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The enhanced antibacterial and antibiofilm properties of titanium dioxide nanoparticles biosynthesized by multidrug-resistant Pseudomonas aeruginosa

BMC Microbiology volume 24, Article number: 379 (2024) Cite this article

Abstract

The emergence of Multidrug-resistant (MDR) bacteria are becoming a major worldwide health concern, encouraging the development effective alternatives to conventional antibiotics. The study identified P. aeruginosa and assessed its antimicrobial sensitivity using the Vitek-2 system. Carbapenem-resistant genes were detected through Polymerase chain reaction (PCR). MDR- P. aeruginosa isolates were used to biosynthesize titanium dioxide nanoparticles (TiO2NPs) and characterized using X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), field emission scanning electron microscopy (FE-SEM). A study involving 78 P. aeruginosa isolates revealed that 85.8% were MDR, with meropenem and amikacin showing effectiveness against 70% of the isolates. The most prevalent carbapenemase gene was blaOXA-48, present in 83% of the isolates. Majority of the isolates formed biofilms, and biosynthesized TiO2NPs were able to reduce biofilm formation by 94%. TiO2NPs exhibited potent antibacterial action against MDR-Gram-negative bacilli pathogens and showed synergistic activity with antibiotics, particularly piperacillin, with a significant fold increase in areas (283%). A new local strain of P. aeruginosa, identified as ON678251 in the World GenBank, was found capable of producing TiO2NPs. Our findings demonstrate the potential of biosynthesized TiO2NPs to manage antibiotic resistance and regulate the formation of biofilms. This presents a promising direction for the creation of novel antimicrobial agents or substitutes for use in clinical settings, particularly in the management of isolates capable of resisting multiple drugs.

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Background

Antimicrobial resistance is a major contributor to the high rates of illness and death caused by bacterial infections worldwide [1]. Pseudomonas aeruginosa exhibits a diverse array of pathoadaptive traits and pathogenicity mechanisms that enhance its capacity to inhabit, survive, and proliferate in different habitats [2]. The incidence of P. aeruginosa infections is on the rise, particularly in hospitalized patients, and there is an emergence of multidrug-resistant strains. Carbapenems have traditionally been reserved as a last-resort treatment for controlling infections caused by MDR-P. aeruginosa. However, it is important to note that carbapenem-resistant P. aeruginosa may also exhibit resistance to other classes of antimicrobial drugs, leaving limited treatment options and resulting in high rates of illness and death, especially among hospitalized individuals and those with weakened immune systems [1]. The incidence of carbapenem-resistant P. aeruginosa in nosocomial infections is increasing, with rates varying from 0–60% [3]. Pseudomonas are known for their ability to rapidly develop resistance to commonly used antibiotics, due to the presence of multiple efflux pumps. Additionally, P. aeruginosa is an extremely virulent bacterium, highlighting the urgent need for new antimicrobials [4]. The production of biofilms presents a major obstacle for the host due to the frequent resistance of these biofilms to antibiotics, phagocytosis, and surfactants. Eliminating the biofilms once they have formed is challenging due to this resistance [2, 5]. Biofilms provide a protective growth form for the bacteria and are crucial for survival in healthcare environments, while also maintaining an inflammatory environment in the host [3]. The sensitivity of bacteria to antimicrobial agents varies between their planktonic and biofilm forms. Biofilm-forming microorganisms are more resistant to antibiotics compared to bacteria that grow as individual cells. The biofilm produced by P. aeruginosa is recognized as a major contributor to treatment failure [6].

