Introduction

One of the major predominant normal flora members in the human is Escherichia coli (E. coli), which can cause broad spectrum of infections. E. coli is a major pathogenic bacteria which are responsible for urinary tract infections (UTIs), accounting for greater than 80% of these infections (Qureshi and Doi 2014). The emergence of multi-drug resistant organisms is associated with the wide-spread use of empirical antibiotics in the hospital setting that causes serious opportunistic infections (Kareem et al. 2021). Many bacteria produce β-lactamases (cephalosporinases) to confer resistance to β-lactam antibiotic (Hammoudi Halat and Ayoub Moubareck 2020). A great threat has been posed after the arrival of extended-spectrum β-lactamases (ESβLs) on the usage of majority of the antibiotics classes, particularly cephalosporins. The β-lactamases production causes resistance to the antimicrobial agents, particularly in the gram-negative bacteria. As a consequence, numerous enzymes are mutated leading to the development of ESβLs in response to increased pressure of antibiotic use (Bahr et al. 2021). The majority of the affiliates of Enterobacteriaceae have acquired mutated plasmids that are capable of producing β-lactamases, whereby conferring β-lactam antibiotics resistance (Kumar and Saikumar 2021). Amongst those members of Enterobacteriaceae, an increased prevalence of ESβL producers has been found to vary between different geographical areas (Padmini et al. 2017). ESβL producers are common in hospital settings; as a result, it is a major aggravating factor for hospitalization, and the outcomes would be seriously critical (Oteo et al. 2010).

Graphene is one of the carbon allotropes that includes charcoal, graphite and carbon nanotubes. It is a two-dimensional (2D) nano-structure consisting of sp2 carbons first discovered in 2004. Graphene has unique chemical, and physical properties such as optical, electrical, mechanical strength and large surface area in addition to the catalytic activity, so it has a potential application in biomedicine such as biosensors, cancer therapy drug delivery and antimicrobial activity. Graphene nanoparticles can be synthesized in a simple method such as electrochemical as mentioned in Hussain et al. (2020). Many studies have recently proved the biocidal properties of graphene nanoparticles and nano-sheets toward microorganisms, including pathogenic bacteria (Ali et al. 2019; Ghanem et al. 2020; Aziz et al. 2021). Its bactericidal effect include physical mechanism induced by the sharp edge of graphene that leading to bacterial membrane damage. The second mechanism is the chemical effect that employs the charge transfer which leading to oxidative stress and ROS generation (Kumar et al. 2019). Despite the fact that Enterobacteriaceae have emerged as the most questionable pathogens of critical health care interest, little is known about these organisms virulence genes which contribute to their pathogenesis (Kareem et al. 2017).

In this study, we aimed to isolate E. coli from clinical samples which were procured from outpatients and inpatients wards from the diagnostic and research laboratory, determine their antimicrobial susceptibility, phenotypic and genotypic detection of ESβL-producing ability, detection of some virulence-related genes, estimate the impact of graphene nano-sheets as antibacterial, and study the adherence-related gene expressions in E. coli isolates.

Materials and methods

Bacterial collection and analysis

1000 clinical samples were collected from different hospitals in Iraq during 2019–2021. The routine microbiological examination was carried out for bacterial isolates. To confirm the identification phenotypically, were used conventional PCR was carried out to these species by using specific primers, for 16SrRNA gene which described by (Wang et al. 2002). The primer sequences are listed in Table 1.

Table 1 List of primers or PCR amplification
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Synthesis of graphene nano-sheets

The electrochemical cell was used to synthesize graphene nano-sheets. (Liu et al. 2019; Hussain et al. 2020). The graphite rods (6.5 × 75 mm) were used in the two electrodes figurs, cathode and anode. An aqueous electrolyte of 50 ml, 0.1 M H2SO4 with 50 ml, 0.1 M KOH solution was used to carray out the electrochemical study. They were washed in a series of steps employing aqueous and organic cleansers. (ethanol and de-ionized water) prior to laying the graphite rods in the cell. Each cleansing stage took 180 s.The D.C voltage was utilized. The voltage between electrodes was 6 V under a current density of 2.11 × 10–3 mA/cm2 for 8 h. After obtaining a brown precipitate, the sample was extracted, bathed with de-ionized water, and left to dry for further processing. Reference (Rheima et al. 2019) describes the procedure for synthesizing and characterizing graphene nano-sheets.

Antimicrobial susceptibility test

Antimicrobial susceptibility test toward cefoxitin, cefepime, ceftazidime, cefotaxime, ertapenem, imipenem, amikacin, gentamicin, ciprofloxacin, and chloramphenicol was carried out by diffusion disks test (Kirby Bauer method). The results were compared with Clinical Laboratory Standards Institute (Clinical and Institute 2017).

