INTRODUCTION:
Klebsiella pneumoniae is one of such clinically significant organisms that
have acquired much public health concern1,2. Klebsiella pneumoniae is a significant Enterobacteriaceae
considered as one of the opportunistic pathogens causing broad spectra of
diseases and showing increasingly frequent acquisition of resistance to
antibiotics, specifically the extended-spectrum β-lactamase
(ESBL)-producing strains3,4,5. Herbal or medicinal plants have proved a good
choice. Medicinal plants have many substances such as peptides, unsaturated
long-chain aldehydes, alkaloidal constituents, essential oils, phenols and
water extracts, methanol and butanol soluble compounds, which show significant
antibacterial properties against a number of human pathogens6,7.
Azadirachta indica (neem) is world-renowned medicinal plant, having long
history of usage in various ailments in Indian traditional medical system8,9 since time immemorial. Each part of neem, including
leaves, bark extracts, oil, and products made from neem have medicinal
properties10,11,12.
Neem products have been proved antihelminthic,
antimicrobial, and act as a contraceptive and sedative agent13. Although neem has been proved antimicrobial in nature14,15,16, to the best of our knowledge, its role has not been
evaluated in ESBLS and biofilm-associated infections. Therefore, the current
study was undertaken to evaluate the role of neem in ESBLS production and
biofilm formation for Klebsiella pneumoniae.
MATERIALES AND METHODS:
Isolation and identification of Klebsiella
pneumonia:
One hundered of Klebsiella
pneumonia isolated from clinical different sources . MacConkey Agar and
nutrient agar slant was prepared following the manufactures instruction and was
used to subculture Klebsiella pneumonia incubated at 37⁰C for 24 h
after which colonial morphology was observed and biochemical test was conducted
to confirm the organisms. All isolates identified using conventional ,and
automated methods using Vitek-2 system . They were maintained on nutrient agar and MacConkey slant under
refrigerated at C temperature for further analysis of neem leaf extract'.
Antibiotics susceptibility:
The susceptibility profiles of K.
pneumoniae were determined using the VITEK 2 method (bioMérieux) in accordance
with the Clinical and Laboratory Standards Institute guidelines17 and (EUCAST)18. All K. pneumoniae
was tested for their resistance against the following 15 antibiotics:
piperacillin/tazobactam (TZP), cefuroxime (CXM), cefoxitin (FOX), ceftazidime
(CAZ), ceftriaxone (CRO), cefepime (FEP), imipenem (IMP), and meropenem (MEM).
Phenotypic detection of ESBLs:
Determination of the production of ESBls
was carried out by modified Hodge test, modified Cephalosporins Inactivation
Method and under the CLSI guidelines17 and as described elsewhere19.
Collection of the plant material:
The leaves of Azadirachta indica were collected from
Rawaa city, Anbar, Iraq. Collected leaves were dried in shade at room
temperature and blended
using dry blender to obtain the powder for more efficient and effective solvent
extraction.
Preparation of the Extracts for Antibacterial Assay:
A total of 100 grams of leaves powder was soaked using ethanol, methanol,
and ethyl acetate as solvents and kept at room temperature for 24 hours, then extracted
according to20.
Determination of minimum inhibitory concentration (MIC) and MBC:
MIC and MBC of Neem extract estimated using Resazurine
–coloured method21.
Biofilm formation Assay:
A quantitative adherence assay was
employed to perform biofilm formation assay22.
Estimation of Extended spectrum 
lactamse enzymes:
β-Lactamase activity was assessed
spectrophotometrically by hydrolysis of nitrocefin23. The assay mixture contained 83 μg of
nitrocefin, 167 μg of BSA, 10% glycerol and 0.33ml
(0.6 μg/mL of albumin) of cell lysate that contained
β-lactamase in a final volume of 1.5ml of 50mM phosphate buffer.
β-Lactamase activity was monitored by measuring the decrease in absorbance
at 390nm for 10 min at 37°C. The enzyme activity was expressed as
μmol of nitrocefin hydrolyzed/min/mg of protein and the calculation was
based upon the molar extinction coefficient of 15 000 M−1
cm−1 for nitrocefin23. E. coli ATCC25922 used as control in this
method.
RESULTS AND
DISSCUSION:
Isolation:
In this study 100 samples were collected ,as were as
following: wounds (13) and burns (9) samples, urine (51) samples,sputum (24)
samples, and blood (3) samples. These samples were distributed among burns
(9%), wounds (13%) samples ,urine samples (51 %), sputum samples (24%), and
blood samples (3 %), as shown figure 1:
Figure1: Percentage of K. pneumoniae according
to sources.