Carbapenem resistance in Pseudomonas aeruginosa is frequently associated with the acquisition of particular genes that encode carbapenemases, which are enzymes capable of degrading carbapenem antibiotics. These genes are commonly found on mobile genetic components, which enables them to easily travel among various bacterial strains and species. The predominant carbapenemases detected in P. aeruginosa include metallo-beta-lactamases (MBLs) such as VIM, IMP, and NDM, as well as serine carbapenemases like the OXA-type enzyme. These enzymes not only provide resistance to carbapenems but also to other antibiotics, making treatment more difficult [2, 7]. The transfer of new resistance genes via horizontal gene transfer can result in resistance to the four main categories of antipseudomonal agents. These include important cephalosporins (including combinations with β-lactamases), carbapenems, aminoglycosides, and respiratory fluoroquinolones (such as ciprofloxacin, levofloxacin, and moxifloxacin), as well as colistin [3]. The emergence and widespread dissemination of acquired carbapenem hydrolyzing enzymes (carbapenemases) are significant concerns in terms of global health. These enzymes pose serious challenges for therapy and infection control, leading to increased mortality rates and longer hospital stays. Consequently, clinicians have become increasingly concerned about promptly identifying bacteria carrying these genes to mitigate or prevent this issue in healthcare facilities [8]. Therefore, there is a need for alternative strategies to combat bacterial infections and block resistance mechanisms. One such strategy is the use of metallic nanoparticles (NPs) [9]. Nanotechnology offers a promising platform for developing NPs with antimicrobial properties that can work synergistically with antibiotics against different bacteria also has anticancer properties to treat human breast cancer [10, 11]. Bacterial NP synthesis is favored among biological entities because of its large production, quick growth rate, and controllability [12]. TiO2NPs, in particular, have attracted significant interest due to their diverse applications and favorable properties [13]. Nanoparticles can disrupt the bacterial outer membrane, inhibit enzymes, induce changes in gene expression, and trigger pathways for cell death. They are also more stable and less toxic than traditional antibiotics, consequently, the medical sector becoming more interesting in employing these NPs as an alternative or co-antimicrobial agent [14]. Inorganic minerals like silver, gold, titanium, copper, and zinc have been explored as alternatives to antibiotics and disinfectants, with applications in preventing catheter-associated infections, inhibiting biofilm formation, and serving as drug delivery systems [15]. Antibiotics are less effective because of their poor intracellular bioavailability and non-target-specific methods of action. Additionally, resistance develops as a result of its broad use. Furthermore, using certain antibiotics can have adverse consequences like cytotoxicity. These issues can be resolved by the creation of novel treatment approaches, such as NP therapy in combination with other antimicrobial medications [16]. The combination of NPs with antibiotics in "combination therapy" has emerged as a promising approach to combat multidrug-resistant bacteria. This approach can overcome bacterial resistance mechanisms and enhance the efficacy of antibiotics [17, 18]. While antibacterial NPs will not only shield drugs from the molecular mechanisms of resistance, their combined actions with antibiotics are expected to prevent mechanisms of bacterial resistance to antibacterial agents. Instead, the delivery mechanism for NPs will confer an antibacterial action by a synergistic combination with nanoparticles, reinforcing traditional antibiotics against multidrug-resistant bacteria [19].

This study investigates MDR in P. aeruginosa and evaluates the antibacterial activity of biosynthesized TiO2NPs against MDR-Gram-negative bacteria, both alone and in combination with antibiotics, and their impact on biofilm production.

Methods

Bacterial isolates

The study encompassed 78 clinical samples of P. aeruginosa that had multidrug resistance (MDR), including resistance to carbapenem. These samples were gathered from both hospital and community settings in Erbil, which is located in Iraq's Kurdistan Region, over the period from January to September 2021. The specimens were obtained from several locations of infection, namely urine (24 specimens), sputum (16 specimens), wound swabs (8 specimens), and blood (3 specimens)). The study involved 78 patients aged 1 month to 80 years, with a median age of 40, with 60.3% being female. The antibacterial activity of biosynthesized TiO2NPs was evaluated against MDR Gram-negative bacilli strains from Rizgari Hospital in Iraq, including E. Coli, Klebsiella pneumonia (K. pneumonia), Acinetobacter baumannii (A. baumannii), and Proteus sp.