Phenotypic detection of ESβL

Screening of E. coli ESβL was done based on the CLSI recommendations using double-disk diffusion methods including augmentin disk (amoxicillin/clavulanic acid) placed in the midst of plate. While ceftazidime and cefotaxime disks were placed at 3 cm from a central disk. The inhibition zones of the augmentin, cefotaxime, and ceftazidime disks were compared after 24 h of incubation at 37 °C. Overlapping between cefotaxime and augmentin disk was considered as EsβL-positive producer. E. coli ATCC 25922 was used as the negative control strain.

Genotypic detection of ESβL

PCR amplification for blaTEM, blaSHV, and blaCTX-M-type ESβL genes was carried out using the boiling method to separate genomic DNA of the bacterial isolates. The amplification system was (GeneAmp PCR System 9700) from (Applied Biosystems, Foster City, CA, USA). Primers were designed for the amplification are listed in Table 1. The results obtained from PCR were analyzed using agarose gel electrophoresis.

Detection of adherence virulence genes

PCR was used to identify adherence virulence genes such as papC and fimH. Table 1 shows the primer sequences and product sizes.

Detection of antibacterial activity of grapheme nano-sheets

The antibacterial activity of the grapheme nano-sheets was screened using the agar well-diffusion test. Few colonies of E. coli isolates from overnight cultures were transferred to a sterile test tube containing 5 mL of normal saline and completed to equal 108 colony-forming unit (CFU)/mL) and cultured on Mueller Hinton agar plates. On agar plates, 5 mm wells were made and loaded with 50 μg/mL graphene nano-sheets. However, normal saline was used as a negative control. Plates were then incubated at 37 °C for 24 h and the inhibition zone diameters were measured, then incubated in 37 °C for overnight to determine the inhibition zone in mm.

Determination of the minimum inhibitory concentration (MIC) of graphene nano-sheets

The MIC was employed using the 96-well microtiter plate for graphene nano-sheets (Jasim et al. 2020). Briefly, 100 μL of Mueller Hinton broth was added to microtiter plate wells, then each well of the first vertical row was loaded with 100 μL (10 mg/mL) of graphene nano-sheets followed by mixing 1:2 serial dilutions. 100 μL from the last well was discarded. After that, all wells (except negative control row A11–H11, which was filled only with MH broth) were filled with 5 μL of diluted suspension of E. coli isolates and then incubated for 24 h at 37 °C. Finally 10 μL of resazurin dye was putted on the wells, then left 4 h in incubation to be ready for reading; the actions of different concentrations of nanoparticles for the bacterial growth were detected using UV–visible spectrophotometer. The assay was carried out in triplicate.

Expression of adherence-related genes in the presence of graphene nano-sheets

Real-time RT-PCR was used to evaluate the effect of graphene nano-sheets by the gene expression of adherence-related genes (papC and fimH genes). TRIzol reagent (Sigma-Aldrich, Switzerland) was used to extract RNA from bacteria treated with graphene nano-sheets, and untreated bacteria served as controls. Following that, cDNA was synthesized using the Superscript III kit (Invitrogen Inc., USA) according to the manufacturer's instructions. PCR reactions were achieved in a 7500 Sequence Detection System using ePower SYBR Green PCR Master Mix (Applied Biosystems, USA) (CFX 96 biorad, USA). The fold change in the expression of the target genes was calculated using the 2−ΔΔCT method and 16S rRNA gene served as the normalizing gene (Livak and Schmittgen 2001). The level of expression of each gene in cells treated with graphene nano-sheets was compared to the expression of untreated bacteria as a control.

Data analysis

The statistical analysis for this study by (SPSS) version 21.0 was used to analyze the data. The t-test and Chi-square tests were used to examine continuous and categorical variables, respectively. The Fisher exact test was used to calculate the difference in drug resistance levels between ESβL-producing and non-ESβL-producing isolates. The data were analyzed at a rate of 95%. (p 0.05 indicates a confidence interval).

Results

Characterization of graphene nano-sheets

Powder XRD was used to analyze the produced graphene nano-sheet at diffraction 2θ range of 10°–80° as showed in Fig. 1. Diffraction peaks of graphene had adjusted about 2θ = 26°, and 42° corresponding to (002) and (100) was referred to as the structure of the carbon in graphene (Wang et al. 2020). The broad peak analogical to the (002) at 2θ = 26° elucidates that the graphene nano-sheets were obtained. The broad X-ray hump obtained in the graphene reveals sp2carbon bonds in this electrolysis synthesized sample.