Identification of K. pneumoniaea:
To confirm the diagnosis, the collected isolates were
initially diagnosed as K. pneumoniae. The bacterial isolates were
cultured on Blood agar, and MacConkey agar
under aerobic conditions followed by other
diagnostic tests figure 2. All isolates showed pink lactose fermenter mucoid
colonies on MacConkey agar (MCA). On Blood agar media they grow as nonhemolytic grey-white, mucoid colonies after
24 h of incubation. Table 1 explain other diagnostic test for K. pneumoniaea
.
A B
Figure 2: Klebsiella pneumoniae on B: MacConky
agar, A: Blood agar.
Table 1: Microscopic and biochemical test .
|
Characteristics
|
Klebsiella
pneumoniae
|
|
Gram stain
|
Negative
|
|
Shape
|
Rod
|
|
Catalase
|
+
|
|
Citrate
|
+
|
|
Indole
|
-
|
|
Oxidase
|
-
|
|
Urease
|
+
|
|
Capsule
|
+
|
Positive: +, Negative: -.
Antibiotic susceptibility testing:
The antibiotic susceptibility test revealed that all
of the studied K. pneumonia (30/100 )from 100 clinical strains were
resistant cephalosporins community of β- lactam and they were resistant to
most antibiotics under test and it showed an elevated resistance to various
groups of β- lactam and non
β- lactam antibiotics. Figure 3.
Figure (3): Antibiotic resistance percentage for K.
pneumoniae based on kirby bauer disk diffusion
Phenotypic detection of ESBLs:
The phenotypic detection of ESBLs by using : modified hodge test, modified cephalosporins inactivation methods for Suspected ESBLs. Phenotypic testing revealed the existence of
the ESBL genes in all resistance isolates. Since the updated Hodge test was
used as a phenotypic confirmatory procedure for ESBLs, this examination yielded
a positive result of (10%).
In this analysis, modified cephalosporins inactivation
methods for suspected ESBLs development were used as phenotypic confirmatory
methods for distinguishing between serine and other metallo—lactamases
production, with the results showing that (95%) was serine beta lactamase and
(5%) as metallo betalactamases. figure 4.
Figure 4: Phenotyping detection of ESBLs, A: Clover
leaf test, B: mCIM, C: Metalo beta lactamase, D: Serine beta lactamase , E:
Double disk synergy.
Effects of Neem extract against ESBLS and Biofilm
production:
Results demonstrated that neem leaves
extract was quite effective in disrupting formation of biofilms and ESBLS
activity at P- value : 
.Results indicated that there was a inhibitory effect
for neem extract against ESBLS as shown in Table(2). K. pneumomieae β-Lactamase
activity was decreased by using neem extract at P- value <0.01. In study Conducted by24 show that neem was shown to be active against
b-lactamase-producing K. pneumoniae isolates for the first time.
In the biofilm mode of growth, K.
pneumomieae produces glycocalyx, an extracellular polysaccharide, and
organic polymer such as alginate, which play an important role in the
structural development of biofilm . Exopolysaccharide is responsible for
binding the cells together in a highly hydrated polymer network. Although
alteration in cell surface hydrophobicity of K. pneumomieae by different
antibiotics have been shown previously, the effect of NE on cell surface
hydrophobicity has not been evaluated yet25.
Table 2: Effects of Neem extract against ESBLS and
Biofilm production. A: Neem against ESBLs, B: Neem against biofilm formation.
|
A
|
ESBLs activity
|
With neem
|
P-value
|
|
Mean
|
0.4577
|
0.5762
|
> 0.01
|
|
Std. Deviation
|
0.2688
|
0.2824
|
|
Std. Error of
Mean
|
0.06010
|
0.06314
|
|
B
|
Biofilm formation
|
Treated with neem
|
P-Value
|
|
Mean
|
0.72
|
0.58
|
> 0.01
|
|
Std. Deviation
|
0.02688
|
0.029
|
|
Std. Error
of Mean
|
0.06010
|
0.05872
|
CONCLUSION:
The results suggest that neem leaves
possess antibiofilm property and reinforce the possibility of employing NE in
the eradication of biofilm infections. Neem either alone or in combination with
antibiotics can be explored as a potent biofilm-eradicating agent. As global
scenario is changing toward the use of nontoxic plant products having medicinal
value, hence extensive research is required on neem for its better economic and
therapeutic utilization.
ACKNOWLEDGEMENT:
Authors gratefully acknowledge Dr. Safaa
Abed lateef , Head of Biotechnology Department .
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