Introduction

Uropathogenic Escherichia coli(UPEC) is the foremost causative agents of urinary tract infections (UTIs). UPEC are able to quickly invade, survive and multiply within the host cells and tissues of the urinary tract (Lüthje and Brauner, 2014; Abdulla et al., 2016). About 60% of women in the United States will have at least one UTI during their lifetime (especially cystitis). Symptoms of an uncomplicated bladder infection (cystitis) can be relatively mild, including frequent or urgent voiding and suprapubic pain (Bent et al., 2002; Colgan et al., 2007). The classical virulence factors were extensively studied while the resistance to metals like copper and silver were studied with less details. Heavy metals such as silver and copper are considered toxic to bacteria and can be used in industry of medical disposable like silver compounds which are used to coat urinary catheters in order to prevent nosocomial UTIs. UPEC have a set of genes that confer resistance to many heavy metals and especially copper and silver (Gupta et al., 2001; Johnson et al., 2006). Copper and silver have an antibacterial (bactericidal) effects and the resistant to them regard as a virulence traits. Copper antibacterial functionality is associated with various mechanisms, including damaging the microbial DNA, altering bacterial protein synthesis and altering membrane integrity (Warnes et al., 2010; Grass et al., 2011; Chaturvedi and Henderson, 2014). The mechanism of antibacterial action of silver depends on their reaction with thiol group in vital enzymes and inactivates them or interacts with DNA, resulting in marked enhancement of pyrimidine dimerization by photodynamic reaction and possible prevention of DNA replication and also generation of toxic free-radicals (Feng et al., 2000; Kim et al., 2007; Chen and Schluesener, 2008; Prabhu and Poulose, 2012).

Generally E. coli, have three systems that confer resesitance or tolerance to copper ions. The first of these, CopA, is an inner membrane P-type ATPase that actively pumps excess copper out of the cytoplasm. The second system consists of the multicopper oxidase CueO, which may protect periplasmic enzymes from copper-mediated damage. Finally, the Cus system is responsible for copper and silver resistance in E.coli (Franke et al., 2003; Rensing and Grass, 2003). Escherichia coli, frequently utilize CusCFBA efflux complexes in the resistance-nodulation-cell division (RND) family to expel diverse toxic compounds from the cell. cusCFBA operon dependent on the concentration of copper and silver ions within the cell. Within the cusCFBA operon CusA encodes an inner membrane protein belonging to the resistance-nodulation-division (RND) family. Phylogenetic analysis shows that CusA belongs to a subgroup of RND proteins involved in heavy metal extrusion (Nies, 2003). CusB is a membrane fusion/adaptor protein (MFP) and CusC is an outer membrane (OM) protein belonging to the outer membrane factor (OMF family). Together, CusABC likely forms a protein complex spanning the inner membrane, periplasmic space and the outer membrane, analogous to the well-studied AcrAB-TolC multidrug efflux pump from E. coli. CusF is a small periplasmic protein that binds copper and silver and may subsequently transport these ions to CusB. Thus, the CusCFBA proteins form a tetrapartite system dedicated to the efflux of heavy metal ions from E. coli (Kittleson et al., 2006; Loftin et al., 2007; Bagai et al., 2008). The current study aims to investigate copper resistance (tolerance) phenotypically and presence of the outer membrane part, cusC, as an evidence for presence of cusCFBA efflux pump.

Materials and Methods

Bacterial Isolates

Twenty seven UPEC isolated and diagnosed previously (By Viteck2 compact system/Biomeruex), were taken from Advance Microbiology laboratory at college of science-university of Babylon, Iraq. All isolates were recovered from women with cystitis.

Determination of Minimum Inhibitory Concentration of Copper

Agar dilution method was used to determine the minimum inhibitory concentration (MIC) of copper against 27 isolates of UPEC according to CLSI 2016. Briefly Muller-Hinton agar plates were supplemented with one of the 20 concentration of copper sulfate pentahydrate (CuSO4x5H2O) (Scharlab/Spain). The concertrations were 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 ppm. A suspension of UPEC isolates were calibrated with 0.5 McFarland and then 100 μl inoculated on Muller-Hinton agar supplemented with copper sulfate pentahydrate and incubated for 24 h at 37^°C. The results of now growth were recorded as resistance and the least concentration of copper sulfate pentahydrate at which now growth appeared were recorder as MIC for copper (Reyes-Jara et al., 2016).

DNA Extraction

Genomic DNA of UPEC were extracted using FavorPrep Genomic DNA Mini Kit (Blood/Cultured Cell) according to instructions of manufactured company (Favorgen/Tiwan).

Primer Design, PCR and Gel Electrophoresis

The primer pairs used in this study were designed using primer 3 at Biology Workbench 3.2 (http://workbench.sdsc.edu). The designed primer pair then checked with primer-blast: (https://www.ncbi.nlm.nih.gov/tools/primerblast). The primer pairs sequence were cusC forward:5′-CGCCTTTAAAGAAGTGGCAG-3′ and cusC reverse:5′-CTGACGGGCATAATTCAGGT-3′. The PCR protocol were designed using Optimase Protocol Writer™. The 20 μl final volume of PCR reaction were mixed in 0.2 thin wall PCR tube containing 5 μl of premix, 5 μl of DNA, 3 μl of each of forward and reverse primer and the 4 μl of nuclease free water (Al-Alaq et al., 2016). The PCR conditions were 95^°C for 2 minutes, 30 cycles of 95^°C for 30 seconds, 58^°C for 30 seconds, 72^°C for 40 seconds. The final extension were 72^°C for 5 minutes. All tubes were place in Prime thermocycler (Techno/UK). The PCR products were electrophoresed using 1.5% agarose gel (Condalab/Spain) stained with SimplySafe, using 100 bp DNA ladder (Eurex/Poland)  and visualized using Quantum ST5 gel doc (Vilber/France).

Results and Discussion

The results of phenotypic investigation of copper resistance (tolerance) revealed that 16 (59.25%) of  UPEC isolated have MIC ˂= 500 ppm while the rest 11(40.75%) have MIC>=1500 ppm (Table 1). Our data disagree with those gathered by Reyes-Jara et al. (2016) who found that 93% of isolates were inhibited with less than 500 ppm of copper. Generally different studies reported different MIC values of copper to E. coli like Sabry et al. (1997) who found that the level of copper resistance was reported to be 2.5 mM. while the copper MIC for Escherichia coli was 10 mM as stated by Roane and Kellogg (1995). Koowatananukul et al. (2010) found that most E. coli (92.2%) had an MIC of 6.4 mM to copper sulfate.

Table 1: Distribution of MIC for copper among UPEC isolates.

Figure 1: 1.5% Agarose gel electrophoresis for cusC amplicon (217 bp). Lane M 100 bp DNA Ladder while U1-U11 represent UPEC isolates.   Click here to View figure

PCR results revealed that absence of cusC gene in  all UPEC isolated (16 (59.25%) that have MIC ˂= 500 ppm while it presence in all those have MIC>=1500 ppm (except U5 and U8) (figure 1). Although it have high MIC but U5 and U8 isolates have no cusC and may have another efflux system like  CopA or CueO (Franke et al., 2001; Grass and  Rensing, 2001; Macomber et al., 2007; Pontel and Soncini, 2009). Our results in accordance with Macomber et al. (2007) who found that E. coli protect it selves from copper-mediated oxidative stress prevent accumulation of significant intracellular concentrations of free copper ions by efflux.

As a conclusion this study state that the CusCFBA efflux system is the main copper extruding that can protect E. coli from oxidative damage by cooper.

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