|Year : 2017 | Volume
| Issue : 1 | Page : 109-112
Mutation pattern in the genome of Neisseria gonorrhoeae and its association with multidrug-resistant isolates from Delhi, India
Divya Sachdev1, Indu Kumari1, Manju Bala2, Vineet Kumar1, Daman Saluja1
1 Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, New Delhi, India
2 Training and Research Centre, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India
|Date of Web Publication||16-Mar-2017|
Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, New Delhi - 110 007
Source of Support: None, Conflict of Interest: None
Mutations in PenA, PorB, MtrE and MtrR genes responsible for antimicrobial resistance were checked in 27 drug-resistant clinical isolates of Neisseria gonorrhoeae (NG). Phenotype PIB (88.88%) and mutation at G120 and A121 positions of porB were recurrent. N122K, a novel mutation, was observed in PorB in three resistant isolates. Substitution H105Y in MtrR was widespread (37% of clinical isolates). The presence of a novel mutation (L33V), along with G45D mutation in MtrR, was associated with less-resistant isolates, in contrast to isolates with G45D mutation alone. African-type penicillinase-producing NG plasmid was observed most frequently (17/27) in penicillin-resistant isolates.
Keywords: Antibiotic resistance, mutations, Neisseria gonorrhoeae
|How to cite this article:|
Sachdev D, Kumari I, Bala M, Kumar V, Saluja D. Mutation pattern in the genome of Neisseria gonorrhoeae and its association with multidrug-resistant isolates from Delhi, India. Indian J Med Microbiol 2017;35:109-12
|How to cite this URL:|
Sachdev D, Kumari I, Bala M, Kumar V, Saluja D. Mutation pattern in the genome of Neisseria gonorrhoeae and its association with multidrug-resistant isolates from Delhi, India. Indian J Med Microbiol [serial online] 2017 [cited 2017 Jun 25];35:109-12. Available from: http://www.ijmm.org/text.asp?2017/35/1/109/202347
| ~ Introduction|| |
Neisseria gonorrhoeae (NG) is one of the major sexually transmitted diseases causing pathogens, with 80%–85% of cases occurring in developing countries. Continuous emergence of multidrug-resistant (MDR) strains as well as strains resistant to new drugs has made treatment difficult as administration of antibiotics is the only treatment regimen till date., Although resistant strains are reported from Delhi and other parts of the country, mutational patterns in genes, known to contribute resistance, is poorly understood. In the present study, we report mutations in penA, mtrR, mtrE and PorB genes in antibiotic resistance clinical isolates from India.
NG was isolated from endocervical swabs and urethral swabs, collected from female and male patients, respectively, visiting STI clinic of Safdarjung Hospital, Delhi, India, during October 2008–March 2009 (No. F. 50-2/Eth. Com./ACBR/11/2109). Antibiotic susceptibility was performed by E-test method (AB Biodisk, Solna, Sweden). Results showed that 13/27 (48.14%) isolates were tetracycline resistant whereas 17/27 (62.9%) showed high resistance towards penicillin. None of the isolates showed resistance or reduced susceptibility to ceftriaxone [Table 1]. All penicillinase-producing NG (PPNG) strains (17/27) were found to harbour African-type plasmid as showed amplicon of 3.2 kb [Figure 1] using primers described by Dillon et al. This is in contrast to the earlier report by Bhalla et al., wherein Asian-type plasmid was observed. The increased global travel may be responsible for this variation in profile over a decade.
|Table 1: Using specific primers all the four genes were amplified by polymerase chain reaction using genomic DNA of each clinical isolate, followed by DNA sequencing and amino acid analysis for identification of mutations|
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|Figure 1: Agarose gel (0.8%) indicating identification of amplified gene of penicillinase-producing plasmid of various clinical isolates using primers according to Dillon et al. (1999). Lane1represents Asian-type plasmid (isolated from WHO N strain), Lane 3 represents African-type plasmid (isolated from WHO M strain), Lane M is 1 Kb marker and Lane 4–6 represents various clinical isolates. All three clinical isolates showed a band of 3.1 kb indicating the presence of African-type plasmid.|
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PCR amplification of the various chromosomal genes was performed using 50–100 ng of purified gDNA as described by Veal et al. (for mtrR and mtrE) and Liao et al. (for penA and porB). The amplicons were gel purified, custom sequenced (Biolinkk, India) and translated to amino acid sequences (Gene Runner 3.0.) and compared with the known amino acid sequence of mtrR (NC_002946), mtrE (AE004969), penA (X07468) and porB (J03017) using clustalW software (http://www.ebi.ac.uk/Tools/msa/clustalw2/) for identification of mutations.
