effect of Carbonyl Cyanide M-chlorophenylhydrazone on Ciprofloxacin Resistance and Biofilm Formation in Hospital-acquired Uropathogenic Escherichia coli and Klebsiella pneumoniae

Antibiotic resistant and biofilm forming uropathogenic enterobacteriaceae are rising. this study was conducted to evaluate the efflux pump and plasmid mediated efflux genes in ciprofloxacin (CiP) resistant hospital acquired uropathogenic Escherichia coli and Klebsiella pneumoniae . Also, to assess the anti-biofilm action of carbonyl cyanide m-chlorophenylhydrazone (CCCP). Uropathogenic E. coli and K. pneumoniae isolates were collected from Mansoura University Hospitals in Mansoura, egypt. the effect of Sub-minimum inhibitory concentration (MiC) of CCCP on CiP MiC was evaluated and the MiC decrease factor (MDF) was calculated. the presence of oqxAB and qepA genes was detected by PCR. the effect CCCP on biofilm was detected in strong biofilm formers. 56 and 47 CiP-resistant uropathogenic E. coli and K. pneumoniae isolates respectively were detected. Significant MDF by CCCP was observed in 55.3% of these isolates. the qepA gene was only present in E. coli . However, oqxAB genes were found only in K. pneumoniae . Biofilm formation was detected in 58.9% and 72.3% of CiP-resistant E. coli and K. pneumoniae isolates, respectively. Biofilm formation was significantly decreased by CCCP. According to these findings, CiP resistance and plasmid-mediated efflux pumps in uropathogenic E. coli and K. pneumoniae are of rising concern. efflux pump inhibitor CCCP represents a possible option to decrease the biofilm formation in these resistant urinary pathogens.


iNtRODUCtiON
Urinary tract infection (UTI) represents a predominant infection in both healthcare settings and community. 1The most common bacterial cause of UTI is Escherichia coli, but many other Enterobacteriaceae such as Klebsiella pneumoniae are also present. 2 T h e M u l t i d r u g -re s i sta nt ( M D R ) Enterobacteriaceae is increasing as a cause of UTI. 3 Biofilm formation plays a significant role in UTIs. 4 When these uropathogens colonize the mucous membrane of the urinary tract, they tend to form biofilm, which favors their persistence for a long time.In addition, microbial biofilms are impermeable to many antibiotics that contribute to the development of MDR bacteria. 5luoroquinolones (FQs) represent one of first-line agents for UTI empirical therapy. 6fflux pumps (EPs); QepA and oqxAB are important contributors of quinolones resistance in Enterobacteriaceae including E. coli, Klebsiella spp.These pumps actively extrude FQs from the cytoplasm.QepA is a proton-dependent transporter that belongs to the superfamily of major facilitators.QepA promotes resistance to quinolone, 7 whereas OqxAB is a multidrug efflux pump with transmissible resistance-nodulation division that decreases bacterial susceptibility to ciprofloxacin (CIP). 8 h e s e E P s c a n b e b l o c ke d b y in vitro inhibitors such as carbonyl cyanide 3-chlorophenylhydrazone (CCCP).The effect of EP inhibitor CCCP against resistant Gram-negative bacteria has been investigated. 9This study was aimed at finding out the role of EP in CIP resistance of E. coli and K. pneumoniae causing hospital acquired UTI.In addition, the prevalence of plasmid-mediated EP genes, biofilm, and the anti-biofilm effect of CCCP against these isolates were investigated.

Bacterial identification
A Prospective cross-sectional study was carried out for 18 months from March 2019 to August 2020 in Mansoura University hospitals (MUHs), Mansoura, Egypt.The Institutional Research Board at faculty of medicine, Mansoura University has approved this study (with a code number R.21.09.1463).Each patient included in this study signed an informed consent.Urine samples were collected from patients with suspected UTIs.Cystine-lactose-electrolytedeficient (CLED) agar (Oxoid-UK) plates were used to culture collected urine samples according to method described before. 10outine microbiological methods were used to identify bacterial isolates, biochemical reactions (Kligler iron agar, oxidase test, lysine iron agar, methyl red, Voges-Proskauer test, citrate tests, motility indole ornithine). 10API 20E (BioMerieux) was used to confirm the bacterial identification.

