Subjects&Methods

- Place of study

This study was conducted at microbiology laboratory, Faculty of Medicine,

Fayoum University.

-Period of study

This study was carried out from November 2017 to Septembe2019 .

- Sample collection and storage

From November 2017 to July 2018, Two hundred K. pneumoniae samples

were collected. K. pneumoniae strains were isolated from hospitalised patients'
urine, pus, sputum, blood, cerebrospinal fluid (CSF), and bronchoalveolar
lavage (BAL) samples. Traditional microbiological procedures were used to
definitively identify the isolates (Seifi et al., 2016).

Conventional microbiological methods-

Isolates had been cultivated on appropriate selective media, sub-cultured,

and biochemical tests had confirmed their identity. MacConKey Agar was used
to identify and confirm K.pneumoniae from the isolated samples (MAC).
K.pneumoniae was confirmed by the colony morphology and cultural
characteristics of the samples on this media.
 :Identification of K. pneumonaie
Gram staining, colony characters, and biochemical assays such as
oxidase, catalase, Simon citrate, MR-VP (methyl red – Voges Proskauer),
urea test, and Triple Sugar Iron (TSI) test were used to identify isolated
lactose fermenting colonies. The isolates of K. pneumoniae were indole
negative, MR negative, VP positive, citrate utilisation positive, urease
positive, catalase and oxidase negative (Cheeesbrough,. 2006). For further
characterisation, K. pneumoniae isolates were kept in 20% glycerol broth at
80°C.

String Test

The production of a mucoviscous string of >5mm by stretching bacterial

colonies grown on an agar plate at 37°C was recognised as a positive string test
result (Lee et al., 2017). Strains with a positive string test result were
categorised as hvK. pneumoniae, while those with a negative result were classed
as classic K. pneumoniae (ckp).

Biofilm formation
Bacterial growth

Biofilm formation was examined in K.Pneumoniae isolates. Strains of

K.Pneumoniae were produced by harvesting two or three morphologically
identical colonies and suspending them in 10mL of sterile Mueller Hinton broth
in sterilised tubes. The specimens were incubated for 24 hours at 37°C (Shehu
et al. 2016).

Biofilm assay

Primarily, 2ml of inoculums were extracted from the universal bottle

aseptically and then placed in a micro cuvette (Eppendorf, Germany). Next that,
Klepsiella Pneumoniae biofilms were created by putting 100μl of adjusted
inoculums into sterilized 96-well plates (Fisher Scientific, UK). A broth devoid
of bacteria was created as a negative control. The media were then taken from
the plate by lightly tapping it. To eliminate free-floating planktonic bacteria, the
plate was rinsed three times using sterile distilled water and then drained off by
inverting to enable it to air dry. For 10 minutes, the biofilms were dyed with
100µl 0.1 percent (w/v) crystal violet. The plate was rinsed three times with
phosphate-buffered saline to remove the crystal violet. Each well received 100 l
of 95 percent ethanol to remove the biofilms. Finally, the solubilized biofilm
formations were measured at 570 nm using a micro plate reader (Tecan Infinite
200 PRO, Austria Gmbh). The experiments were carried out three times. The
biofilm formation was classified using the formulas below. No biofilm
=ODODC)], weak biofilm= ODCOD(2ODC), moderate biofilm
=(2ODC)OD(4ODC), and strong biofilm SA=(4ODC)OD) (Al-kafaween et al,.
2019).

Where OD: Optical density.

-Antibiotic susceptibility testing:

-Antibiotic susceptibility by Kirby–Bauer disk diffusion method:

Antibiotic susceptibility pattern of K. pneumoniae isolates was performed

according to the Kirby–Bauer disk diffusion method. Mueller–Hinton agar
(OXOID, United Kingdom) medium was used for this purpose. The
susceptibilities of the isolates to clinically relevant antibiotics (amikacin)(30
µg), cefotaxime(30 µg), ceftazidime(30 µg), ceftriaxone(30 µg), cefepime(30
µg), cefoperazone/sulbactam(75/30 µg), chloramphenicol(30 µg),
ciprofloxacin(5µg), levofloxacin(5 µg), gentamicin(10µg), Piperacillin(100 µg)
piperacillin/tazobactam(100/10 µg), tetracycline(30 µg), imipenem(10 µg),
meropenem (10 µg), Aztreonam (15 µg) and Amoxicillin/Clavulanic acid
(20/10 µg). All of the inoculated plates were aerobically incubated at 37°C for
18–24h. Principles of the Clinical and Laboratory Standards Institute (CLSI)
were used and results were interpreted based on the same guidelines (CLSI,
2016).

