Molecular characterization of silver resistant E. coli strains isolated from patients suffering from diarrhea

Silver nanoparticles (AgNPs) are considered a good alternative for antibiotics due to emerging Multidrug Resistance (MDR) crisis. Resistance to AgNPs is approximately limited among Gram-positive and Gram-negative pathogens. Low toxicity to human cells permits its safe use as a new antimicrobial with broad spectrum. Bacterial cells are used as a factory for AgNPs synthesis supplying a powerful antimicrobial with eco-friendly way. In this study, MDR Escherichia coli strains were recovered from patients attending Minia University hospital. Biogenic synthesis of AgNPs was performed using E. coli cells. Transmission Electron Microscopy (TEM) was used to characterize AgNPs size and shape. Antibacterial activity of AgNPs was tested against the MDR E. coli isolates. Screening for Sil and Omp genes was done using polymerase chain reaction (PCR). A total of 13 MDR E. coli bacterial culture supernatant isolates were recovered from patients under study. Biosynthesis of AgNPs was observed after addition of supernatant to AgNO3 by color change from yellow to brown. TEM characterization indicated the presence of silver nanoparticles with 15-75 nm particle size range. Eleven of MDR E. coli isolates were sensitive to biogenic AgNPs under study. SilB and SilE genes were encoded by the two AgNPs-resistant E. coli isolates which were negative for OmpF and OmpC genes, respectively demonstrating the role of Sil efflux pump genes and porin deficiency in AgNPs resistance. As indicated, the emergence of silver resistance due to the wide spread of biocides including silver has become a great challenge for the treatment of different infections.


INTRODUCTION
Bacterial infections caused by Escherichia coli are the most common between hospitalized patients including septicemia, urinary tract infections, enteritis and neonatal meningitis (Allocati et al., 2013).Prevalence of antimicrobial resistance among clinical bacterial isolates is growing everyday representing a great medical challenge for microbiologists and health care professionals all over the world (Prestinaci et al., 2015).Trials for finding effective alternatives for routinely used antibiotics are being made to overcome antimicrobial resistance crisis.AgNPs are promising alternatives which possess great antibacterial and antiviral activity.Antibacterial activity of AgNPs was greater against Gramnegative bacteria than Gram-positive ones, due to *Corresponding author.E-mail: dr_rehab010@yahoo.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License attraction between negative charges on Gram-negative lipopolysaccharide and weak positive charge on AgNPs (Franci et al., 2015).Green synthesis using bacterial cells is considered a friendly approach for creating AgNPs in a more economic and safe way (Ghorbani, 2013).Bacterial cell enzymes such as nitrate reductase reduce Ag + ions to AgNPs, which are indicated by colorimetric reaction (Rajesh et al., 2014).Antibacterial activity of biosynthesized AgNPs has been screened against different Gram-negative isolates including E. coli indicating observable inhibitory activity (Abu-Zaid, 2016).Resistance to AgNPs can be correlated to decrease permeability either due to porin deficiency in Gramnegative cell wall or due to presence of Sil genes which encodes efflux pumps (Gupta et al., 1999).Porins (outer membrane proteins) are encoded by Omp genes.OmpF and OmpC are two major proteins of E. coli outer membrane, these proteins have a role in small hydrophilic molecules passive diffusion through bacterial membrane (Matsuyama et al., 1986).Sil efflux genes consist of SilRS (transcriptional regulatory system which consists of two components), this system controls SilE (protein binds to periplasm).Another components are two efflux pumps SilP (a P-type ATPase) and SilCBA (three protein chemiosmotic RND Ag (I)/H* exchange system) (Silver, 2003).In the present study, AgNPs were biosynthesized using E. coli cells, antibacterial activity of AgNPs against clinical MDR E. coli isolates, recovered from patients suffering from gastroenteritis, was studied using agar well diffusion method and the presence of OmpC, OmpF, SilB and SilE, SilP, SilS genes were screened among recovered isolates.

