Investigation for the Ciproﬂoxacin Resistance Genes Gyr A and Par C in E. Coli Isolates from Urinary Tract Infected Patients

Background and Objective: Gyr A and Par C genes, are known to cause resistance especially in Alterations in proteins of ﬂuoroquinolones. Here, we investigated human pathogens Escherichia coli causing Urinary Tract Infection [UTI] to explore the possible link between the abundance of mutations, and the exposure to ﬂuoroquinolones. In this study, we investigated the occurrence of Gyr A and Par C gene producers among Quinolones resistant [QR] Escherichia coli isolated. Methods: 148 Urine samples were collected from a patients with UT infections. Phenotypically, Ciproﬂoxacin resistance was screened by micro broth dilution method. Multiplex PCR was carried out to determine the mutations in Gyr A and Par C genes. We have determined partial sequences of the Gyr A and Par C genes of E. coli including the regions analogous to the quinolone resistance-determining region of the E. coli Gyr A gene. Results: Out of 148 urine samples, 100 E. coli were isolated and identiﬁed. We analysed 20 quinolone-resistant strains for alterations in Gyr A and Par C . Of these, 11 Gyr A -positive isolates were identiﬁed using the Gyr A speciﬁc primers and were clearly Ciproﬂoxacin resistant. The other 9 Ciproﬂoxacin-resistant isolates were found to have Par C genes using speciﬁc primers. We observed an unexpectedly high prevalence of Gyr A than Par C in patients attending a tertiary care hospital by PCR with an estimation of 9.0% (95% conﬁdence interval). This study demonstrated that the number of mutations in QRs of Gyr A and/or Par C was signiﬁcantly associated with the MICs of quinolones (P<0.01). Conclusion: The Gyr A and Par C genes were detected predominantly in E. coli . The data emerging out of this study helps in understanding the dynamics of this infection and provide inputs for antibiotic policy in the treatment of urinary tract infections. based detection (9) , and single-stranded conformation polymorphism (SSCP). The present study is to check the prevalence, demonstrate the virulence factors and study the antibiotic susceptibility pattern of E. coli in our clinical settings. Studies on the prevalence of drug resistant E. coli have been done in the hospital. The data emerging out of this study helps in understanding the dynamics of this and provide inputs for antibiotic policy in the treatment of such infections.


Introduction
Escherichia coli is an important pathogen causing septicemia, wound, and urinary tract infections. Fluoroquinolones (FQs) are synthetic compound derivatives of quinolones that are currently one of the main classes of agent used for treatment of many types of bacterial infection, including E. coli infection (1) . FQs form complex with bacterial DNA Gyr Ase and topoisomerase IV, two crucial enzymes used during DNA-replication process, thereby inhibiting bacterial growth (2) . Ciprofloxacin is among the most frequently prescribed FQs, which was introduced into clinical use more than 30 years ago. It has been widely used to treat infections caused by bacteria due to its effective inhibitory activity against Gram-positive and Gram-negative bacteria, especially the Enterobacteriaceae (3) .
A plasmid-mediated Ciprofloxacin resistance gene, Gyr A, harbored by E. coli isolated from animals and hospital inpatients, was first reported in China (4) . Then it became popular all over the world, demonstrating a horizontal transfer mechanism for Ciprofloxacin resistance (5) . Additional novel plasmid-encoded Ciprofloxacin resistance genes were identified as well: Par C, identified in E. coli isolates and sharing 76.7% of nucleotide identity with Gyr A; identified in porcine E. coli isolates and sharing 45.0% of nucleotide sequence identity with Gyr A. (6) These genes (mention them) encode a phosphoethanolamine transferase family protein that modifies the lipid A component of LPS and confers a low level of Ciprofloxacin resistance (MIC=4-8mg/L) (7) .
Presently, nucleotide-sequencing analysis is a common method for the detection of mutations of Gyr A and Par C in the QRDRs. However, conventional sequencing is time-intensive and expensive. Various alternative methods to replace sequencing have been proposed, including polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) (8) , multiplex real-time (RT) based detection (9) , and single-stranded conformation polymorphism (SSCP).
The present study is to check the prevalence, demonstrate the virulence factors and study the antibiotic susceptibility pattern of E. coli in our clinical settings. Studies on the prevalence of drug resistant E. coli have been done in the hospital. The data emerging out of this study helps in understanding the dynamics of this infection and provide inputs for antibiotic policy in the treatment of such infections.

Methodology and research design
This is a prospective study conducted in the department of microbiology, Saveetha Medical College and Hospital, Thandalam, Chennai.

