High Prevalence of Ciprofloxacin Resistance in Community Associated Staphylococcus aureus in a Tertiary Care Indian Hospital

Bhavya Chakrakodi, Sushma Prabhakara, Savitha Nagaraj, Jerome Etienne, Gayathri Arakere

Reference Centre for Staphylococci, Hospices Civils de Lyon, Lyon, France;Faculte de Medicine Lyon Est, University of Lyon, Inserm U851, Lyon, France.
Society for Innovation and Development, Indian Institute of Science, Bengaluru, India.
St. John’s Hospital and Medical College, Bengaluru, India.

DOI: 10.4236/aim.2014.42018                

Abstract

We have studied the nature of ciprofloxacin resistance in methicillin sensitive and resistant Staphylococcus aureus among patients in a tertiary care hospital in Bengaluru, South India. All the isolates were highly resistant to ciprofloxacin. Molecular characterization of these samples performed using Staphylococcal Cassette Chromosome typing and multilocus sequence typing showed that 37.5% of total isolates and 59% of MRSA were sequence type (ST)772 and the rest were other STs. This indicates high prevalence of CA-MRSA in this tertiary care hospital serving the Indian community. Mutations responsible for ciprofloxacin resistance among these isolates in DNA gyrase (gyrA and gyrB) and topoisomerase IV (grlA and grlB) were analyzed by PCR amplification of specific fragments and sequencing. We found that for ST772 and five other STs present in this collection, single mutation in the gyrA gene, Ser-84→Leu, was sufficient for the high resistance. In vitro generation of ciprofloxacin resistance in two sensitive ST772 isolates by exposure to increasing antibiotic concentrations also resulted in the same single mutation of gyrA. The factors responsible for high ciprofloxacin resistance are varied and are dependent on the genetic background of the isolates and the environment. This is the first report on the mechanism of ciprofloxacin resistance among the most prevalent Indian CA-MRSA.

