Frequency of Chromosomally Encoded gyrA and parC Genetic Determinants of Fluoroquinolone Resistance in A. baumannii

Fluoroquinolones are administered as routine drugs of choice for treating complicated urinary tract infections caused by multidrug resistant Acinetobacter baumannii strains. It is now a world-wide issue that gyr and par induced quinolone resistance as one of the major drug resistance mechanisms. This investigation is thus aimed to assess the prevalence of quinolone resistance and to characterize the gyrA and parC producing strains of A. baumannii. Genomic DNA from 50 fluoroquinolone resistant A. baumannii were screened for gyrA and parC by PCR for the genetic relatedness with fluoroquinolone resistance, with sequencing of the representative strains. All the strains were positive for gyrA(100%) and 82% (n=41) for parC. Presence of parC was observed in 56.09% (n=23) ciprofloxacin resistant A. baumannii with 43.90% (n=18) in levofloxacin resistant A. Original Research Article Singh et al.; JPRI, 32(16): 18-24, 2020; Article no.JPRI.59689 19 baumannii. The findings of the present study showed the prevalence of fluoroquinolone resistance among A. baumannii in urinary tract infections and the frequency of gyrA and parC in inducing the resistance.


INTRODUCTION
Acinetobacter baumannii is considered as an ubiquitous gram negative non-fermentative bacilli documented as one among the six nosocomial pathogens by WHO [1]. Severe systemic complications associated with A. baumannii, among hospitalized patients in intensive care units manifesting various infections like pneumonia, urinary tract infections, endocarditis, post-operative wound infections and septicaemia have been documented world-wide [2,3]. Emergence of A. baumannii as a multi-drug resistant (MDR) strain worsens the treatment strategy and further control. Our earlier reports have documented the multi-drug resistance of A. baumannii against beta lactams and carbapenems which are induced by plasmid mediated genetic determinants [4,5]. The high level of resistance exhibited by A. baumannii against the routine antibiotics of choice such as cephalosporins, trimethoprim-sulfamethoxazole and carbapenem group of drugs in the recent decade had led to the implementation of fluoroquinolones as an empiric treatment especially for urinary tract infections [6].
Fluoroquinolone treatments for A. baumannii associated treatment encompass the administration of ciprofloxacin and levofloxacin, that targets the vital enzymes DNA gyrase and topoisomerase IV which are involved in bacterial viability. DNA damage and bacterial cell death is induced by fluoroquinolones by the formation of a topoisomerase-quinolone-DNA complex that has the ability to break the double stranded DNA blocking the DNA replication [7]. These enzymes are encoded by gyrA, gyrB, parC and parR respectively. In recent years, resistance to quinolones is slowly emerging and alarming due to the stepwise initial mutations in the gyrA and parC

PCR Amplification of gyrA and parC
PCR reaction mixture [15 µl] was prepared by adding 7.8 µl of 2x master mix [Taraka, Japan] in 5.6 µl of double distilled water with 0.31 µl of 100 pmol/ml concentration of the specific F'primer and R'primer [Eurofins Genomic India Pvt Ltd, Bangalore] of gyrA and parC genes. 1 µl of the DNA was added to the master mix and the amplification was performed with the PCR conditions as given in Table 1. PCR amplification was carried out in Eppendorfthermocycler, Germany. The resulting PCR amplicons were examined in 1% agarose gel electrophoresis containing ethidium bromide and was visualized by gel documentation system. The 100 bp DNA ladder was used to assess the PCR amplicon size.

Sequencing of the Genetic Determinants for Amplicon Confirmation
The amplicon product of gyrA and parCwere bidirectionally sequenced using Big-Dye terminator cycle sequencing kit and 3730XL Genetic  In the present investigation, we have selected a total of 50 isolates as fluoroquinolone resistant strains that showed 100% positivity for gyrA PCR. This result correlates with the earlier study conducted in Iran documenting 90% positivity for the gyrA mediated ciprofloxacin resistant strains of A. baumannii [13]. In addition, gyrA mediated ciprofloxacin resistance is high when compared to the efflux pump mediated resistance which is considered as a secondary mechanism in inducing ciprofloxacin resistance among A. baumannii [14]. This documents the high-level resistance against fluoroquinolones mediated by gyrA and parC which is normally associated with a double mutation rather than multiple mutations recorded in other gram negative bacilli such as E.coli [15,16].

RESULTS AND DISCUSSION
In the present study, we characterized the gyrA and parC from genomic DNA rather than plasmid DNA as most of the studies portrayed the fluoroquinolone resistance was mainly due to the chromosomal mutations in the quinolone determining regions (QRDRs) representing the intracellular targets for fluoroquinolones [17]. We also observed parC to be present in 82% of the strains which correlates with the earlier study that has documented a 100% presence in the ciprofloxacin resistant A. baumannii [18]. This might be associated with the mutations in parC playing a vital role in inducing fluoroquinolone resistance with a MIC >32μg/mL [6]. High levels of fluoroquinolone resistance documented in this study and in the earlier studies have also added the co-existence of efflux pumps to achieve these high levels of fluoroquinolone resistance [19].
Among the 50 isolates, 27 were characterized as ciprofloxacin resistant and 23 were as levofloxacin resistant by performing standard Kirby Bauer disc diffusion method as per CLSI guidelines (unpublished data). In a developing country like India, high levels of fluoroquinolone resistance have been documented by many reports based on the antibiotic susceptibility tests ranging from 65% to 83% [20]. Thus, the selection of the resistant strains for fluoroquinolone resistance was performed as per standards for further screening of the gyrA and parC genetic determinants. Among the fluoroquinolone resistant genetic determinants screened, co-occurrences of the gyrA and parC is also not uncommon. 14.6% (n=6) of the strains showed the presence of both gyrA and parC. Earlier studies have also documented the co-occurrences of the genetic determinants mediating fluoroquinolone resistance along with resistance for other groups of antibiotics too resulting in the emergence of multidrug resistant strains of A. baumannii

CONCLUSION
Present investigation emphasizes the frequency of the gyrA and parC mediated fluoroquinolone resistance amidst the A. baumannii urinary tract infections.

CONSENT
It is not applicable.
; Article no.JPRI.59689 gene from the present that was available in the database. The deleted regions ), mismatch as gap ( ) and conserved sequences as star (*) Present investigation emphasizes the frequency mediated fluoroquinolone A. baumannii isolates from

ETHICAL APPROVAL
It is not applicable.

ACKNOWLEDGEMENT
The author would like to acknowledge the help and support rendered by the Department of Microbiology and BRULAC of Saveetha Dental College and Hospital and the management for their constant assistance.