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Indian Journal of Medical Microbiology, Vol. 26, No. 2, April-June, 2008, pp. 148-150 Brief Communication Molecular characterization of genes encoding the quinolone resistance determining regions of Malaysian Streptococcus pneumoniae strains Kumari N, Subramaniam G, Navaratnam P, Sekaran SD Department of Medical Microbiology, Faculty of Medicine, University of Malaya Kuala Lumpur - 50603 Date of Submission: 08-Jan-2007 Code Number: mb08042 Abstract Genes encoding the quinolones resistance determining regions (QRDRs) in Streptococcus pneumoniae were detected by PCR and the sequence analysis was carried out to identify point mutations within these regions. The study was carried out to observe mutation patterns among S. pneumoniae strains in Malaysia. Antimicrobial susceptibility testing of 100 isolates was determined against various antibiotics, out of which 56 strains were categorised to have reduced susceptibility to ciprofloxacin (≥2 μg/mL). These strains were subjected to PCR amplification for presence of the gyrA, parC , gyrB and parE genes. Eight representative strains with various susceptibilities to fluoroquinolones were sequenced. Two out of the eight isolates that were sequenced were shown to have a point mutation in the gyrA gene at position Ser81. The detection of mutation at codon Ser81 of the gyrA gene suggested the potential of developing fluoroquinolone resistance among S. pneumoniae isolates in Malaysia. However, further experimental work is required to confirm the involvement of this mutation in the development of fluoroquinolone resistance in Malaysia. Keywords: Fluoroquinolones resistance; gyrA; Streptococcus pneumoniae Streptococcus pneumoniae is an important bacterial pathogen and is a major cause of otitis media, pneumonia, sinusitis, meningitis and septicaemia and is frequently associated with significant morbidity and mortality. [1] The development of multidrug resistance in S. pneumoniae has prompted the need for alternative therapies in the management of pneumococcal infections. This has led to the development of antipneumococcal fluoroquinolones such as sparfloxacin, levofloxacin, gatifloxacin and moxifloxacin. However, the broad usage of fluoroquinolones has been followed by emergence of resistance to fluoroquinolones, which is mainly due to point mutations in genes encoding the subunits of the drugs′ target enzymes, DNA gyrase and topoisomerase IV. Quinolones are thought to form a topoisomerase-drug-DNA ternary complex [2] that cellular processes convert into a lethal lesion, possibly a double-stranded DNA break. [3] Mutations may occur in a short discrete segment of the DNA gyrase, gyrA and gyrB genes and analogous parts of the topoisomerase IV, parC and parE genes, termed the quinolone resistance-determining region (QRDR). [4] This region has the highest sequence conservation between gyrA and parC . Previous studies have shown hot spots within the gyrA and parC genes causing quinolone resistance in S. pneumoniae , at gyrA Ser81 to Phe or Tyr and of parC Ser79 to Phe or Tyr. [4],[5] Therefore, in this study, we report the molecular characterization of the QRDRs genes of S. pneumoniae isolates in Malaysia Materials and Methods One hundred pneumococcal isolates were obtained from clinical samples processed in the Microbiology Laboratory of the University Malaya Medical Centre from March 1999 to December 2003. The isolates were obtained from invasive and non-invasive sites of both paediatric and adult patients. Samples were streaked onto 5% horse blood agar and incubated at 37°C with 5% CO 2 . Bacterial cultures isolated from these clinical specimens were stored in brain heart infusion broth supplemented with 10% glycerol at −80°C without antibiotics till further use. Each stock culture was kept in triplicates to avoid multiple passaging. The source of the isolates included blood, nasopharyngeal secretion, tracheal secretion, sputum and bronchoalveolar lavage. The identification of the strains was confirmed by sensitivity to ethylhydrocupreine disc (optochin), whereby all the 100 strains showed a diameter measurement of ≥14 mm. [6] The strains also showed to be positive by bile solubility testing and were catalase negative. The antibiotic susceptibility of the strains was tested on Mueller Hinton agar (Oxoid) plates containing 5% sheep blood (Oxoid), incubated at 37°C with 5% CO 2 using the agar dilution method described by the Clinical and Laboratory Standards Institute. [6] The antimicrobial agents used were penicillin, cefotaxime, ceftriaxone and erythromycin in powder forms and were obtained from Sigma Aldrich (Sigma Chemical Co, St. Louis, MO, USA). S. pneumoniae ATCC 49619 was used as control. Strains were also tested against ciprofloxacin (Bayer, Q.F. Barcelona, Spain), moxifloxacin (Bayer,Q.F, Barcelona, Spain), gatifloxacin (Bristol-Myers Squibb, New Jersey, USA) and levofloxacin (Daiichi, Japan). The criteria for resistance to ciprofloxacin were determined as ≥2 μg/mL. Genomic DNA was extracted from pure