotechnology and Environment (ICBE 2018)
Alexandria-Egypt

Bacterial recovery from 21(48.8%) swabs samples showed single specie (mono-
microbial infection), the S.aureus was the predominant 20 (95.2%), on the other hand
a 22(51.1%) swabs samples showed multiple bacterial species being Staph.aureus
accompanied with other Gram negative bacteria as presented in table (2),
Staph.aureus and P.merabilis 9(40.9%), S.aureus and E.coli 6(27.3%); S.aureus and
K.penumonia 3(13.6%); P. merabilis and K.pneumoniae 2(9%) and K.pneumoniae
with E.coli 2(9%) indicating that S.aureus was the predominant in both mono-
microbial and poly-microbial infection table(1). Diabetic foot infection can be mono-
microbial or poly-microbial, however researches have documented that DFI is poly-
microbial in nature (19) the percentage of poly-microbial infection recorded in this
study are within the range (43%-83%) which has been published in other
studies(20,21.22), on the other hand the mono-microbial infection has been found high in
some studies(23) it has been proposed that in the early stage of infection, the mono-
microbial state prevails and as the time run, a poly-microbial state arises(22).

58.4
60

50

40

30 18.4
20
12.3 10.7
10

0
Staphylococcus proteus spp E. coli klebsiella spp
aureus
BACTERIAL SPECIES ISOLATED FROM DIABETIC FOOT
ULCER INFECTION

Figure (1): Different bacterial species isolated from diabetic foot infection

Table (1): Monomicrobial and poly-microbial of diabetic foot infection

Monomicrobial No. (%) Polymicrobial infection No. (%)

infection
Staph. aureus 20(95.2) Staph. aureus +E.coli 6(27.3)

P.merabilis 1(4.8) Staph. aureus + Proteus 9(40.9)

E.coli 0(0) Proteus + Klebsiella 2(9.0)

K.pneumionae 0(0) Klebseilla + E.coli 2(9.0)

Staph. aureus +Klebsiella 3(13.6)

Sub-Total 21(48.8) 22(51.1)

Total 43
 Proceeding of The 2nd International Conference of Biotechnology and Environment (ICBE 2018)
Alexandria-Egypt

The results of PCR for further confirmative identification of Staph.aureus revealed

that all isolates were positive and showed bands of 16S-rRNA of Staph.aureus with
expected size 125bp according to molecular marker figure (2). Among 38 S.aureus
isolates, the (pvl) has been detected in 27(71%) most of these isolates (77.8%)
obtained mono-microbial infection of diabetic foot ulcer infection figure (3). Pantone-
valantine leuckocicidne considered an important virulence factor especially in soft
tissue infection, Dunyach-Remy et al. (24) found that two categories of S.aureus
implicated in DFI these are toxogenic and non-toxogenic, the toxogenic strains are
related with more sever grad of DFI and systemic involvement. The detection rate of
pvl in this study are higher than those mentioned by Nabiel and Barakat. (25) although
there is scariness in investigation of the PVL-producing clones isolated from DFI
there prevalence are varied among countries(22).

1 2 3 4 5 6 7

Figure(2): Agaros gel electrophoresis (1.5%) of amplified 16S-rRNA of Staph

Figure (1): recovery percentage of different bacterial species isolated from
aureus . after 1 hour at 100V, stained with ethidum bromide and visualized by a
diabetic foot ulcer infection.
UV transilluminator. Lane 1: Molecular Marker (2000 bp ladder) Lanes 2, 3, 5 :
positive. Lanes 4,6,7 negative control

1 2 3 4 5 6 7 8 9

400 bp

Figure (3): Agaros gel electrophoresis (1.5%) of amplified. after 1 hour at 100V,
stained with ethidum bromide and visualized by a UV transilluminator. Lane 1:
Molecular Marker (2000 bp ladder) Lanes 2,3,5,7,8 : positive
Proceeding of The 2nd International Conference of Biotechnology and Environment (ICBE 2018)
Alexandria-Egypt
Table (2): Results of Antibiogram of Staph. aureus Isolated from Diabetic Foot
Infection
Antibiotics Susceptible Intermediate Resistance
Amikacin 9(23.7) 3(7.8) 26(68.42)
Clindamycin 5(13.16) 8(21) 25(65. 8)
Gentamycin 12(31.6) 10(26.3) 18(47.36)
Imipenem 34(89.5) 4(10.5) 0(0)
Vancomycin 31(81.6) 5(13.1) 2(5.26)
Cefotaxim 14(36.84) 15(39.5) 9(23.68)
Ceftazidime 16(42.1) 16(42.1) 6(15.78)
Cefoxtin 29(76.3) 4(10.5) 5(13.15)
Amoxicillin-clavulonic acid 10(26.3) 9(23.7) 16(42.1)
Azteornam 18(47.4) 5(13.15) 15(39.47)

Antibiogram analysis showed that methicillin resistant staphylococcus aureus

(MRSA) constitute only 5(13.15% resistant to cefoxtin) of total isolates. In this regard
finding of this study are lower than those recorded by other studies like Perim et al
.,(26) who reported 59% and Amini1 et al .(27) in Iran who reported 66% . however, our
fining are inconsistence with global range of MRSA prevalence in diabetic foot (15-
30%)(28) this difference could be attributed to that all patient attended to diabetic
canter for carbotheraby and with no prolong stay in hospital. The higher resistance
rate are seen for amikacin and clindamycin (68.4% and 65.8% respectively), while the
most effective antibiotic are Imipenem, and vancomycin.it has been documented that
vancomycin is the drug of choice for diabetic foot infection treatment(29)
Majority of isolates 25(56.8%) showed resistance to at least four antibiotic with multi
drug resistance index 0.4 and eight isolates (21%) showed resistance to three
antibiotics (MDRI= 0.3). In conclusions, multi-drug resistance Staph. aureus
prevalent in DFI most of these isolates are harbouring virulence determinant for soft
tissue destruction and worsen the diabetic foot infection also these strains are the
causes of cases and not only transit coloniser.

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