Collection of samples
A total of 30 stool samples were collected from patients suffering from bacterial gastroenteritis attending department of accident and emergency in Minia University hospital (Minia, Egypt), samples were collected from March 2018 to April 2018.

Isolation of E. coli strains
Fecal samples were processed as follow: 3 g of fecal samples were mixed with normal saline, and centrifuged.The supernatants were discarded and the deposit was suspended in peptone water.Aliquots were used for the inoculation of MacConkey agar (Oxoid, UK), followed by an overnight incubation at 37°C.E. coli positive cultures were confirmed using biochemical tests (Himedia, India) (catalase positive, methyl red positive, indole positive, nitrate reduction positive and citrate negative) and the formation of metallic sheen on Eosin methylene blue agar.

Antimicrobial susceptibility testing
Amoxycillin/clavulanic acid, norfloxacin, azithromycin, cefoperazone levofloxacin, ceftriaxone, sulfamethoxazole/trimethoprime, cefuroxime, imipenem, tetracycline, cefipime, amikacin were used for testing antimicrobial activity against E. coli isolates.Disk method using Muller-Hinton agar plates was applied for antimicrobial susceptibility testing.By inoculating nutrient agar plates with suspension of inoculum and streaking of bacterial suspension using cotton swab.Antibiotic discs (Oxoid, UK) were applied after drying of inoculated nutrient agar plates.Diameters of inhibition zones were measured indicating sensitive or resistant isolates according to CLSI standards (CLSI, 2008).

Silver nanoparticles biosynthesis
E. coli culture was obtained from Microbiology and Immunology Department, Faculty of Pharmacy, Minia University.Nutrient broth was used to inoculate E. coli isolate.Supernatant was obtained by centrifugation of E. coli culture for 15 min at 5000 rpm and added to the flask of sterile aqueous AgNO3 (1 mM).The reaction was performed for 10 min in bright conditions (Ghorbani, 2013).

Silver nanoparticle isolation and purification
By centrifugation of mixture of supernatant and AgNO3 for 20 min at 10000 rpm, AgNPs were isolated.The AgNPs pellet was washed by centrifugation with sterile distilled water twice for 20 min at 1000 rpm, so the culture filtrate and excess silver ions were removed.Freeze drying was used to obtain AgNPs pellet as powder for further characterization and applications (Rajesh et al., 2014).

AgNPs characterization
Transmission electron microscope (TEM) (JEM1010, JEOL,Tokyo, Japan) was used to characterize shape and size of the isolated AgNPs.A nanoparticle solution drop was placed over carboncoated copper grids and left for water evaporation.Then, size and shapes of AgNPs were examined (Rajesh et al., 2014).

AgNPs antibacterial activity
Antibacterial screening was performed using disc diffusion method by preparing nutrient agar and nutrient broth culture of tested isolates.Both nutrient agar and broth cultures were mixed together.The mixture was poured onto sterile petridishes.AgNPs discs were prepared by sterilizing filter paper discs in autoclave, dipping into AgNO3 solution (10 µg/ml) and drying in air in sterile area.AgNPs discs were placed onto the nutrient agar after seeding with broth culture.After incubation for 24 h at 37°C, inhibitory activity of AgNO3 discs was indicated by presence of inhibition zones around the discs (Rosoanaivo and Ratsimamamanga-Urvery, 1993;Malabadi et al., 2005).Data entry and analysis were all done using software called Statistical Package for Social Science (SPSS) (IBM, U.S.A).

DNA extraction
The DNA template was prepared by boiling of suspension of bacterial pellet for 10 min and directly used in the PCR assay.Genomic DNA was extracted from E. coli overnight culture by method described by Wilson (1987).

PCR primers and condition
PCR reaction was performed in a total of 50 µl reaction as follows: DNA extract (5 µl), each primer (50 pmole), Go Taq Green Master

Prevalence of isolates
In the present study, out of thirty stool samples, 13 (43.3%)E. coli isolates were recovered.