Keywords

Ciprofloxacin Resistance; Indian ST772 Staphylococcus Aureus; gyrA Mutations

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	H. F. Chambers and F. R. DeLeo, “Waves of Resistance: Staphylococcus aureus in the Antibiotic Era,” Nature Reviews Microbiology, Vol. 7, No. 9, 2009, pp. 629-641. http://dx.doi.org/10.1038/nrmicro2200
[2]	F. D. Lowy, “Antimicrobial Resistance: The Example of Staphylococcus aureus,” Journal of Clinical Investigation, Vol. 111, No. 9, 2003, pp. 1265-1273.
[3]	G. A. Jacoby, “Mechanisms of Resistance to Quinolones,” Clinical Infectious Diseases, Vol. 41, No. S2, 2005, pp. S120-S126. http://dx.doi.org/10.1086/428052
[4]	D. C. Hooper, “Emerging Mechanisms of Fluoroquinolone Resistance,” Emerging Infectious Diseases, Vol. 7, No. 2, 2001, pp. 337-341. http://dx.doi.org/10.3201/eid0702.010239
[5]	L. Fererro, B. Cameron and J. Crouzet, “Analysis of gyrA and grlA Mutations in Stepwise-Selected Ciprofloxacin Resistant Mutant of Staphylococcus aureus,” Antimicrobial Agents and Chemotherapy, Vol. 39, No. 7, 1995, pp. 1554-1558. http://dx.doi.org/10.1128/AAC.39.7.1554
[6]	E. Y. Ng, M. Trucksis and D. C. Hooper, “Quinolone Resistance Mutations in Topoisomerase IV: Relationship to the flqA Locus and Genetic Evidence that Topoisomerase IV Is the Primary Target and DNA Gyrase Is the Secondary Target of Fluoroquinolones in Staphylococcus aureus,” Antimicrobial Agents and Chemotherapy, Vol. 40, No. 8, 1996, pp. 1881-1888.
[7]	S. S. Costa, C. Falcão, M. Viveiros, D. Machado, M. Martins J. Melo-Cristino, et al., “Exploring the Contribution of Efflux on the Resistance to Fluoroquinolones in Clinical Isolates of Staphylococcus Aureus,” BMC Microbiology, Vol. 11, No. 10, 2011, pp. 241-252. http://dx.doi.org/10.1186/1471-2180-11-241
[8]	R. Sharma, C. L. Sharma and B. Kapoor, “Antibacterial Resistance: Current Problems and Possible Solutions,” Indian Journal of Medical Science, Vol. 59, No. 3, 2005, pp. 120-129.
[9]	R. S. Phakade, G. Nataraj, S. K. Kuyare, U. S. Khopkar and P. R. Mehta, “Is Methicillin Resistant Staphylococcus aureus Involved in Community Acquired Skin and Soft Tissue Infections? Experience from a Tertiary Care Centre in Mumbai,” JPGM, Vol. 58, No. 1, 2012, pp. 3-7.
[10]	F. J. Schmitz, M. E. Jones, B. Hofmann, B. Hansan, S. Scheuring, M. Luckefahr, et al., “Characterization of grlA, grlB, gyrA, and gyrB Mutations in 116 Unrelated Isolates of Staphylococcus aureus and Effects of Mutations on Ciprofloxacin MIC,” Antimicrobial Agents and Chemotherapy, Vol. 42, No. 5, 1998, pp. 1249-1252.
[11]	S. Shambat, S. Nadig, S. Prabhakara, M. Bes, J. Etienne and G. Arakere, “Clonal Complexes and Virulence Factors of Staphylococcus aureus from Various Cities in India,” BMC Microbiology, Vol. 12, No. 5, 2012, pp. 64-73. http://dx.doi.org/10.1186/1471-2180-12-64
[12]	N. D’Souza, C. Rodrigues and A. Mehta, “Molecular Characterization of Methicillin-Resistant Staphylococcus aureus with Emergence of Epidemic Clones of Sequence Type (ST) 22 and ST 772 in Mumbai, India,” Journal of Clinical Microbiology, Vol. 48, No. 5, 2010, pp. 1806-1811. http://dx.doi.org/10.1128/JCM.01867-09
[13]	S. Nadig, N. Velusamy, P. Lalitha, S. Kar, S. Sharma and G. Arakere, “Staphylococcus aureus Eye Infections in Two Indian Hospitals: Emergence of ST772 as a Major Clone,” Clinical Optholmology, Vol. 6, No. 1, 2012, pp. 165-173.
[14]	D. Baird, “Staphylococcus: Cluster Forming Gram Positive Cocci,” In: J. G. Collee, A. G. Fraser, B. P. Marmion and A. Simmons, Eds., Mackie and McCartney Practical Medical Microbiology, Vol. 2, 1996, pp. 245-261.
[15]	Clinical and Laboratory Standards Institute, “Performance Standards for Antimicrobial Susceptibility Testing,” 19th Informational Supplement. M100-S19, Clinical and Laboratory Standards Institute, Wayne, 2009.
[16]	G. Arakere, S. Nadig, G. Swedberg, R. Macaden, S. Amarnath and D. Raghunath, “Genotyping of Methicillin Resistant Staphylococcus Aureus Strains from Two Hospitals in Bangalore, South India,” Journal of Clinical Microbiology, Vol. 43, No. 7, 2005, pp. 3198-3202. http://dx.doi.org/10.1128/JCM.43.7.3198-3202.2005