Antibacterial susceptibility test

The antimicrobials that were analyzed comprised imipenem and meropenem (carbapenems), trimethoprim/sulfamethoxazole (sulfonamides), gentamicin and amikacin (aminoglycosides), ciprofloxacin and levofloxacin (quinolones), and piperacillin and piperacillin-tazobactam (penicillins), along with tigecycline (tetracycline). The susceptibility of P. aeruginosa isolates was evaluated using the Vitek-2 automated method, and resistance profiles were utilized to assign MDR [3]. The ATCC P. aeruginosa 27853 strain served as the quality control reference strain.

Biofilm formation assay

The study employed the microtiter plate test method to identify the isolates' ability for biofilm formation [3] The isolates were incubated overnight in tubes containing 5 mL of trypticase soy broth (TSB) (Merck, Germany) at a temperature of 37°C. The expansion was subsequently reduced in a new solution at a ratio of 1 part growth to 100 parts fresh medium. Subsequently, 200 μL of the diluted cultures were introduced into 96-well polystyrene microtiter plates (Costar/USA) and subjected to incubation at 37°C for 24 h under static conditions. Parafilm was applied to the edge of the plate to inhibit evaporation while it was being incubated [2]. Subsequently, the wells were rinsed thrice with 200 μL of Phosphate Buffer Saline at a pH of 7.2 to eliminate the broth, followed by air-drying. Subsequently, the wells were treated with 200 μL of 0.1% crystal violet solution for 30 min at ambient temperature. Subsequently, the plates were rinsed with distilled water to eliminate any dye that was not attached and left to air dry. The stain that was attached was dissolved by adding 200 μL of 95% ethanol, and the optical density (OD) of the dissolved crystal violet was measured at 630 nm using an ELISA reader. Biofilm formation was deemed unfavorable when the optical densities (ODs) were below 0.12, moderately favorable when the ODs ranged from 0.12 to 0.24, and highly favorable when the ODs exceeded 0.24. The experiment was conducted on three separate occasions, and the average of the recorded numbers was reported as the outcome [20].

Molecular analysis of carbapenemase genes

Carbapenemase genes, specifically MBLs (blaNDM, blaVIM, and blaIMP), OXA-48 (blaOXA-48), and KPC (blaKPC) genes, were screened in P. aeruginosa utilizing primers from earlier research [7] Carbapenemase genes, specifically MBLs (blaNDM, blaVIM, and blaIMP), OXA-48 (blaOXA-48), and KPC (blaKPC) genes, were screened in P. aeruginosa utilizing primers from earlier research through the utilization of polymerase chain reaction. Multiplex PCR was utilized to identify the presence of carbapenemase genes. The genomic DNA extraction process involved the utilization of a commercial extraction kit (DNAL and Scientific Cat No. GG2001) to extract the entire DNA content from bacterial cultures during the logarithmic phase. The extraction was performed according to the instructions provided by the manufacturer. The PCR amplification stages were conducted using a thermocycler machine (Techne, UK) with a total volume of 25 μl. The reaction mixture consists of 12.5 μl of Gotaq Green Master Mix (Promega/USA), 3 μl of genomic DNA, 1.5 μl of each primer, and 6.5 μl of nuclease-free water. Thermocycling conditions included a 1 min warm-up at 95 °C, 30 cycles of 30 s at 95 °C, 30 s at 55 °C, and 1 min at 72 °C, culminating in 7 min extension step at 72 °C. the DNA fragments were visualized by electrophoresis in a 2% agarose gel [4, 7].

Biosynthesis of TiO2NPs

Pseudomonas aeruginosa cells were cultured in sterile TSB medium and incubated at 37 °C with shaking at 120 rpm for 24 h. A transformation mixture was prepared by centrifuging 50 mL of the culture broth at 8000 rpm for 10 min, and mixing 20 mL of culture supernatant with 20 mL of 0.025 M TiO2 (Sigma Aldrich) to achieve a 1:1 ratio. The mixture was heated in a water bath at 80 °C for 10–20 min until white deposition appeared at the bottom, indicating transformation. The solution was cooled and incubated at room temperature. After 12–48 h, distinct coalescent white clusters were observed at the bottom of the flask. The resulting suspension was centrifuged at 2000 rpm for 20 min, and the supernatant liquid was removed. The residue was washed multiple times with deionized water to remove impurities. The precipitate was dried in an oven at 40 °C for 30 min. The synthesized TiO2NP samples were then subjected to characterization [21].

