Removal of Ciprofloxacin ( CIP ) by bacteria isolated from hospital effluent water and identification of degradation pathways

Most antibiotics are metabolized incompletely by patients after administration and enter the municipal sewage with the patients’ excretion. Therefore, studies on the biodegradability of some clinically important drugs can be taken as a very first step of an environmental risk assessment. The present study reports the biodegradation of CIP by Lactobacillus gesseri, Enterobacter sp., Bacillus sp., Bacillus subtilius and Micrococcus luteus which were isolated as CIP resistance, non pathogenic bacteria. The presence of antibioticresistant bacteria was identified using the 16s rRNA sequencing. A 0.5ml of overnight starved bacterial suspensions was introduced into medium containing CIP at 5 ppm. Triplicate samples were incubated at 28 0 C with shaking at 100ppm. A 0.5 ml of subsamples was removed at 2 days interval for a period of 14 days. Samples were subjected to High Performance Liquid Chromatography (HPLC) analysis. Fourier Transform Infrared Spectroscopy (FTIR) analyses were carried out for each sample at the end of the 14 days to find structures of by-products. Complete degradation of CIP by L. gasserri was detected at the end of 14 days of incubation with average degradation rate of 0.182 ±0.15μg /day. Descending degradation rates were followed by Enterobacter sp. (0.75 ±0.03 d -1 ) and Bacillus sp. (0.41±0.02d -1 ) at 8 and 6 days respectively. However, clear cut degradation of CIP was not detected for B.subtilis and Micrococcus luteus respectively. Further, FTIR spectrum revealed that incubation of L. gesseri, Enterobacter sp. and Bacillus sp., changed the piperazine ring and quinolone part in the CIP structure while degradation occurred. Keywords— Ciprofloxacin, Degradation, Fourier Transform Infrared Spectroscopy (FTIR), High Performance Liquid Chromatography (HPLC), Lactobacillus gesseri.


Removal of Ciprofloxacin (CIP) by bacteria isolated from hospital effluent water and identification of degradation pathways
Thus, the aims of the present study were to isolate the CIP degradation bacteria and study the degradation kinetics of CIP along with determination of structural changes during the degradation pathways.Further, Ciprofloxacillin was selected to represent flouroquinolones, because they are the most widely used antibiotic at present in most part of the world.

II.
MATERIALS AND METHODS Chemicals and reagents CIP standards (98%), HPLC and Bacteriological grade chemicals were purchased from Sigma Aldrich, USA.

Sample collection and enrichment study
Triplicate effluent water samples were collected from 75 sampling locations: including teaching hospitals, general hospitals, base hospitals and divisional hospitals.50ml of effluent water from each sampling sites were enriched spiking, CIP antibiotic at final concentration of 60ppm in 100 ml erlenmeyer flasks and the final volume was topped up to 100 ml with sterile water and then the flasks were subjected to incubate at 28 0 C ±

Screening of CIP degrading bacteria
The identified non pathogenic CIP resistance bacteria were transferred into 5 ml of liquid LB medium and incubated overnight at 28 0 C. Then followed centrifugation to remove carbon source from the LB media and then the bacteria suspension was subjected to the starvation procedure, in phosphate buffer solution (PBS).Thereafter, equalized the turbidities of the bacteria suspension at A 590 = 0.35 (Manage et al., 2009).150 μl of equalized bacterial suspension was inoculated into micro wells of the MT2 plates.The oxidization of the The concentration of CIP was analyzed using the equations given below.The peak area of the sample, (peak area of CIP = A CIP ), and the slope and intercept of each calibration curve was used to calculate the concentration of antibiotic in unknown samples (concentration of CIP =C CIP ).C CIP = (A CIP -10.451) / 1.5672

FTIR analysis
The structural analysis of degradation product was obtained from FTIR in each sample at the end of 14 days of incubation (Wackerlig, & Schirhagl., 2015).

