Agrawal Parul, Kapila Ketoki, Kumar Satish, Ghosh A N, Maurya Anil Kumar
Department of Microbiology, Armed Forces Medical College, Pune 411 040, India.
Indian J Pathol Microbiol. 2010 Jul-Sep;53(3):509-12. doi: 10.4103/0377-4929.68299.
Enteric fever is an ongoing problem in the developing nations. Resistance and reduced susceptibility to ciprofloxacin narrows the therapeutic options in enteric fever. The present study was carried out with the objective of determining molecular basis of resistance to fluoroquinolone among the clinical isolates of Salmonella enterica serovar Typhi from different parts of India.
A total of 60 S.Typhi clinical isolates were subjected to antimicrobial susceptibility testing and determination of minimum inhibitory concentration (MIC) to ciprofloxacin and nalidixic acid. Polymerase chain reaction (PCR) for GyrA gene followed by restriction fragment length polymorphism (RFLP) with restriction enzyme (RE) SSiI was performed to detect mutation at position Ser83. Further confirmation of mutation was done by nucleotide sequencing of GyrA gene.
Isolates showed 100% sensitivity to first-line drugs ampicillin, chloramphenicol, and cotrimoxazole. Twelve of the 60 isolates (18%) were susceptible to nalidixic acid (NASST) and the remaining 48 (82%) were resistant to nalidixic acid (NARST). Of these 48 NARST strains, 46 (97.5%) had reduced susceptibility to ciprofloxacin (MIC 0.25-1.0 microg/mL), whereas 2 strains (2.75%) were resistant to ciprofloxacin (MIC 4.0 microg/mL). In RFLP analysis, all the NASST strains showed 3 fragments, whereas all the NARST strains showed 2 fragments due to the loss of 1 restriction site as a result of mutation. All the NARST strains with reduced susceptibility to ciprofloxacin (n = 46) had a single mutation in gyrA gene (Ser 83-->Tyr or Ser 83-->Phe), whereas double mutations (Ser 83-->Phe and Asp 87-->Asn) were found in each of the 2 ciprofloxacin-resistant strains. None of the NASST strains (n = 12) revealed any mutation.
Our study exemplifies the correlation between nalidixic acid screening test, MIC values, and the detection of mutation in GyrA gene by PCR-RFLP with a novel RE SSiI.This was further confirmed by nucleotide sequencing.
伤寒热在发展中国家仍然是一个持续存在的问题。对环丙沙星的耐药性和敏感性降低限制了伤寒热的治疗选择。本研究旨在确定来自印度不同地区的肠炎沙门氏菌伤寒血清型临床分离株对氟喹诺酮耐药的分子基础。
共对60株伤寒沙门氏菌临床分离株进行抗菌药物敏感性测试，并测定其对环丙沙星和萘啶酸的最低抑菌浓度（MIC）。进行GyrA基因的聚合酶链反应（PCR），随后用限制性内切酶（RE）SSiI进行限制性片段长度多态性（RFLP）分析，以检测第83位丝氨酸的突变。通过GyrA基因的核苷酸测序进一步确认突变。
分离株对一线药物氨苄西林、氯霉素和复方新诺明显示出100%的敏感性。60株分离株中有12株（18%）对萘啶酸敏感（萘啶酸敏感伤寒沙门氏菌，NASST），其余48株（82%）对萘啶酸耐药（萘啶酸耐药伤寒沙门氏菌，NARST）。在这48株NARST菌株中，46株（97.5%）对环丙沙星的敏感性降低（MIC为0.25 - 1.0微克/毫升），而2株（2.75%）对环丙沙星耐药（MIC为4.0微克/毫升）。在RFLP分析中，所有NASST菌株显示3个片段，而所有NARST菌株由于突变导致1个限制性位点缺失而显示2个片段。所有对环丙沙星敏感性降低的NARST菌株（n = 46）在gyrA基因中都有一个单一突变（Ser 83→Tyr或Ser 83→Phe），而在2株环丙沙星耐药菌株中均发现了双重突变（Ser 83→Phe和Asp 87→Asn）。12株NASST菌株（n = 12）均未显示任何突变。
我们的研究例证了萘啶酸筛选试验、MIC值与用新型RE SSiI通过PCR - RFLP检测GyrA基因突变之间的相关性。核苷酸测序进一步证实了这一点。