Resistant(mm) Intermediate(mm) Sensitive(mm)

Piperacillin ≥ 14 15-20 ≥21
Gentamycin ≥ 12 13-14 ≥ 15
Piperacillin/tazobactam ≥ 14 15-20 ≥21
Aztreonam ≥ 17 18-20 ≥21
Chloramphenicol ≥ 12 13-17 ≥ 18
Cefotaxime ≥ 14 15-22 ≥23
Ceftriaxone(13-21) ≤ 13 14-20 ≥21
Ceftazidime (14-18) ≤14 15-17 ≥18
Tetracycline (14-19) ≤14 15-18 ≥19
Cefoperazone (14-18) ≤14 15-17 ≥18
Ciprofloxacin(15-21) ≤15 16-20 ≥21
Amikacin (14-17) ≤14 15-16 ≥17
Imipenem (15-19 ) ≤15 16-18 ≥19
Meropenem (15-19) ≤15 16-18 ≥19
Cefepime(14-18) ≤14 15-17 ≥18
Amoxicillin-clavulanate ≤19 19 ≥20
(19-20)
Tegacyclin
Fosfomycin
Colistin
Levofloxacin (13-17) ≤13 14-16 ≥17

-Disc diffusion/Kirby–Bauer method

 Antibiotic susceptibility pattern of K. pneumoniae isolates was performed
according to the Kirby–Bauer disk diffusion method. Mueller–Hinton agar
(OXOID, United Kingdom) medium was used for this purpose. Agar plates are
enriched with bacterial inoculum and incubated for 18–24 h. These colonies are
then resuspended in broth or saline to a standardised concentration and applied
with a cotton swab on an agar plate. Subsequently, the paper discs are placed
and the plates are incubated overnight. An inhibitory zone arises where the
concentration of the diffused antibiotic particles is high enough to inhibit
bacterial growth.

-E test

The E test is an extended version of the agar diffusion method and performed
similarly to the disc diffusion method. Instead of the paper discs, a predefined
exponential gradient of antibiotic agent is applied to the bottom of aplastic strip,
which is subsequently placed on an agar-based medium to generate diffusion of
the drug. After 24 h of incubation with the bacterial inoculum, the exact MIC of
a drug that is necessary to stop bacterial growth is easily read on the
strip.Another technique with comparable functionality is the spiral gradient
endpoint technique, where the gradient is produced by a spiral plate that
deposits a stock solution on the agar plate. The distance from the centre of the
plate to the endpoint of growth enables the determination of the MIC 31
(Doddangoudar et al, 2010).

3.7- Phenotypic Characterization of ESBL

- Screening for ESBL Isolates resistant to ceftazidime, cefotaxime and

ceftriaxone in antibiotic susceptibility test were identified as possible ESBL
producers [19]. ESBL productions by these isolates were confirmed by
confirmatory test following CLSI 2012.
 -Double disk diffusion [Double disk approximation/double disk synergy
test (DDST)]

MHA plates were inoculated with 0.5 McFarland matched test inoculums.
On inoculated plate amoxicillin-clavulanate (20 µg/10 µg) was placed at center
and on sides ceftadizime (30 µg) and cefotaxime (30 µg) were placed 30 mm
apart from center to center. They were incubated at 37°C for 18-24 hrs. Those
inoculums which exhibited an enhanced zone of inhibition, the synergism in
between Amoxacillin/clavulanic acid and cefotaxime or ceftadizime was
identified as confirmed ESBL producers. In doubtful cases distance between
amoxicillin-clavulanate, ceftadizime and cefotaxime was reduced to 20 mm
apart from center to center [20,21]

Molecular identification and characterization of

hypermucoviscous K. pneumoniae isolates:
DNA extraction:
DNA extraction was done by the Thermo Scientific GeneJET Genomic
DNA Purification Kit (Thermo Fisher Scientific Baltics UAB Company,
Vilnius, Lithuania) according to the Gram-Negative Bacteria Genomic DNA
Purification Protocol (Fig ….).

Steps:

1. Isolates were harvested up to 2109 bacterial cells in a 1.5 or 2 mL

microcentrifuge tube by centrifugation for 10 min at 5000 g. The
supernatant was discarded.
2. The pellet was resuspended in 180 μL of digestion solution. Twenty (20)
μL of proteinase K Solution was added and mixed thoroughly by
vortexing or pipetting to obtain a uniform suspension.
3. The sample was incubated at 56°C while vortexing occasionally, rocking
platform were used until the cells were completely lysed (30 min).
4. Twenty μL of RNase A Solution was added, mixed by vortexing and the
mixture was incubated for 10 min at room temperature.
5. Two hundred μL of Lysis Solution was added to the sample, mixed
thoroughly by vortexing for about 15 s until a homogeneous mixture is
obtained.
6. Four hundred μL of 50 ethanol were added and mixed by pipetting or
vortexing.
7. The prepared lysate was transferred to a GeneJET Genomic DNA
Purification Column inserted in a collection tube. The column was
centrifuged for 1 min at 6000  g. The collection tube containing the
flow-through solution was discarded. The GeneJET Genomic DNA
Purification Column was placed into a new 2 mL collection tube.
8. Five hundred μL of Wash Buffer I (with ethanol added) was added and
centrifuged for 1 min at 8000  g. The flow-through was discarded and
the purification column was placed back into the collection tube.
9. Five hundred μL of Wash Buffer II (with ethanol added) to the GeneJET
Genomic DNA Purification Column, centrifuged for 3 min at maximum
speed (≥12000  g). If residual solution was seen in the purification
column, it was optional to empty the collection tube and re-spin the
column for 1 min. at maximum speed. The collection tube containing the
flow-through solution was discarded and the GeneJET Genomic DNA
Purification Column was transferred to a sterile 1.5 mL microcentrifuge
tube.
10. Two hundred μL of Elution Buffer were added to the center of the
GeneJET Genomic DNA Purification Column membrane to elute
genomic DNA. It was incubated for 2 min at room temperature and
centrifuged for 1 min at 8000  g.
11.The purification column was discarded. The purified DNA was used
immediately in downstream applications or stored at -80 °C.
Figure (…): A diagram for Gram-Negative Bacteria Genomic DNA
Purification Protocol.
 Polymerase Chain Reactions:
1. Identification of Klebsiella pneumonaie by PCR technique:
The K. pneumoniae Pf/ K. pneumonia Pr1 primer pair (Table 3) was used for the
molecular identification of K. pneumoniae (Liu et al., 2008) in a simplex PCR
reaction.

Total DNA (2 µL) from each isolate was subjected to momoplex PCR in a 20
µL reaction mixture containing:

 10.0 µL master mix (MyTaq™ Red Mix, Bioline, United Kingdom).

 0.5 µL of each primer (Table 3).
 7.0 μl distilled water to complete the reaction volume.
Thermocycler conditions were: 94⁰ C for 3 min, followed by 30 cycles of 94 ⁰
C for 30 s, 58 ⁰ C for 30 s, 72 ⁰ C for 45 s and a final extension at 72 ⁰ C for 10
min.

PCR name Primer Sequence (5´–3´) Product Annealing Reference

size (bp) Temperature

1. Identification of klebsiellapneumonaie by PCR technique:

58 °C
K. Pf ATTTGAAGAGGTTGCAAACGAT 130 Liu et al.,
pneumoniae K. TTCACTCTGAAGTTTTCTTGTGTTC 2008
Identification Pr1

2. Detection of genes encoding important b-lactamases:

Two multiplex PCRs were modified from protocols published by (Dallenne et

al., 2010).
 a) Multiplex I ( blaTEM, blaSHV and blaOXA-1-like)

Total DNA (2µL) from each isolate was subjected to multiplex PCR in a 20µL
reaction mixture containing:

 10µL master mix (MyTaq™ Red Mix, Bioline, United Kingdom).

 0.4µL of each primer (Table 3).
 5.6μl distilled water to complete the reaction volume.

group 1, group 2 and group 9) and (blaCTX-M group 8/25) b) Multiplex II

Total DNA (2µL) was used for each multiplex PCR in a 20µL reaction mixture
containing:

 10µL master mix (MyTaq™ Red Mix, Bioline, United Kingdom).

 0.4µL of each primer (Table 3).
 4.8μl distilled water to complete the reaction volume.
Amplification in the two multiplex reactions was carried out as follows: initial
denaturation at 94⁰C for 10 min; 30 cycles of 94⁰C for 40 s, 60⁰C for 40 s and
72⁰C for 1 min; and a final elongation step at 72⁰C for 7 min.
 PCR Primer name Sequence(3-5) Anneali Amplic Primer Referen
name ng on concentrat ce
position size(bp) ion
(pmol/mL)
Multipl Multi TSO-T-for CATTTCCGTGTCGCCCTT 13-34 800 0.4
Dallenne 
ex I ATTC
et al.,
TEM ,S 2010
HV and
OXA- .

1-like
Multi TSO-T- CGTTCATCCATAGTTGC 812-791 0.4
Rev CTGAC
Multi TSO- AGCCGCTTGAGCAAATT 71-91 713 0.4
S_for AAAC
Multi TSO- ATCCCGCAGATAAATCA 783-763 0.4
S_Rev CCAC
Multi TSO- GGCACCAGATTCAACTT 201-222 564 0.4
O_for TCAAG
Multi TSO- GACCCCAAGTTTCCTGT 764-743 0.4
O_Rev AAGT
Multipl MultiCTXMGp1 TTAGGAARTGTGCCGCT 61-80 688 0.4
ex II _for GYA
CTX-
Mgroup
1,
group2
and
group9
MultiCTXMGp1 CGATATCGTTGGTGGTR 748-728 0.2
-_rev CCAT
MultiCTXMGp2 CGTTAACGGCACGATGA 345-362 404 0.2
_for C
MultiCTXMGp- CGATATCGTTGGTGGTR 748-728 0.2
2_rev CCAT
MultiCTXMGp9 TCAAGCCTGCCGATCTG 299-317 561 0.4
_for GT
MultiCTXMGp9 TGATTCTCGCCGCTGAA 859-842 0.4
_rev G
CTX-M CTX- AACRCRCAGACGCTCTA 172-189 326 0.4
group Mg8/25_for C
8/25
CTX-Mg8/25- TCGAGCCGGAASGTGTY 497-479 0.4
rev AT
 Multiplex PCR for detection of some acquired extended spectrum beta
lactamases (ESBL) of k.pneumonia:

A multiplex PCR was used. The following genes were targeted: magA
(K1serotype), rmpA, entB, kfu, mrkD, and the K2 capsular serotype. (19)
(Table….).

Multiplex PCR was carried out in a 20 ul volume. The final reaction mixture
contained a 1 x PCR mixture (which consists of preoptimized concentrations of
hot start DNA polymerase, MgCl2, dinucleoside triphosphate [dNTP], and PCR
buffer), various primer concentrations (Table…), and 2 ul of the crude DNA
extract.

The PCR conditions were as follows: initial activation at 95°C for 15 min,
followed by 30 cycles at 94°C for 30 s, 60°C for 90 s, and 72°C for 60s, and a
final extension at 72°C for 10 min. The amplicons were separated at 100 V for
2h in a 1.5 % (wt/vol) agarose gel containing ethidium bromide.

Pcr name Primer name Sequence(3-5) Amplicon Primer

size(bp) concentration
(pmol/mL)
mrkD_ mrkD_for AAGCTATCGCTGTACTTCCGGC 340 0.1 (C
A
mrkD_rev
GGCGTTGGCGCTCAGATAGG

entB entB_for GTCAACTGGGCCTTTGAGCCGT 400 0.1

C
entB_rev
TATGGGCGTAAACGCCGGTGAT

K2 K2_for CAACCATGGTGGTCGATTAG 531 0.4

K2_rev TGGTAGCCATATCCCTTTGG

Kfu kfu_for GGCCTTTGTCCAGAGCTACG 638 0.075

kfu_rev GGGTCTGGCGCAGAGTATGC

magA magA_for GGTGCTCTTTACATCATTGC 1.283 0.3

magA_rev GCAATGGCCATTTGCGTTAG

.
 -Detection of some hypervirulence genes using PCR technique (peg344,
iucA, iroP, rmpA):

Total DNA (5 µL) from each isolate was subjected to momoplex PCR in a
20 µL reaction mixture containing:

 10.0 µL master mix (MyTaq™ Red Mix, Bioline, United Kingdom).

 0.5 µL of each primer (Table 3).
 4.0 μl distilled water to complete the reaction volume.

The PCR conditions were 95°C for 5 min, followed by 30 repeated

cycles of 30 s at 95°C, 30 s of annealing at 56°C and 1 min of extension at
72°C, followed by 7 min as a final extension at 72°C. 

Pcr name Primer name Sequence(3-5) Amplico Annealing

n temperature
size(bp) (ºC)
 Peg344 GCGGGAAAGGACAGAAAGCCAGTG 332 56
Peg344-f

Peg344-rev GAGGGAAGATGAGAAATACGAGC

iucA iucA-for AATCAATGGCTATTCCCGCTG 239 62

iucA_rev CGCTTCACTTCTTTCACTGACAGG

rmpA rmpA_for CATAAGAGTATTGGTTGACAG 461 53

rmpA_rev CTTGCATGAGCCATCTTTCA

N.B.
 All PCR reactions were done using (A200 Gradient LongGene thermal
cycler, LongGene Scientific Instruments Ltd, China).

 In all the PCR reactions, Amplicons were separated using (Cleaver

electrophoresis system, Cleaver scientific Ltd , United Kingdom) in a 1.5
% agarose gel (TopVision Agarose, Thermo Fisher Scientific Baltics,
UAB company, Vilnius, Lithuania) in 1×TAE buffer (Thermo Fisher
Scientific Baltics, UAB company, Vilnius, Lithuania) contained: (40
mmol/L Tris–HCl, 2 mmol/L acetate, 1 mmol/L EDTA) with 20 µl/L
ethidium bromide (Carl Roth GmbH + Co. KG - Karlsruhe, Germany)
added.
 The size of the amplicons was estimated by comparison with a size
ladder (GeneRuler 100 bp DNA Ladder, Jena Bioscience, Germany) and
visualized using (Clear View UV Transilluminator, Cleaver scientific
Ltd, United Kingdom).
 All oligonucleotide primers were synthesized at (Thermo Fisher
Scientific Company, United Kingdom).