Fig. 1
figure 1

XRD pattern of the graphene nano-sheets

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Figure 2 shows the UV–Vis absorption spectrum in the graphene nano-sheet with 0.5 mg/mL de-ionized ultrasonic dispersion for 15 min. In the UV–Vis spectra, a peak at 274 nm was observed, indicating π → π*transition of C–C bonds of the graphene sheet. It appears the graphene π → π* network with the restoration of electronic conjugation of the sp2 hybridized carbon network (Selvakumari et al. 2020).

Fig. 2
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The UV–visible spectrum of graphene nano-sheets

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The nano-morphology of the synthesized graphene sheets can be analyzed with the TEM images shown in Fig. 3A. A significant number of scrolls and wrinkles appear on the sheet surface, and TEM images display a clear multilayer formation. The graphene demonstrates multi-layered morphology. It says the layered graphene structure in sheet format. Figure 3B shows the SEM images of the synthesized graphene sheets show a layered structure that supplies ultrathin and homogeneous graphene films. The edges of the sheets of silk are crumbled and folded back. Hairy, as the surface of blurry arcs, are the graphene nano-sheets. It is made of a layered graphene sheet structure. The more layers that fit the XRD results are observed. The SEM images display a random orientation with a wavy look. The layered and flaky structures have a silky look, with the scale-like arrangement of nano-sheets (Selvakumari et al. 2020).

Fig. 3
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Images of the synthesized graphene nano-sheets by A TEM and B SEM

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Bacterial demographic characterization and susceptibilities

Table 2 summarizes the demographic characteristics such as age, gender, reality, and any interventions. The average age of the total study participants was 9–56 years. The means ± SD age of patients with ESβL positive and negative was 47 ± 17.5 years and 47.5 ± 12.8 years, respectively (p = 0.093). Out of the 511 patients, 412 (80.1%) were females and 101 (19.8%) were males (p = 0.0001). The results have shown that the majority of patients were old and belonged to different Iraqi areas. During the research, a total of 511 E. coli were isolated. From those 511 positive cultures, 360 (69.9%) were extended-spectrum β-lactamase (ESβL) producing E. coli (Fig. 4). Males had a higher incidence of ESβL than females. Figure 5 depicts the frequency of extended-spectrum β-lactamase-producing E. coli isolates from various samples (n = 359). According to the findings, 64% of E. coli were resistant to amoxicillin and cefixime. Furthermore, a significant drop of 30–40% in susceptibility to all cephalosporins was observed. E. coli, on the other hand, demonstrated a disparate sensitivity rate to chloramphenicol, ofloxacin, gentamicin, and amikacin, with 94%, 98%, 97%, and 97%, respectively. Regarding the PCR detection of ESβL genotypes, it was discovered that all of the ESβL-positive E. coli isolates had one or more of the ESβL genes tested in the current study. Overall, one or more ESβL genes were found in 83.56% (300/359) of E. coli isolates. The PCR assay results revealed the following ESβL genotype frequencies: 85% for the blaCTX-M gene, 26% for the blaSHV gene, and 28% for the blaTEM gene.

Table 2 The demographic characteristics for the bacterial isolates
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Fig. 4
figure 4

Bar graph showing extended-spectrum β-lactamase-producing E. coli

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Fig. 5
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Graph showing number of specimens of β-lactamase-producing E. coli

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Data availability statement

The research data are not accessible because of institutional rules and regulation policy.

Antibacterial activity and MIC of graphene nano-sheets

The antibacterial activities of the graphene nano-sheets against ESβL-producing E. coli isolates has been investigated. The growth curve of E. coli isolates was determined in the presence of graphene nano-sheets or its absence as a control. The mean of inhibition zone for the graphene nano-sheets in the agar well-diffusion method was 15 mm in diameter as cleared in Fig. 6. Furthermore, the MIC demonstrate that graphene nano-sheets has high-antibacterial achievement mean values of 25 μg/mL. These results represent the remarkable enhanced antibacterial activity of the graphene nano-sheets.

Fig. 6
figure 6

Antibacterial activity in the agar well-diffusion method shows inhibition zone of G-sheet against ESβL-producing E. coli (in right) with normal saline as a control (in left)

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Modulation of adherence-related genes expression

The expression of fimH and papC genes were measured with and without presence to graphene nano-sheets. In the presence of graphene nano-sheets, the expression of fimH and papC adhesion genes were significantly reduced by 4.6-fold (p = 0.001) and 3.4-fold (p = 0.001), respectively, as cleared in Table 3.