Antimicrobial Susceptibility testing
The antibiotic susceptibilities of E. coli and K. pneumoniae isolates were determined by disc diffusion method according to guidelines of Clinical and Laboratory Standards Institute(CLSI). 11he tested antibiotics include: gentamicin, ciprofloxacin, cefuroxime, cefotaxime, ceftazidime, cefoxitin, amoxicillin/clavulanic, piperacillin/ tazobactam, imipenem, trimethoprim/ sulfamethoxazole, and aztreonam.Quality control was done using E. coli ATCC®25922TM strain.Criteria for multidrug-resistant (MDR) phenotypes were defined as described before. 12

Determination of CiP and CCCP Minimum inhibitory Concentrations
Broth micro dilution assay was done for determining CIP and CCCP minimum inhibitory concentrations (MICs) in E. coli and K. pneumoniae isolates.The procedure and break points interpretation were done according to CLSI recommendations. 13Pure powders of CIP and CCCP were acquired from Sigma Aldrich (St. Louis, MO, USA).E. coli ATCC®25922TM strain was used as Quality control.

evaluating the effect of eP inhibitor CCCP on CiP Resistance
To detect the EP mechanism, the effect of EP inhibitor CCCP on CIP MIC was determined. 14IP MIC in CIP resistant isolates was re-measured in the presence of a sub-MIC of CCCP (i.e.0.5 × MIC) using serially rising concentrations of the antibiotics.
The MIC decrease factor (MDF) was calculated for each isolate using this formula; MDF = CIP MIC in the absence of CCCP/ CIP MIC in presence of CCCP. 14With MDF value of 4 or above, significant inhibition of EP by CCCP was considered (in the presence of CCCP, the CIP MIC drops fourfold or more). 14

Plasmid Mediated eP Genes
Plasmid EP genes were assessed in CIP resistant isolates as previously described by two PCR: one PCR for oqxAB genes7 and the other PCR for the detection of qep A gene8 (Table 1).

Detection of Biofilm
Biofilm formation assays were performed for CIP resistant isolates as previously described. 15vernight culture of isolates was adjusted to turbidity 0.5 McFarland standard.Then for each isolate, 125 microliters of 1:100 diluted bacterial suspensions were put in sterile flat-bottomed 96well microtiter plates and incubated at 37°C for 24 hours.The wells were washed thrice with 300 mL of sterile phosphate buffered saline (PH 7.3) and dried inverted at room temperature.For staining of the formed biofilm, 125 µL crystal violet of 0.1 percent was added for 10-15 minutes followed by rinsing with distilled water for three times.Uninoculated medium was used as control.The experiment was done in triplicates.A microtiter plate was read at the optical density (OD) 600 nm.The strains were then categorized using the following criteria: The cut-off OD (ODc)= mean OD of the negative control+ three standard deviations (SD).Biofilm were classified as strong biofilm producer when (OD more than 4×ODc); OD of non biofilm producer (OD equal to or less than ODc); weak biofilm producer (ODc< OD≤2x ODc); moderate biofilm producer (2 x ODc< OD≤4 x ODc). 16

the effect of Sub-MiCs of CCCP on Biofilm Formation
The effect of CCCP on the formation of biofilm were assessed on strong biofilm producing CIP resistant isolates of K. pneumoniae and E. coli.
The biofilm assays were done as above but with the addition of sub MIC CCCP concentration of each strain compared to biofilm formation in absence of CCCP as control.The microtitre plate was read at the OD600nm after 24 h incubation at 37°C.

Statistical Analysis
After the KS test for normality, quantitative data will be described as mean (SD) or median (range).parametric tests were used for normally distributed data, while non parametric tests were used for non normally distributed data.A P value is considered statistically significant when it is < 0.05.

Antibiotics Sensitivity testing
The highest resistance among E. coli isolates was cefuroxime (86.4%) followed by amoxicillin / clavulanic acid (76.1%) and the least was gentamicin (27.3%).Regarding K. pneumoniae, the highest resistance was found in amoxicillin / clavulanic acid (78.9%) and cefuroxime (76.1%) and the least was in piperacillin-tazobactam (47.9%).CIP resistance was reported in 63.6% of E. coli and 66.2% of K. pneumoniae isolates (Table 2).93% of the E. coli and 95% of K. pneumoniae were MDR.