Silver nanoparticles synthesis
Silver nanoparticles formation were detected by the change of color of AgNO 3 solution after addition of bacterial supernatant to yellow, orange then brown (Figure 1).

Silver nanoparticle characterization
AgNPs characters were detected by TEM, the TEM micrographs of biogenic AgNPs showing that particles are spherical with size ranges from (15-75) nm (Figure 2).

Antibacterial activity of AgNPs against MDR E. coli isolates
Our study shows that out of 13 E. coli isolates tested, 11 (84.6%) were sensitive to AgNPs which indicated by presence of inhibition zone with a diameter ≥ 1.6 cm around prepared AgNPs discs (Table 3).

Screening of Omp and Sil genes
Figures 3a and b show that among 13 E. coli isolates tested, one isolate was positive for SilB gene and another was positive for SilE gene but No strain was positive for SilP or SilS genes.Figure 4 shows that 11 E. coli isolates tested were positive for OmpC and OmpF genes and only two isolates were negative for OmpF (SilB positive) and OmpC (SilE positive).All isolates were negative for SilS and SilP genes.

DISCUSSION
Silver was known for longtime by its antibacterial and antifungal activity.By increasing the incidence of antimicrobial resistance between clinical pathogens, silver use as AgNPs has found a great application in healthcare facilities such as wound dressings and antimicrobial agents (Shutterlin et al., 2012).Bacterial cells are found to supply a good factory for AgNPs synthesis (Ghorbani, 2013).In the present study, AgNPs  were synthesized biogenically from E. coli strain by addition of bacterial supernatant to AgNO 3 solution and the reaction color change to brown within 10 min in the light suggesting that nitrate reductase enzyme of E. coli reduced Ag + ions to AgNPs.Formation of brown color was suggested to be due to surface plasmon vibration excitation in AgNPs.Extracellular reduction by E. coli was applied for AgNPs synthesis (Ghorbani, 2013).Lactobacillus acidophilus culture was filtered and used for AgNPs synthesis by Ag + reduction at room temperature within one day time (Rajesh et al., 2014).Also, Nitrate reductase of B. licheniformis has a role in AgNPs synthesis (Kalimuthu et al., 2008).AgNPs isolated in this study and characterized by TEM micrographs showed spherical shape with size ranges from 15-75 nm.Smaller size AgNPs shows antimicrobial activity greater than larger ones (Rai et al., 2012).Also, shape of AgNPs is of great influence as rod-shaped and spherical AgNPs are less effective as biocidals against E. coli than triangular ones (Pal et al., 2007;Sharma et al., 2009).Particle size (4-50 nm) with spherical shape also indicated for AgNPs synthesized from Lactobacillus acidophilus (Rajesh et al., 2014).In another study, it was reported that AgNPs size ranges from 10-100 nm (Ghorbani, 2013).
The previous findings are in agreement with the results obtained in the present study as AgNPs were biosynthesized extracellularly with particle size ranges from 10-80 nm.As illustrated in our study, AgNPs discs showed inhibitory activity against 11 (84.6%)MDR E. coli clinical isolates.Gram-negative bacteria such as Klebsiella pneumonia, E. coli and Gram-positive such as Staphylococcus aureus and Bacillus subtilis are affected by antibacterial activity of AgNPs (Malabadi et al., 2012).Confirmation with high antimicrobial activity of biogenic AgNPs against MDR E. coli was reported (Abu-Zaid, 2016).It was indicated that K. pneumoniae was affected    by Lactobacillus acidophilus-synthesized AgNPs antibacterial activity (Rajesh et al., 2014).AgNPs antibacterial activity against E. coli was reported (Cunha et 2016;Li et al., 2010).Mode of action of AgNPs as antimicrobial may be attributed to free radicals generation, also after penetration of AgNPs into the cells, oxidative stress was reported (Hussain et al., 2006).Particle size of AgNPs is influential in its antibacterial M action as E. coli was killed by low concentration of 16 nmsize AgNPs, which was found inside bacteria and it is cell wall adherent (Raffin et al., 2008).Also, E. coli membrane disruption