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Subjects&Methods: Place of Study
Subjects&Methods: Place of Study
- Place of study
-Period of study
String Test
Biofilm formation
Bacterial growth
Biofilm assay
-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).
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]
Steps:
Total DNA (2 µL) from each isolate was subjected to momoplex PCR in a 20
µL reaction mixture containing:
58 °C
K. Pf ATTTGAAGAGGTTGCAAACGAT 130 Liu et al.,
pneumoniae K. TTCACTCTGAAGTTTTCTTGTGTTC 2008
Identification Pr1
Total DNA (2µL) from each isolate was subjected to multiplex PCR in a 20µL
reaction mixture containing:
Total DNA (2µL) was used for each multiplex PCR in a 20µL reaction mixture
containing:
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.
K2_rev TGGTAGCCATATCCCTTTGG
kfu_rev GGGTCTGGCGCAGAGTATGC
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:
Peg344-rev GAGGGAAGATGAGAAATACGAGC
iucA_rev CGCTTCACTTCTTTCACTGACAGG
rmpA_rev CTTGCATGAGCCATCTTTCA
N.B.
All PCR reactions were done using (A200 Gradient LongGene thermal
cycler, LongGene Scientific Instruments Ltd, China).