Computational protocol: Exploring quinolone resistance-determining region in Neisseria gonorrhoeae isolates from across India

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Protocol publication

[…] From November 2010 to October 2013, a total of 670 urethral swabs from male patients and 3500 endocervical swabs from female patients were collected and processed as a part of a multicentric study for the surveillance of AMR in N. gonorrhoeae. The six centres included (no. of isolates) All India Institute of Medical Sciences (AIIMS), New Delhi (n=43); Apex Regional STD Teaching, Training & Research Centre, VMMC and Sajdarjung Hospital, New Delhi (n=39); Government Medical College, Srinagar (n=1); Institute of Microbiology, Madurai Medical College, Madurai (n=6); Grant Medical College and Sir JJ Group of Hospitals, Mumbai (n=6) and Regional Institute of Medical Sciences, Imphal (n=9). Dr. Ram Manohar Lohia Hospital and Lal Bahadur Shastri Hospital (LBSH) served as additional sample collection sites (n=9) for AIIMS, New Delhi. LBSH was included only in the third year of the study to obtain data from NCR East region as well as border area of Uttar Pradesh. AIIMS, New Delhi, coordinated the network and also conducted the molecular studies. Culture and identification of N. gonorrhoeae was done as per standard protocol. The study was approved by the Institutional Ethics Committee, AIIMS, New Delhi.Antimicrobial susceptibility testing: Antimicrobial susceptibility testing was done using disc diffusion method as per Calibrated Dichotomous Sensitivity (CDS) technique against the following antibiotics - penicillin (0.5 IU), tetracycline (10 µg), nalidixic acid (30 µg), ciprofloxacin (1 µg), spectinomycin (100 µg), ceftriaxone (0.5 µg) and cefpodoxime (10 µg) (Oxoid Limited, UK). Azithromycin (15 µg) was initially tested as per Clinical and Laboratory Standards Institute guidelines but from August 2011 as per CDS technique. The minimum inhibitory concentration (MIC) of ciprofloxacin was determined for all isolates using E-test strips of ciprofloxacin as per the manufacturer’s instruction (bioMerieux, France). Based on the MIC values, the isolates were categorized as susceptible (MIC ≤0.03 µg/ml), less susceptible (MIC 0.06-0.5 µg/ml) and resistant (MIC 1-3 µg/ml). High-level resistance (HLR) was defined by MIC ≥4 µg/ml. World Health Organization (WHO) 2008 N. gonorrhoeae reference strains such as WHO F (ciprofloxacin-S), WHO M (ciprofloxacin-R) and WHO K (ciprofloxacin-HLR) were used as controls.Mutation studies: The molecular studies were carried out in all ciprofloxacin susceptible and less susceptible N. gonorrhoeae isolates and in about 50 per cent of the resistant and high-level resistant isolates. Genomic DNA was extracted by boiling method. DNA extraction was followed by amplification of gyrA and parC gene. The gyrA PCR was performed in 50 μl of reaction mixture containing reaction buffer (10 mM KCl & 10 mM HCl), 1.5 mM MgCl2, 200 µM of dNTP mix, 2 unit Taq DNA polymerase, 12 picomoles of each of the two primers (forward and reverse) and 8 μl of DNA. Reaction conditions were as follows: initial incubation at 93°C for five minutes, followed by 35 cycles of 93°C for 30 sec, 56°C for one minute and 72°C for one minute and finally a hold at 72°C for five minutes. These primers amplified gyrA gene from nucleotides 160-438. The amplified PCR product (278 bp) was separated by electrophoresis on 1.5 per cent agarose gel. Similar reaction mixture and reaction conditions except primers were used for amplification of parC gene. The primers amplified parC gene from nucleotides 166-420. The amplified PCR product (255 bp) was separated by electrophoresis on 1.5 per cent agarose gel and visualized by ethidium bromide staining.Gel extraction was performed by Qiagen MinElute Gel Extraction kit (Qiagen, The Netherlands), according to the manufacturer’s procedural directives. Purified PCR products of gyrA and parC genes of all the isolates and WHO reference strains F, M and K were subjected to DNA sequencing by the dideoxy chain termination method, using the BigDye Terminator v3.1 cycle sequencing kit and model 3130XL Genetic Analyzer (Applied Biosystems, USA). Sequence analysis was performed by online available bioinformatics tools BLASTX (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastx&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) and LALIGN (www.ch.embnet.org/software/LALIGN_form.html).Statistical analysis: Fisher’s exact test was used to evaluate the variation between mutation pattern in gyrA and parC genes in N. gonorrhoeae isolates collected from within and outside Delhi. Further, it was used to determine the correlation between mutations and the level of resistance. All statistical analyses were done using STATA 12.1 (StataCorp LLC, USA) and results were considered significant at P<0.05. […]

Pipeline specifications

Software tools BLASTX, LALIGN
Application Nucleotide sequence alignment
Organisms Neisseria gonorrhoeae, Homo sapiens
Diseases Infection
Chemicals Ciprofloxacin