[17]	D. C. Oliveira and H. de Lencastre, “Multiplex PCR Strategy for Rapid Identification of Structural Types and Variants of the Mec Element in Methicillin-Resistant Staphylococcus Aureus,” Antimicrobial Agents and Chemotherapy, Vol. 46, No. 7, 2002, pp. 2155-2161. http://dx.doi.org/10.1128/AAC.46.7.2155-2161.2002
[18]	Y. Kondo, T. Ito, X. X. Ma, S. Watanabe, B. N. Kreiswirth, J. Etienne, et al., “Combination of Multiplex PCRs for Staphylococcal Chromosome Mec Type Assignment: Rapid Identification System for mec, ccr, and Major Differences in Junk Yard Regions,” Antimicrobial Agents and Chemotherapy, Vol. 51, No. 9, 2007, pp. 264-274. http://dx.doi.org/10.1128/AAC.00165-06
[19]	C. Milheirico, D. C. Oliveira and H. de Lencastre, “Update to the Multiplex PCR Strategy for the Assignment of Mec Element Types in Staphylococcus Aureus,” Antimicrob Agents Chemotherapy, Vol. 51, No. 9, 2007, pp. 3374-3377. http://dx.doi.org/10.1128/AAC.00275-07
[20]	G. Lina, Y. Piemont, F. Godail-Gamot, et al., “Involvement of Panton—Valentine Leukocidin-Producing Staphylococcus Aureus in Primary Skin Infections and Pneumonia,” Clinical Infectious Diseases, Vol. 29, No. 5, 1999, pp. 1128-1132. http://dx.doi.org/10.1086/313461
[21]	P. Gilot, G. Lina, T. Cochard and B. Poutrel, “Analysis of the Genetic Variability of Genes Encoding the RNA IIIActivating Components Agr and TRAPb in a Population of Staphylococcus Aureus Strains Isolated from Cows with Mastitis,” Journal of Clinical Microbiology, Vol. 40, No. 11, 2002, pp. 4060-4067. http://dx.doi.org/10.1128/JsCM.40.11.4060-4067.2002
[22]	B. Shopsin, M. Gomez, S. O. Montgomery, D. H. Smith, M. Waddington, D. E. Dodge, et al., “Evaluation of Protein A Gene Polymorphic Region DNA Sequencing for Typing of Staphylococcus aureus Strains,” Journal of Clinical Microbiology, Vol. 37, No. 11, 1999, pp. 3556-3563.
[23]	M. C. Enright, N. P. Day, C. E. Davies, S. J. Peacock and B. G. Spratt, “Multilocus Sequence Typing for Characterization of Methicillin-Resistant and Methicillin-Susceptible Clones of Staphylococcus Aureus,” Journal of Clinical Microbiology, Vol. 38, No. 3, 2000, pp. 1008-1015.
[24]	H. Ito, H. Yoshida, M. Bogaki-Shonnai, T. Niga, H. Hattori and S. Nakamura, “Quinolone Resistance Mutations in the DNA Gyrase gyrA and gyrB Genes of Staphylococcus aureus,” Antimicrobial Agents and Chemotherapy, Vol. 38, No. 9, 1994, pp. 2014-2023. http://dx.doi.org/10.1128/AAC.38.9.2014
[25]	J. I. Yamagishi, T. Kojima, Y. Oyamada, K. Fujimoto, H. Hattori, S. Nakamura, et al., “Alterations in the DNA Topoisomerase IV grlA Gene Responsible for Quinolone Resistance in Staphylococcus aureus,” Antimicrobial Agents and Chemotherapy, Vol. 40, No. 5, 1996, pp. 1157-1163.
[26]	J. A. Lindsay, “Hospital Associated MRSA and Antibiotic Resistance—What Have We Learnt from Genomics?” International Journal of Medical Microbiology, Vol. 303, No. 6-7, 2013, pp. 318-323. http://dx.doi.org/10.1016/j.ijmm.2013.02.005
[27]	H. M. Blumberg, D. Rimland, D. J. Carroll, P. Terry and K. I. Wachsmuth, “Rapid Development of Ciprofloxacin Resistance in Methicillin-Susceptible and Resistant Staphylococcus aureus,” Journal of Infectious Diseases, Vol. 163, No. 6, 1991, pp. 1279-1285. http://dx.doi.org/10.1093/infdis/163.6.1279
[28]	F. B. Marangon, D. Miller, M. S. Muallem, A. C. Romano and E. C. Alfonso, “Ciprofloxacin and Levofloxacin Resistance among Methicillin-Sensitive Staphylococcus Aureus Isolates from Keratitis and Conjunctivitis,” American Journal of Ophthalmology, Vol. 137, No. 3, 2004, pp. 453-458. http://dx.doi.org/10.1016/j.ajo.2003.10.026
[29]	T. Takenouchi, C. Ishii, M. Sugawara, Y. Tokue and S. Ohya, “Incidence of Various gyrA Mutations in 451 Staphylococcus aureus Strains Isolated in Japan and Their Susceptibilities to 10 Fluoroquinolones,” Antimicrobial Agents and Chemotherapy, Vol. 39, No. 7, 1995, pp. 1414-1418. http://dx.doi.org/10.1128/AAC.39.7.1414
[30]	M. T. G. Holden, L. Hsu, K. Kurt, L. A. Weinert, A. E. Mather, S. R. Harris, et al., “A Genomic Portrait of the Emergence, Evolution, and Global Spread of a Methicillin-Resistant Staphylococcus aureus Pandemic,” Genome Research, Vol. 23, No. 4, 2013, pp. 653-664. http://dx.doi.org/10.1101/gr.147710.112