Table 3 Gene expression of fimH and papC target genes of E. coli
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Discussion

The medical community depends on the published guidance and clinical expertise to help the physicians with different options in empirical therapy for the infections that are mostly based on system classification, including: urinary tract infections and community-acquired pneumonia. E. coli produce extended-spectrum β lactamases, which was firstly recognized from the late 1990s and appeared in the community settings as a significant reason of urinary tract infections (Pitout 2010). Extended spectrum β-lactamases are the major causes of βlactam antibiotics resistance, particularly in E. coli. The increasing frequency of the co-existence of ESβLs is a serious threat to successful treatment interventions of bacterial infections producing such defending enzymes (Adegoke et al. 2020; Nzima et al. 2020; Adekanmbi et al. 2020).

Gram-negative bacteria infections may be accompanied with the appearance of β-lactamase enzymes that play a major role in the spread of hospital infections, which represents a crucial concern. Some serious issues related to diagnosis and treatment have been concurrently occurred due to the prevalence of such strains that possess several resistance enzymes (Al-Kadmy et al. 2020; Marjani et al. 2020). Generally, AmpC β-lactamases and ESβL-producing isolates are susceptible to imipenem (Kuo et al. 2021). Ejaz et al. (2013) have reported a frequency of 68% for ESβL-producing gram-negative bacteria using the DDST method and a 100% using the CLSI method. These results altogether suggest that the method of detection might have an impact on the reported values as a result of the differences in the analytical sensitivity and specificity. Therefore, the need is pressing to give more attention to this kind of bacterial infections, and proper precautions should be taken to prevent the spread of them to other patients. ESβL-producing E. coli is a huge health concern, which is evolved primarily due to drug resistance, limited management options, increased mortality, and cost of management. In this context, most ESβLs evolved from genetic mutations in β classic lactamases (TEM-1, TEM-2 and SHV-1). These lactamases originated from the various ESβLs of TEM and SHV types (Liakopoulos et al. 2016). The present study has evaluated the frequency of extended-spectrum β-lactamases E. coli isolated from clinical samples in Iraq. The study has also documented an increased frequency of extended-spectrum β-lactamase-producing E. coli. In the present study, the antimicrobial sensitivity arrays were detected in all isolates, and the results of the antimicrobial susceptibility test against E. coli revealed that isolated bacteria differed in their sensitivity to the tested antimicrobial drugs. Carbapenems are frequently the last choice of defense in treating infections caused by MDR Enterobacteriaceae (Elshamy and Aboshanab 2020; Zheng et al. 2021). Based on previous research, imipenem and meropenem, which have been identified as the most effective antibiotics against ESβL-producing isolates, demonstrated 100% sensitivity. This is an important finding from the current study because carbapenems can be used to treat a variety of infections. Our findings indicated that 33% and 35% of the ESβL-producing isolate were susceptible to cefotaxime and ceftazidime, respectively. We also observed that blaTEM and blaSHV genes were less common in our settings with 28% and 26%, respectively. In recent years, new approaches to antibacterial treatment have been developed, such as the combinations of antibiotics, and the formulations based on the antibacterial nanoparticles (Aziz et al. 2021; Mulani et al. 2019; Yeh et al. 2020; Hetta et al. 2021). Several studies have recently characterized the enhanced antimicrobial activity of graphene nano-sheets as a topically applied treatment versus pathogenic microorganisms. The roles of graphene nano-sheets against bacteria, including ESβL-producing isolates based on the direct action of graphene nano-sheets on the bacterial cell membrane because of the crystalline sharping corners and edges. Importantly, the current study considering the first in Iraq that proves the efficiency of graphene nano-sheets to reduce the gene expression of some adherence -related genes (fimH and papC). Our findings have demonstrated that 25 µg/mL of graphene nano-sheets significantly reduced the expression of adherence-related genes. However, further researches are recommended to evaluate the drug resistant patterns of bacterial isolates using rapid molecular diagnostic methods that are essential for effective clinical management. Moreover, continuous epidemiological surveillance is mandatory as well as necessary for the control of drug resistant E. coli. Finally, the inability of in vivo experiments are the limitations for the current study, which we recommend in future researches.

Conclusion

The current study has helped us to better understand the characteristics of ESβL-producing E. coli. The analysis of the isolates proved their higher antibiotics resistance, harboring a sheaf of adherence-related genes. On the other hand, the current study concluded the potential ability of graphene nano-sheets to reduce bacterial growth, multiplication, and the expression of adherence-related genes. To the best of our knowledge, few studies in Iraq showed the activity of graphene nano-sheets in inhibiting ESβL-producing E. coli isolates in vitro. Furthermore, the current study showed further step to understand the mechanisms by which graphene nano-sheets can conflict the bacterial virulence and resistance. However, further studies are necessary to understand the mechanisms by which graphene nano-sheets interfere with the microbial spread and resistance.