Characterization of TiO2NPs

The process of NP formation was monitored by observing the change in color of the culture supernatant. This change was verified by utilizing a dual-beam UV–visible spectrophotometer (Perkin Elmer Lambda model 35USA), to measure the peak exhibited by the TiO2NPs. The UV–visible spectra of 2 ml samples of the culture supernatant were measured in the wavelength range of 300–800 nm, using double distilled water as a reference [22]. The properties of the functional groups in TiO2NPs were analyzed using Fourier-transform infrared spectroscopy (FT-IR). The FT-IR study was conducted using the Spectrum 4600 instrument from JASCO. The analysis focused on the spectral range of 4000–400 cm − 1 with a resolution of 4 cm − 1 [23].

X-ray diffraction (XRD) measurements were conducted using the PAN analytical X’Pert PRO instrument to analyze the crystalline structure and chemical content of the TiO2NPs. The XRD analysis employed Cu K (k = 1.5406) radiation, with the apparatus configured at 40 mA and 40 kV. The diffraction intensities were measured within the angular range of 20°–80°, specifically in the 2θ region. TiO2NPs in dry powder form were placed on the XRD grid for investigation [24, 25]. The particle size distribution and morphology of the TiO2NPs were analyzed using advanced transmission electron microscopy (TEM) (TEM; Titan3 G2 60–300, Cs corrector: image and probe, FEI, USA) and field emission scanning electron microscopy (FESEM) (Quanta 4500). The sample for TEM examination was made by drop casting onto a grid coated with Cu TEM holey carbon and then air-dried, resulting in a thin film. An investigation using Field Emission Scanning Electron Microscopy (FESEM) was performed on a sample that was prepared on a copper grid coated with carbon. Furthermore, the chemical composition of the TiO2NPs was examined using energy dispersive X-ray spectroscopy (EDX) in combination with field emission scanning electron microscopy (FESEM). The specimens were processed and dehydrated on a carbon-coated copper grid [26].

Antimicrobial activity of biosynthesized TiO2NPs

  1. 1.

    Antibiotic susceptibility testing by disc diffusion method

    The Kirby-Bauer disc diffusion assay was used to assess the susceptibility of MDR Gram-negative bacilli isolates (E. coli, Klebsiella sp., A. baumannii, Proteus sp. and chosen P. aeruginosa) to β-lactam antibiotics. The antibiotics used were ceftazidime (30 μg), cefoxitin (30 μg), piperacillin (100 μg), and imipenem (10 μg). The McFarland standard solution was used to standardize the inoculum density. A sterile swab was used to inoculate the suspension, which was then streaked on a Muller-Hinton agar (MHA) plate. Antimicrobial discs were placed on each plate after a 5-min incubation at room temperature. The plates were incubated at 37 °C for 18–24 h and the results were compared to the standard levels specified in the CLSI (2020) documentation [27].

  2. 2.

    The antimicrobial susceptibility of TiO2NPs alone and in combination with antibiotics

    The antibacterial activity of TiO2NPs was assessed using a disc diffusion assay. A suspension of MDR- E. coli, Klebsiella sp., A. baumannii, Proteus sp., and P. aeruginosa isolates was prepared at a concentration of 1.5 × 108 CFU/mL and swabbed onto MHA plates. Plain TiO2NP discs were created by adding 10 µl of TiO2NPs (5000 µg/mL) to 6 mm diameter Whatman paper discs [28]. The effect of TiO2NPs in combination with antibiotics was assessed by adding 10 µl of the defined concentration of TiO2NPs to standard discs of ceftazidime, piperacillin, cefoxitin, and imipenem. A disc soaked in sterile distilled water was used as a negative control, and various discs were inoculated onto MHA plates and incubated at 37 °C for 24 h. The disc diffusion assay was conducted in triplicate, measuring and comparing inhibition zones with the standard antibiotic inhibition zones published by CLSI (2020) [27].

  3. 3.