III. RESULTS AND DISCUSSION
Quinolone antibacterial drugs have many characteristics, such as broad antibacterial spectrum, high bactericidal activity, low toxicity and unique mechanism.Norfloxacin, enoxacin, ciprofloxacilin and ofloxacin are the representatives of the third generation antibiotics and have wide antibacterial spectrum (Luo et al., 2011).Based on 16S rRNA gene sequence analysis with morphological characteristics and biochemical tests, the CIP degrading bacterial strains were identified as Enterobacter sp.(KM504128), Lactobacillus gasseri (KM4055978), Bacillus sp.(KM504129), B. subtilis and Micrococcus luteus respectively.Due to an incomplete elimination, antibiotics have been introduced to the environment and their residues have been found both in waste and urban waters.Mathew and Unnikrishnan, (2012), reported that the CIP concentrations in the effluents waste water treatment plant was up to 31mg/l in pharmaceutical industries in India.During the wastewater treatment, 80 -90% of CIP is removed via sorption to sludge, which stabilizes the substance (Mathew and Unnikrishnan, 2012).Golet et al., (2003) found that 83±14% of CIP in the anaerobically digested sludge and may have resulted in the accumulation of antibiotics in the bio-solids treated soil.2010).Thus, in the present study, this may be the reason to get low degradation rates at the beginning and with the time, degradation rates increased (Table 1).The infrared spectra of samples at 0 day and at 14 days are shown in figure 2.  Regarding the fluoroquinolones, several studies on pure water solutions revealed that photodegrdation takes place mainly via substitution on the piperazinyl ring in the position 7 (figure 1) (Paul et al., 2010) where, additional experiments revealed that the presence of organic and microbiological matter in water solution significantly affects the degradation of fluroquinolones (eg: CIP) (Parshikov et al., 1999;Paul et al., 2010).The compounds containing a β-lactam ring (cefotian hydrochloride, penicillin G and etc) could have been degraded chemically by hydrolactic ring opening without oxygen consumption.Thus, it was clear that the most common degradation pathways for the CIP, may be mediated by hydroxyl radical attack.It is known that the degradation of CIP occurs at least at two functionalities of the CIP molecule; the piperazinyl substituents and quinolone moiety (Lissemore et al., 2006;Dewitte et al., 2008Santoke et al., 2009;Wei et al., 2013) The obtained spectrums (figure 2) were compared to those previously reported results in the literature, and the spectrums correspond for bacterium L. gesseri, Enterobacter sp. and Bacillus sp. were found to be very similar to CIP degradation products recorded by others' studies (Prieto et al., 2011;Parshikov et al., 2001).These compounds correspond to the partial and complete elimination of the piperazynilic ring of the ciprofloxacin molecule which were found in the present study (Figure 2 (b), (c), (d)).The decrease of the vibrations that correspond to piperazine ring compared with initial FTIR spectrum of CIP (figure 2 (a)] suggest that multiple sites on the piperazine ring were oxidized and potentially forming carboxy groups.Guo et al., (2013), recorded in their experiment that the antibacterial activity in the sample was generally decreased by the bacteriological treatment and the products of the quinolone ring structure remained.These results also suggested that the cleavage of the piperazinyl ring is capable of eliminating the antibacterial activity of CIP.However, complementary analytical techniques, such as nuclear magnetic resonance (NMR), and/or use of authentic standards are needed in future work to further confirm the structure of CIP transformation product.

IV.
CONCLUSION As one of the third-generation antimicrobial quinolones, ciprofloxacilin, possessing broad spectrum of antimicrobial activity, wide application in clinical treatment of infections and diseases caused by gram negative and positive bacteria.L. gasseri efficiently degraded (100%) CIP resulting in a complete removal of CIP from the sample while Enterobacter sp., Bacillus sp., B. subtilius, M. luteus degraded 96%, 74%, 70%, 56% respectively.The FTIR spectrums of each bacterium showed the changes in antibacterial active sites (piperazine ring and quinolone part) of CIP and confirmed the degradation of CIP by the bacteria isolated.Therefore, it can be concluded that the five bacterial strains are the potent candidates that can be introduced into waste water effluents to remove CIP in effluent water before reaching natural environment.

Fig. 1 :
Fig.1: Degradation of CIP at 5 ppm .When error bars are not shown, standard deviation was less than the width of symbol (Open square; Enterobacter sp., Closed square; L. gasseri, Closed triangle; Bacillus sp., Open triangle; B. subtilis; Closed circle; M. luteus; Open circle; control)

Fig. 2 :
Fig.2: FTIR spectrums (a) Initial sample (0 day), (b) Treatment with L. gesseri at 14 days, (c) Treatment with Enterobacter sp. at 14 days, (d) Treatment with Bacillus sp. at 14 days, (e) Treatment with B. subtilis at 14 days, (f) Treatment with M. luteus at 14 days [ black arrows showed changing of functional groups from initial spectrum]

Figure 2 (
a) indicated the FTIR spectra for initial sample.Among the vibrations, those at 1473vibrations are attributed to carbonyl and hydroxyl in carboxyl group respectively.The 1396 cm -1 and 941 cm -1 vibration correspond to the bending of O-H and around 800 cm -1 indicate the absorption peak of secondary amine.Overall, the spectrum is indicative of CIP, since it is similar to standard spectrum of CIP containing HCl (Kummar and Min., 2011; Tan et al., 2012; Yan et.al., 2012) It can be observed in figure 2 (b) that the peaks in 3403 cm while intensity of peaks in 2921 cm -1 , 1396 cm -1 and 941 cm -1 increased compared to the initial spectrum, which accounts for bacteria degradation.Two new stretching absorption peaks International Journal of Medical, Pharmacy and Drug Research (IJMPD) [Vol-2, Issue-