Ciprofloxacin Resistance
Ciprofloxacin MIC values of isolated E. coli and K. pneumoniae ranged from 0.125 to 256 mg/L using the broth microdilution method.One hundred and three isolates were CIP resistant.CIP MIC estimates ranged from 1-256 mg/L.Sixty-four isolates (62.1%)of the isolates were high-level CIP resistance (MIC ≥ 32 mg/L), the MIC 50 and MIC90 were 32 mg/L and 256 mg/L, respectively.

Activity of eP
Reduction of fourfold or more CIP MIC in the presence of CCCP (MDF value of higher than 4) was detected in 57(55.3%)CIP resistant isolates.

Plasmid Mediated eP Genes in Quinolone Resistant isolates
As regard resistant K. pneumoniae, both OqxA and OqxB genes were detected together in 8 isolates, while OqxA gene only was detected in 12 isolates.OqxA nor OqxB was not detected in any of E. coli isolates.qepA was detected in 12 isolates of E. coli (Figure 1, 2).

Biofilm Formation in CiP Resistant isolates
Among CIP resistant isolates, biofilm formation was detected in 33 (58.9%) of E. coli and 34(72.3%)ofK. pneumoniae isolates.Six of E. coli in addition to 8 K. pneumoniae isolates expressed strong biofilm production capacity (Table 3).MICs of CCCP, CIP and resistance genes among strong biofilm producers were summarized in Table 4.

effects of CCCP sub-MiC on Biofilm Formation
The effect of sub-MIC concentration of CCCP was evaluated on biofilm forming capacity of strong biofilm producing isolates (six E. coli and eight K. pneumoniae isolates).The results showed that sub-MIC of CCCP significantly inhibited the formation of E. coli and K. pneumoniae biofilms (P equal 0.002 and 0.0001 respectively) (Figure 3).