bacterial cultures using a previously described method. [7] Bacterial colonies suspended in 15 μL of distilled water containing 50 μg/mL lysostaphin (Sigma Chemical Co, St. Louis, MO, USA) were incubated at 37°C for 10 minutes. This was followed by addition of 10 μg/mL Proteinase K and 0.1 mM Tris HCL pH 7.5 and incubated at 37°C for another 10 minutes. Subsequently, the suspension was boiled for 5 minutes and finally centrifuged at 13000 rpm for 2 minutes. The supernatant obtained was used as the template in the PCR reaction. The primers used in this study were adapted from previously published sequences. [8] The optimal PCR condition for a 50 μL reaction included 1x PCR buffer, 1.5 mM MgCl 2 0.2 mM dNTP mix, 2U Taq Polymerase (Fermentas) and 20 pmol of each primer. The PCR cycling parameters were as follows: An initial denaturation step at 95°C for two minutes, 15 cycles of amplification performed as follows: denaturation at 94°C for 15 seconds, annealing temperature at 52°C for 30 seconds and extension temperature at 72°C for 15 seconds and finally completed with an extension at 72°C for two minutes. PCR products were purified using the PCR Purification Kit (Qiagen) and PCR DNA sequencing was carried out using an automated DNA sequencer (an ABI Prism 377 DNA sequencer, Perkin Elmer ABI). Results The pneumococcal strains were assigned as fluoroquinolone susceptible strains or fluoroquinolone reduced susceptibility strains based on the MIC values tested against ciprofloxacin, which was used as a marker of resistance in this study. Out of the 100 strains tested to ciprofloxacin, 48 strains had MIC value of 2.0 μg/mL, 8 strains had MIC value of 4.0 μg/mL, 38 strains had MIC value of 1.0 μg/mL and 6 strains had MIC value of 0.5 μg/mL. Ninety three per cent of the strains were sensitive to moxifloxacin, while 6% of the strains were intermediately sensitive to this drug. Sensitivity to levofloxacin was seen in 95% of the strains and intermediate sensitivity to levofloxacin was seen in 5% of the strains, while 88% were sensitive to gatifloxacin and 12% of the strains had intermediate sensitivity to this drug. All the 100 isolates were screened for the presence of gyrA , gyrB , parE and parC gene using PCR. Both fluoroquinolone susceptible and reduced susceptibility strains showed amplification of these genes. Eight representative strains were chosen and sequenced to identify mutations within the QRDR of the four genes. Sequencing of representative strains with various MIC levels to ciprofloxacin, levofloxacin, moxifloxacin and gatifloxacin identified mutations within the gyrA and parC gene. There were no mutations detected within the gyrB and parE gene. Only two strains (strains 98 and 99) had point mutation at position Ser81, with an amino acid substitution of serine to phenylalanine which has been previously reported to confer fluoroquinolone resistance. Strains S64 (MIC-CIP: 0.5 μg/ml0), 10 (MIC-CIP: 2.0 μg/ml0), 91 (MIC-CIP: 1.0 μg/ml0) and 1 (MIC-CIP: 1.0 μg/ml0 ) showed no mutation within the gyrA gene while strains 96, 97, 98 and 99 which had (MIC-CIP: 4.0 μg/ml0 ) had multiple mutation sites, some of which were silent with no amino acid substitutions. Other hot spots identified within the gyrA gene of strain 96 had substitution of amino acid sequences at position 99 (Met-Ile), 170 (Thr-Pro), 214 (Asp-Asn) and 224 (Lys-Asn). Strain 97 had amino acid changes at position 201 (Val-Met) and 202 (Thr-Pro) [Table - 1]. Sequencing of the parC gene showed multiple mutation sites in these strains with mutation site Asn38-Ser being common in all the strains. Strain 10 had two additional mutations within the parC gene which were Ala115-Val and Arg124-Ile, while strain 1 had the same additional mutation at position Arg124-Ile. Discussion The data revealed that fluoroquinolone resistance is yet to be detected among S. pneumoniae isolates in Malaysia. However, recent reports of resistance in other neighboring countries may be a heralding sign of emergence of fluoroquinolone resistance in Malaysia. The detection of the mutation at codon Ser81 of the gyrA gene does not confirm its contribution towards the development of fluoroquinolone resistance but could suggest that these strains could evolve to develop resistance upon further antibiotic pressure. Preliminary screening of S. pneumoniae strains for the specific mutation at Ser81 within the gyrA gene should be carried out to monitor the development of fluoroquinolone resistance in Malaysian S. pneumoniae strains. A prospective study should be carried out to study the evolution of these strains and further experimental work is required to confirm the involvement of this mutation in the development of fluoroquinolone resistance in Malaysia.Acknowledgement This work was supported by a grant from the Ministry of Science and Technology, Malaysia: 36-02-03-6027.References
Copyright 2008 - Indian Journal of Medical Microbiology The following images related to this document are available:Photo images[mb08042t1.jpg] |
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