International Journal of Medical, Pharmacy and Drug Research (IJMPD) [Vol-2, Issue-3, May-Jun, 2018] https://dx.doi.org/10.22161/ijmpd.2.3.1 ISSN: 2456-8015 (
G.Y. Liyanage and Pathmalal M. Manage* Centre for Water Quality and Algae Research, Department of Zoology, University of Sri Jayewardenepura, Sri Lanka Corresponding author:-pathmalal@sjp.ac.lk*Sahinturak et al., 2016).In fact, the occurrence of CIP in the output of a treatment water plant, in hospital wastewater and in surface waters has been reported (Shaheen et al., (Kummerer, 2009)., 2006)nce recently, the concern about potential ecological impacts of synthetic antibiotics has been increasing, because they may inhibit key environmental processes of nutrient regeneration, carbon and nitrogen cycles and pollutant degradation mediated by microorganisms (Rajilic-stojanovic et al., 2013).However, since mid-1990s, usage of these compounds was widespread and new analytical technologies were developed to quantify the amount of antibiotics(Lissemore et al., 2006).The introduction of these compounds into the environment through anthropogenic sources which can constitute a potential risk for aquatic and terrestrial organisms was documented(Kummerer, 2009).Quinolones represent a highly potent group of modern antibiotics, which were initially employed in the treatment of urinary tract infections in humans and animals(Avarin et  al., 2013).They are very effective against gram negative bacteria via inhibition of the DNA gyrase responsible for preservation of DNA.The 6-fluorinated piperazinyl derivatives namely fluoroquinolones, are a more effective second generation of this family (Avarin et al., 2013).

International Journal of Medical, Pharmacy and Drug Research (IJMPD) [Vol-2, Issue-3, May-Jun, 2018] https://dx.doi.org/10.22161/ijmpd.2.3.1 ISSN: 2456-8015 analyte
(Garland & Mills, 1991)colour reaction taking place in the well by the reduction of tetrazolium dye which can be measured and quantified spectroscopically(Garland & Mills, 1991).Each bacterial strain (150µl) was tested against 5 ppm of CIP in triplicates.The control wells in the BIOLOG MT2 plate contained CIP and sterile PBS in triplicates.Then the plate was wrapped with wet tissue Degradation kinetics of the isolated bacteria Among 22 bacterial isolates, five bacterial strains namely Lactobacillus gesseri, Enterobacter sp., Bacillus sp., Bacillus subtilius and Micrococcus luteus showed high absorbance values for the BIOLOG MT2 plate assay.Further selection for degradation kinetics studies were based on the results obtained from the BIOLOG MT2 plate assay.A 0.5 μl of equalized bacterial suspension was inoculated into filter-sterile freshwater, containing CIP at a final concentration of 5 ppm respectively.All flasks were incubated at 28°C with continuously shaking at 100 rpm.A 0.5ml of sub samples were collected at two days intervals for a period of 14 days.Then the subsamples were centrifuged (12000 rpm) and supernatant of each sample was subjected to immediate freezing and stored at -20°C.

Table .
1: Degradation rates of CIP concentration in 5 ppm along with incubation time (given as mean value of triplicates)

International Journal of Medical, Pharmacy and Drug Research (IJMPD) [Vol-2, Issue-3, May-Jun, 2018] https://dx.doi.org/10.22161/ijmpd.2.3.1 ISSN: 2456-8015
Nasland et al., 2008).Therefore, findings of the degradation processes are important in order to decrease the CIP contaminant amount in the waste water before it reached the natural environment.Once fluoroquinolones (eg: CIP) reach the aquatic environment, they are exposed to multiple environmental factors and may undergo various degradation processes.Bacillus sp. and B. subtilis (figure1) are unable to degrade the complex cyclic structure of CIP more than 75% and this has resulted in retarded degradation rates for CIP.Presence of benzene ring and piperazinyl ring in CIP could be the possible cause for the failure of complete removal of the antibiotic.This was slightly different from the amoxicillin degradability of the species in same genera.According to Liyanage and Manage (2015), B. cereus removed amoxicillin (AMX) completely from the samples after 8 days of incubation.
Limited number of research studies have found that some fungus and bacterial species could significantly transform CIP but at relatively low rates (Prieto et al., 2011; Parshikov et al., 2001).Prieto et al., (2011) reported that white rot fungi and brown rot fungi degraded more than 60% of CIP in tested samples.Many previous studies reported low biodegradation rates for antibiotics in their findings they degraded more than 50% of CIP from the samples (Kummerer et al., 2000; Rajilic-stojanovic et al., 2013).In the present study L. gasseri.M. luteus, Enterobacter sp., Bacillus sp. and B. subtilius were identified as potent CIP degradares.Unlike L. gasseri (100%) and M. luteus (96%); Enterobacter sp., It was recorded that CIP mainly reduced the microbial activity at the beginning, because it is a biostatic compound that targets growing microorganisms (Vasconcelos et al., 2009; An et al.,