In the present study, 23/27 clinical isolates carried one or more mutations at G120 or/and A121 position of the loop 3 regions of PorB, well documented for increased MDR., PIB phenotype was more prevalent (24/27; 88.88%) than PIA phenotype (3/27; 11.11%). Among penicillin-resistant isolates (n = 17), substitution G120K, A121N/G/D (9/17; 52.94%) was most frequent double mutation while A121S/N (5/17; 29.41%) and G120D/K/N (2/17; 11.76%) mutations were observed in five and two penicillin-resistant isolates, respectively. Strangely, mutations were also observed in penicillin-susceptible isolates. No mutation was observed in isolates carrying PIA allele [Table 1]. Analysis of PorB mutations with tetracycline-resistant isolates (n = 12) indicated G120K, A121N/G/D double substitutions in three isolates (3/12; 25%) while five isolates carried A121S/N (5/12; 41.66%) mutations. G120N was observed in one resistant isolate whereas three tetracycline-resistant isolates carried wild-type genotype. Curiously, double mutations were also observed in tetracycline sensitive isolates (5/8; 62.5%) [Table 1]. A novel mutation N122K (Genebank number: KF939134.1) was found in three MDR isolates in conjunction with A121S mutation and N122K resides in loop 3 of PorB. The loop plays an important role in pore constriction formed by trimer of PorB, and mutations in the loop could alter the anionic properties of the pore and hence its function.
Mutations in the mtr operon are known to be responsible for MDR.,, In the present study, we observed 81.4% isolates (22/27) carried mutations either in the promoter region or in the coding region of MtrR. H105Y was most frequent (12/27) and was coupled with A/T deletion in promoter [11/12, [Table 1]. Although 8/12 H105Y mutants were penicillin resistant and PPNG positive, four isolates were PPNG negative and penicillin sensitive. A novel mutation L33V (Gene Bank number: KF685745) is observed in two penicillin-reduced susceptible and tetracycline-resistant clinical isolates coupled with G45D. These two isolates showed high resistance towards ciprofloxacin but were sensitive for azithromycin [Table 1].
We also studied the mutations in mtrE, a structural gene of the operon, and found K181E (residing in non-conserved region) in all clinical isolates, suggesting that K181E substitution has no direct contribution to antimicrobial resistance. Another mutation K191R was observed at the lining of intra-protomer groove of the pump, close to the amino acids which interact with the MtrC, the other protein of efflux pump. Comparing the MIC values for tetracycline in PPNG-positive isolates (isolate numbers 4461 and 2160 with 550 and 642), we found that isolates with K181E, K191R double mutants showed higher MIC values in comparison to K181E alone and K181E/I429S double mutant [Table 1].
We also analysed the mutation pattern of PenA gene encoding penicillin-binding protein that is known to impart multidrug resistance by altering acylation rate of β-lactams. [Table 1] shows the presence of mutations F504K, A510V and A516G and insertion of aspartic acid at 346th position in all the clinical isolates. P551 L was found in 14/27 (51.8%) isolates while none showed P551S substitution reported by Whiley et al. Polymorphic forms IX (11/26; 42.3%) and II (10/26; 38.46%) were most prevalent patterns in isolates from Delhi, in contrast to previous reports from outside India., Majority of the isolates having P551 L mutation were penicillin resistant and PPNG positive (11/14; 78.5%) or showed reduced susceptibility towards penicillin (2/14), except 3 (21%) that were penicillin sensitive. We also found three novel mutations, G550R, N562I (in penicillin-sensitive isolates) and S567T (in penicillin-resistant isolate), and observed new substitution at I566N along with the previously reported mutations [Table 1].
To conclude, plasmid-mediated resistance to penicillin is one of the major mechanisms of acquiring penicillin resistance in NG isolates in Delhi with African type of plasmid in all the resistant isolates. The pathogen has acquired multiple mutations in different genes to become resistant to various clinical drugs and is continuously acquiring new mutations such as N122K in porB, L33V in mtrR and G550R, N562 L and S567 in penA. Since some the mutations were present both in antibiotic-resistant and sensitive isolates, it is envisaged that several hitherto unexplored genes may also be playing a role in imparting high resistance.
Fellowship from CSIR (DS and VK) and ICMR (IK and DS) is greatly acknowledged. Funding from UGC (Grant No. 36-254/2008) to Prof. Daman Saluja is gratefully acknowledged. We are indebted to all the patients for their cooperation and Mrs. Leelamma Peter for technical assistance.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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