DiSCUSSiON
Treatment of MDR bacteria in UTI represents a challenge in healthcare settings. 3MDR organisms expressed simultaneous resistance to common antibiotics that are used in the treatment of UTI such as aminoglycosides, beta-lactams and FQs that are considered a great concern. 17Plasmid exchange of resistance aggravates the problem and represents major threat to health care system. 18n this study, 93% and 95% of isolated urinary E. coli and K. pneumoniae were MDR, respectively.Similar high percentage of MDR in uropathogenic E. coli in a study that was conducted in Egypt, 90.85% were MDR, 19 but lower percentage of MDR E. coli was reported in previous studies in Saudi arabia 20 and Australia. 21esistance to broad spectrum 2 nd generation (Cefuroxime) cephalosporin and amoxicillin-clavulanic acid was found to be the highest in uropathogenic E. coli representing (86.4%, 76.1%) and in K. pneumoniae representing (76.1%, 78.9%).Resistance to 3 rd generation cephalosporin cefotaxime and ceftazidime in E. coli was 75%and 68.2%, respectively and in K. pneumoniae was 71.8% and 77.5%, respectively.Imipenem resistance was presented in about forty percentages (43.2%) of isolated uropathogenic E. coli and about seventy percentages (72.5%) of K. pneumoniae isolates.Resistance to beta lactam antibiotics is a global phenomenon, but occurrence rates are variable.Matching with the result of this study, high resistance rate to 2 nd and 3 rd generations cephalosporin was found by Tandogdu 22 in Klebsiella and by Esmaeel et al. 23 in uropathogenic E. coli isolates in Egypt.Similarly, high prevalence of amoxicillin-clavulanic acid resistance was detected in K. pneumoniae, 24 and in E. coli in Asian area. 22However, lower rate of resistance was detected in K. pneumoniae in Tunisia. 25bout 64.8% of the isolates were CIP resistant including 62.1% of the isolates that showed high CIP resistance (MIC >32).This result consistent with previous report. 23High rates of CIP resistance 75% and 91.4% was reported in urinary isolates of E. coli in other studies in tertiary hospitals.High CIP resistance was described previously. 26Lower rate of CIP resistance (33%) was reported in urinary K. pneumoniae. 24his high rate of resistance in this study to beta-lactam antibiotics and FQ could be due to their wide use as an empirical therapy for community acquired UTIs and gastrointestinal infection representing a selective pressure for development of resistance in patient's flora, which represents the main source of UTI infection.In addition, the use of these agents empirically to treat UTIs with the absence of antibiotic policy in the hospital may represent another factor for this high rate of resistance. 27umerous mechanisms are responsible for FQ resistance for example decreases in membrane permeability, efflux systems, and topoisomerase mutations. 28EP genes that transfer quinolones outside the bacterial cytoplasm are involved in plasmid-mediated quinolone resistance.Two of them are well known; qepAB and oqxAB. 29By transporting antibiotics across the membrane, these EP systems OqxAB increased CIP and norfloxacin MICs 32-and 64-fold, respectively, above the control strain.Because OqxAB conferred CIP resistance on other Enterobacteriaceae, the spread of this FQ resistance mechanism is very likely.
The over expression of OqxAB and QepA mediates resistance to many antibiotics including quinolone.This plasmid-borne MDR-EP may represent a major problem, as it may pose resistance to many antimicrobial agents in addition to the spread of this resistance via horizontal transfer. 30he efflux systems as a cause of drug resistance in Enterobacteriaceae is growing. 9EP inhibitor CCCP was used to reveal the contribution of EPs in CIP resistance and MIC of CIP was assessed in the presence of sub-inhibitory concentrations of CCCP.Reduction in CIP MIC (MDF ≥ 4) was as a principle for significance, efflux activity was present in 57 (55.3%) of CIP resistant isolates.Likewise, the role drug EP was detected in 47.5% of CIP resistant isolates of K. pneumoniae. 31While the role of EP was at lower rate that range from 12.1% and 18.3% of isolates of Enterobacteriaceae in other study. 14Higher rate of EP inhibition by CCCP was previously reported with CIP MIC reduction ranged from 2-64 fold in 93% of MDR k. pneumoniae. 32s regard plasmid-mediated EPs, QepAwas found in E. coli , while oqxAB was detected in K. pneumoniae only.OqxA gene was the highest prevalence in K. pneumoniae (20 isolates) including eight isolates had both OqxA and OqxB genes.qepA was found in 12 isolates of E. coli.The same was reported in a study conducted in CIP resistant isolates in Egypt. 14Also efflux pump qepA was detected in 12.3% of CIP resistant bacteria isolated from UTI. 33 Higher rate of oqxAB was detected in K. pneumoniae but it was not detected in E. coli.Moreover, QepA was not detected in any of Enterobacteriaceae species in a study conducted in South Africa. 34Also, QepA was not detected in other studies in India and South Africa. 26,35n this study, biofilm was produced in 60% and 75.5% of quinolone resistant E. coli and K. pneumoniae, respectively.A similar higher rate was reported in Egypt, 19 Ethiopia 36 and Iran, 37 while, a lower rate of biofilm production Klebsiella and E. coli was also described. 38,39his study assessed the ability of EP inhibitor CCCP to inhibit biofilm formation in strong biofilm forming isolates with EP activity.Biofilm formation was inhibited by sub-MICs of CCCP in both E. coli and K. pneumoniae.The same was previously in a study showed inhibition of biofilm formation of Salmonella typhimurium by CCCP. 40Also, a study investigates K. pneumoniae biofilm showed CCCP inhibition of biofilms in a dose-dependent effect.With the effective inhibitory concentration of CCCP towards K. pneumoniae EP was 10 μg/mL. 41

CONClUSiON
CIP resistance in hospital acquired uropathogenic E. coli and K. pneumoniae is of high prevalence and plasmid mediated EP is of rising concern.Biofilm development is a critical virulence determinant in these organisms.EP inhibitor CCCP represents a possible option to decrease the biofilm formation in these resistant urinary pathogens.

Figure 3 .
Figure 3. Impact of CCCP on the biofilm formation after 24h of incubation compared to control (a) E. coli (b) K. pneumoniae

table 1 .
Primers of plasmid EP genes

table 2 .
Antibiotic resistance pattern of uropathogenic E. coli and K. pneumoniae

table 4 .
MIC and plasmid mediated EP genes among strong biofilm producing CIP resistant E. coli and K. pneumoniae