Sample size and sampling techniques
Continuous sampling method was used in the study, urine samples received in the clinical microbiology laboratory during the period of three months (July 2019 -October 2019) were included in the study. E. coli isolates were collected from urine specimens of hospitalized patients with suspected UTI, who had not yet received antibiotics, during the study period. To avoid testing multiple isolates from a single patient, E. coli was isolated in only one urinary culture from each patient. Urine specimens were collected by clean-catch midstream or from catheter in catheterized patients. In this interpretation informed consent is obtained by generally accessible information.

Sample processing
Microscopy Smears were prepared by placing a loopful of the sample on a clear glass slide and gram staining was done for microscopic examination.

Culture
All the samples were inoculated onto Blood agar and MacConkey agar and the plates were incubated at 37ºC. Biochemical Identification of E. coli was done with IMVIC tests.
Virulence factor testing -bio film [tissue culture plate assay (10) Isolates from fresh agar plates were inoculated in Trypticase Soy Broth and incubated for 24 hours at 37 • C, then diluted with fresh Trypticase Soya Broth in 1 in 100 dilution. Individual wells of sterile, polystyrene, 96 well & #8209; flat bottom tissue culture plate (TCP) wells filled with 0.2 mL aliquots of the diluted cultures and only broth served as control to check sterility https://www.indjst.org/ and nonspecific binding of media. The TCP was incubated for 18-24 h at 37 • C. After incubation content of each well was gently removed by tapping the plates. Then wells were washed four times with 0.2 ml of PBS (pH 7.2) to remove free & #8209; floating "planktonic" bacteria. Wells were stained with crystal violet (0.1%). Excess stain was rinsed off by washing with deionized water, and the plate was kept for drying. If bio film is formed by organisms, then wells are uniformly stained with crystal violet. Optical density (OD) of stained adherent bacteria was determined with a micro ELISA auto reader at a wavelength of 570 nm (OD 570 nm). Experiment was repeated thrice, and the data then were averaged, and standard deviation was calculated. The mean OD value obtained from media control was deducted from all the test OD values.

Minimum inhibitory concentration -broth dilution method (mic)
For Ciprofloxacin susceptibility testing, the isolates (n=105) were subjected to the broth microdilution (BMD) method, with susceptible E. coli ATCC 259226. The antibiotic pure substance, Ciprofloxacin sulphate powder, was obtained from Sigma-Aldrich.

Determination of minimum bactericidal concentration (mbc)
The minimum bactericidal concentration (MBC) is the amount of agent that will prevent growth after subculture of the organism to antibiotic free medium. An aliquot from each wells of microtitre plate was inoculated and streaked on to nutrient agar plate. The plates were incubated at 37ºC for 24 hours and the minimum concentration at which the bactericidal activity occurred were determined.

PCR (Polymerase Chain Reaction):
PureFast ® Bacterial DNA mini spin purification kit (Kit contains Lysozyme, Lysozyme digestion buffer, Proteinase-K, Binding buffer, Wash Buffer-1,Wash Buffer-2,Spin columns with collection tube and elution buffer. HELINI 2X ReDdye PCR Master Mix, Agarose gel electrophoresis consumables and Gyr A and Par C Primers are from HELINI Biomolecules, Chennai, India.
Primers used for PCR assay

Data Analysis
We calculated the frequency of identification of Gyr A and par C genes and their antibiotic resistance pattern for quinolones mutated genes positive bacteria. Pivot table function of Microsoft Excel 2016 was used to calculate the descriptive analysis (as a percentage), and the prevalence of Gyr A and par C harboring strains among the total strains including 95% confidence intervals (CIs), the total number of resistant isolates (number of resistant isolates/total number of positive isolates from same species) to each individual antimicrobial drug.
From the 100 E. coli strains 20 (3.80%) were found to be resistant to the Ciprofloxacin by broth dilution method, according to the CLSI guidelines.
Out of 100 E. coli, 4 isolates were in the MIC range of 0.25µg, 5 isolates were in the MIC range of 0.5µg, 3 isolates were in the range of 1µg, 3 isolates were in the MIC range of 2µg, 4 isolates were in the MIC range of 4µg, 1 isolates were in the MIC range of 8µg, 0 isolates were in the MIC range of 16µg.
Out of 100 E. coli, 6 isolates were in the MBC range of 0.25µg, 1 isolates were in the MBC range of 0.5µg, 3 isolates were in the MBC range of 1µg, 4 isolates were in the MBC range of 2µg, 2 isolates were in the MBC range of 4µg, 1 isolates were in the MBC range of 8µg, 3 isolates were in the MBC range of 16µg.

