Molecular characterisation of antibiotic resistant genes and development of real time loop- mediated isothermal amplification for rapid detection of virulence gene (speB) in group a streptococci
Group A streptococcus is one of the major human pathogens responsible for over 600 million infections annually. This “flesh-eating bug” has caused more than 600,000 deaths per annum globally. Heightened concerns have been escalating among the scientists and physicians due to its ability to cause...
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| Format: | Thesis |
| Language: | English |
| Published: |
2020
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| Online Access: | http://ethesis.upm.edu.my/id/eprint/16175/1/FPSK%28p%29%202021%202%20T.pdf |
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| Summary: | Group A streptococcus is one of the major human pathogens responsible for
over 600 million infections annually. This “flesh-eating bug” has caused more
than 600,000 deaths per annum globally. Heightened concerns have been
escalating among the scientists and physicians due to its ability to cause serious
invasive disease and long-term sequelae. Loop mediated isothermal
amplification (LAMP) has been considered as a novel approach for its cost -
effectiveness, good reliability, high sensitivity results, and can be performed in a
rural setting with less sophisticated equipment. Thus, this study is aimed to
develop a rapid, reliable and cost -effective diagnostic approach for the detection
of streptococcal pyrogenic exotoxin B (speB) from GAS isolates by RT-LAMP
technique. Forty-three GAS isolates were obtained from stock culture with 31
(72.1%) and 12 (27.9%) isolates from non-invasive and invasive samples
respectively. Re-identification of these isolates was carried out using several
conventional methods and confirmed with 16s rRNA. Antimicrobial susceptibility
testing was performed using Kirby-Bauer disk diffusion method and interpreted
according to CLSI guidelines. Preliminary screening for antibiotic resistance
genes (tetM, lnuA, ermA, ermB, mefA) and virulence genes, (speB, and prtF1)
were performed using PCR amplification to choose variables for real time-LAMP
development. Some categorical variables were tested by Chi-square analysis
and p values less than 0.05 were considered significant. All GAS isolates (100%)
were sensitive to erythromycin and azithromycin. Twenty-five (58.1%) and 7
(16.3%) of them exhibited resistance to doxycycline and clindamycin
respectively. There were no inducible MLSB (iMLSB) or constitute MLSB
(cMLSB) or MS phenotypes detected among GAS isolates but an L-phenotype
7 (16.3%) was observed. Regarding virulence genes, 43 (100 %) and 20 (46.5%)
of GAS isolates carried speB and prtF1 genes, respectively. In addition, tetM
and lnuA genes were detected in all doxycycline and clindamycin-resistant isolates (100% for each). speB, the major cause of pathogenicity in GAS that
was detected up to 100% in the preliminary gene, was used as a marker to
progress with the development of real -time loop mediated isothermal
amplification (RT-LAMP). ATCC 19616, (S. pyogenes, positive control) and no
DNA template as negative control) were used after optimization of primers, time
and RT- LAMP reagents for the initial development. The optimal RT- LAMP
condition was obtained at 63◦C for 45 min using laboratory heating block and
Real-time turbidimeter machine (LA- 500 Eiken Company, Japan) respectively.
Forty- three clinical isolates of S. pyogenes as described earlier were also used
to verify the possibility of the RT-LAMP assay in the detection speB gene
followed by nine bacteria strains including Streptococcus pyogenes and other
non- GAS bacteria strains (from American Type Culture Collection) and clinical
isolates in evaluating the specificity and sensitivity following serial dilutions of 10-
2 to 10-6ng/μl. The detection limit of our RT-LAMP was 0.001ng/μl of the template,
showing higher sensitivity than conventional LAMP and PCR detection limit of 0
.000001 ng/μl and 0.001 ng/μL making it 100,000- folds more sensitive than PCR
and 100-fold more sensitive than conventional LAMP assay. The detection rate
of speB using RT-LAMP 100% while PCR was 93% with conventional PCR
primer set.
In conclusion, the improved rapidity for detection of the speB which contribute
greater percentages in GAS virulence among other factors by the RT-LAMP
technique with its specificity and sensitivity using less sophisticated equipment,
simple to perform and cost-effective, is expected to be a new frontier in the
reliable method for the diagnosis of S. pyogenes infection and will soon replace
other time- consuming and costly molecular assays. The technique particularly
is suitable for rural or community hospitals in developing nations with middle
income level. |
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