by 20 nm-size AgNPs was illustrated after few minutes' exposure indicating effect of large surface area of AgNPs on its antimicrobial efficiency (Raffin et al., 2008).Yeast, Staphylococcus aureus and E. coli were inhibited and killed by AgNO 3 and Ag + ions (Kim et al., 2007).Heat shock protein expression of E. coli was altered by short AgNPs exposure (Lok et al., 2006).Due to safe AgNPs use against human cells and effective low concentration, AgNPs are more applicable as antimicrobial agent (Abo-Neima and El-Khaly, 2016).In addition to antibacterial activity, also cells infected with HIV were killed due to AgNPs antiviral activity (Sun et al., 2005).It was found that combination of AgNPs with antibiotic increase its antibacterial activity (Ingle et al., 2009, Rathod andRanganath, 2011).MDR E. coli and Streptococcus species showed great response to AgNPs antimicrobial action (Lara et al., 2010).All previous reports agree with our results as AgNPs showed good antibacterial activity against MDR isolates.In another finding, E. coli isolates showed no response to AgNPs indicated by absence of inhibition zone which is in a disagreement with results obtained in our study (Inbaneson et al., 2011).Our study shows that 11 isolates were positive for OmpC and OmpF genes as it confirms the presence of porin channels among 11 AgNPs-sensitive tested E. coli isolates.Also, absence of OmpC and OmpF genes is observed among AgNPsresistant E. coli isolates.Ag+ ions enter the bacterial cell thorough porins and in case of porin protein mutation, E. coli becomes resistant to silver antibacterial activity (Li et al., 1997).When OmpF or OmpC porins are absent due to mutation, E. coli becomes 4-8 times less susceptible to the antibacterial activity of AgNPs (Radzig et al., 2013).The previous reports agree with our findings with respect to the role of Omp genes in E. coli response to antibacterial activity of AgNPs.The present study illustrated the absence of SilB and SilE genes among 11 tested E. coli isolates and only two isolates were positive for SilB and SilE genes for each.These two isolates were resistant to AgNPs antibacterial activity while other 11 isolates were AgNPs sensitive as confirmed by the absence of Sil genes.Bacteria resist silver by uptake decrease, cell membrane alteration and efflux pump that cause silver to be expelled out of cell (Silver and Phung, 2005).SilB is a fusion protein in periplasmic space which links SilA (pump protein present in inner membrane) to SilC (porin channel).SilB gene is an indicator for resistance of Gram-negative to silver (Silver et al., 2006).Whether in presence or absence of nanosilver, SilB gene is constitutively expressed among Salmonella senftenberg (Losasso et al., 2014).SilE gene was common among E. coli isolates producing CTX-M after exposure to silver (Shutterlin et al., 2014).The previous findings are in agreement with our results as Sil operon plays a role in AgNPs resistance.As illustrated in our study, one E. coli isolate was found negative for OmpF and positive for SilB and another E. coli isolate was found negative for OmpC and positive for SilE, these two isolates were AgNPs resistant confirming the role of efflux pump encoded by Sil gene and the effect of porin absence in E. coli resistance to AgNPs.In another report, Sil genes were not correlated to resistance of E. coli to silver (Shutterlin et al., 2012) which contradicts with the findings of the present study.

Conclusion
Although silver resistance was not frequent among bacterial isolates for long time, the presence of Sil genes and absence of Omp genes have a great influence in E. coli response to AgNPs antibacterial activity indicating that silver resistance among MDR isolates is a possible behavior which threatens the power of silver as a strong old biocidal.

Table 1 .
Primers in the study with corresponding amplification products.

Table 2 .
Antimicrobial susceptibility of the tested E. coli isolates.

Table 3 .
activity of AgNPs against tested E. coli isolates. E.

coli isolated in the study Zone of inhibition (cm)
Mean = 1.39,Standard deviation = 0.59.