Molecular detection of Gyr A and Par C gene:
From 100 clinical E. coli isolates, we identified 20 isolates that exceeded the Ciprofloxacin resistance breakpoint (>2 mg/mL) using the agar dilution method. [ Figure 3 a.b] Full gene sequencing confirmed that all these 11 strains encoded Gyr A. The other 9 Ciprofloxacin-resistant isolates were found to have Par C genes using specific primers.
The MICs value and GenBank accession numbers of the E. coli strains have been mentioned in Table 3. The choice of E. coli in this study was to attribute its prevalence as a urinary tract infection. https://www.indjst.org/

Statistical analysis:
Overall, the Gyr A prevalence determined by both culture and PCR methods was estimated to be 9.0% (95% confidence interval of 5.7%-13.7%, Wilson score interval). This was statistically significant * (p=0.005).

Discussion
In the present study, during a period of six months from Nov 2019 to Feb 2020, 100 samples received to Clinical Microbiology Laboratory of Saveetha Medical College and Hospital were included. In this study, 100 Escherichia coli strains were collected. (100%) were from urine. A study done by N Prim et al, Barcelona, Spain (13) closely related to our study, in that study they isolated 76 Escherichia coli between January 2012 to March 2012 66 (90.85%) were from urine, 10 (9.5%)were from wound swab.
Biofilm producing bacteria are responsible for many recalcitrant infections and are difficult to eradicate. Biofilm production in E. coli promotes colonization and lead to increased UTI. Such infections may be difficult to treat as they exhibit multiple drug resistance. Ponnusamy et al. showed 69% isolates as biofilm producers by TM and TCP methods. Congo red method showed 59.4% strains to be biofilm producer. (14) Significant production of biofilm was seen in 67.5% isolates of E. coli in a study conducted by Sharma et al. by TCP method. In our study 49 were bio film producers and 51 were non bio film producers by tissue culture assay. (15) Antibiotic susceptibility testing was done by the following antibiotics-Amikacin, Ampicillin, Cefazolin, Cefoxitin, Cefotaxime, Cefepime, Cefoperazone sulbactam, Ciprofloxacin, Cotrimoxazole, Ciprofloxacin, Ertapenem, Gentamicin, Imipenem, Meropenem, Nitrofurantoin, Norfloxacin, Ofloxacin , Piperacillin tazobactam and Polymyxin B. The percentage of susceptibility were towards Amikiacin 78%. The percentage of susceptibility Gentamicin was 54%. The percentage of susceptibility for Ertapenem 47%.
The highest Resistance was noted for Ampicillin 78%. The resistance to Cotrimoxazole was 64% and resistance to Ciprofloxacin 60%. In the study of Kareem et al., (16) The antimicrobial agents tested included meropenem, imipenem, tigecycline, Ciprofloxacin, aztreonam, amikacin, levofloxacin, cefoperazone-sulbactam, cefotaxime, cefepime, and trimethoprim/sulfamethoxazole. The highest susceptibility were found to be towards the Cefoperazone-sulbactam 48% and the highest rate of resistance were found to be towards Amikiacin 82% which is least similar to our study.
When we talk about Ciprofloxacin resistance exclusively in our study Escherichia coli isolates were 1% resistance towards Ciprofloxacin by antibiotic susceptibility testing. The MIC showed 1% of the strains were resistance to Ciprofloxacin.
Out of 100 Escherichia coli isolates, four (4%) were resistance in MIC to Ciprofloxacin by Broth dilution method. Among 100 isolates four (3.8%) isolates which were showing resistance to Ciprofloxacin by MIC method were sent for molecular detection for Gyr A and Par C gene all 4 isolates were positive for both the genes. This study correlates Kareem et al., (16) the Gyr A gene was detected by PCR.

Conclusion
To conclude, since Disk diffusion and Vitek interpretation for Ciprofloxacin is not recommended, Minimum inhibitory concentration is the only method by which the susceptibility of Ciprofloxacin can be reported. Incidence of Ciprofloxacin resistance might be higher among MDR isolates for which Ciprofloxacin might be used in treatment. Hence, judicial use of this drug will help in preserving this drug usage in infections with multi drug resistant strains. Misuse and overuse of antibiotics can be prevented by constant monitoring of the antibiotic susceptibility testing for the bacterial isolates in the hospital and by framing antibiotic policy and initiating antibiotic stewardship program.