    Evaluation of the synergistic effect of TiO2NPs with antibiotics

    The study evaluated the synergistic effect between TiO2NPs and antibiotics by calculating the fold increase in the diameter of the inhibition zone for each antibiotic when combined with TiO2NPs. The calculation used the equation: Fold increase = (B-A)/A × 100, where (A) represents the inhibition zone of the antibiotic alone and (B) represents the inhibition zone of the antibiotic combined with nanoparticles [29].

Antibiofilm potential of TiO2NPs

The study used a 96-well microtiter plate method to evaluate the effectiveness of biosynthesized TiO2NPs in biofilm formation [3]. The plates were filled with 180 μL of MH broth and inoculated with 10 μL of an overnight culture. 10 μL of TiO2NPs were added to achieve a concentration ranging from 19.531 to 5000 μg/mL. The plates were incubated at 37 °C for 24 h, then washed three times with PBS (pH 7.2) to eliminate free-floating bacteria. The biofilms formed by adherent organisms were fixed with 2% sodium acetate and stained with 0.1% crystal violet dye. The excess stain was rinsed off with sterilized Millipore water, and the plates were dried. After drying, 200 μL of 95% ethanol was added to each well. The absorbance at 620 nm was measured using an ELISA reader (Multiskan® EX, Thermo Scientific, Finland), and the percentage of biofilm inhibition was calculated using an equation: % biofilm inhibition = [(1 − OD620 of cells treated with TiO2NPs/OD620 of non-treated control) × 100]. The experiment was conducted three times. A bacterial cell-free filtrate (10 μL) used as a positive control in the preparation of TiO2NPs.

Sequencing of 16S rRNA gene

The PCR products were sent to the National Instrumentation Center for Environmental Management in Seoul, Korea for sequencing using primers PA-SS-F (GGGGGATCTTCGGACCTCA) and PA-SS-R (TCCTTAGAGTGCCCACCCG) [30]. The nucleotide sequence of the P. aeruginosa strain was submitted to the National Centre for Biotechnology Information (www.ncbi.nlm.nih.gov), assigned GenBank accession number ON678251. The 16S rRNA partial gene sequencing was analyzed using the Basic Local Alignment Search Tool (BLAST), which compares the sequence with other biological sequences to identify similarities with the P. aeruginosa isolate. Similarities between bacterial strains were aligned using the CLUSTAL W phylogenetic tree and extracted from nucleotide sequence databases. The Molecular Evolutionary Genetics Analysis (MEGA) version 11 software was utilized to construct a neighbor-joining tree.

Analysis of statistics

The data was analyzed using GraphPad Prism and a chi-square test, with a statistically significant p-value of less than 0.05.

Results and discussion

Characteristics of the bacterial isolates and their demographics

The study analyzed 78 MDR P. aeruginosa isolates from various clinical specimens, finding no significant association between gender and clinical specimens, despite a significant gender-related association (p > 0.05) (Table 1).

Table 1 Epidemiological and clinical characteristics of the Pseudomonas aeruginosa isolates
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Among the discovered P. aeruginosa isolates, the majority (60.3%) were from females, while 39.7% were from men. The highest infection rate was observed among individuals aged 60–80 years (51.2%). This result aligns with a prior investigation conducted by Subramaniyan and Sundaram, which revealed that 31.1% of the samples were male and 68.8% were female [31]. The majority of isolates (35, 44.8%) were obtained from sputum (35, 44.8%), followed by urine samples (18, 23%), swabs (15, 19.2%), cerebrospinal fluid (8, 10.2%), and blood (2, 2.5%) (Table 1). The largest number of clinical isolates was obtained from sputum, indicating the presence of respiratory tract diseases. The findings align with the research conducted by others, who similarly observed a significant occurrence of P. aeruginosa in tracheal aspirates [31, 32].

Antimicrobial resistance

The Vitek-2 automated system test indicated that all 78 P. aeruginosa isolates exhibited significant resistance to the antimicrobials that were evaluated. A total of 13 antibiotics belonging to seven different classes were chosen for antibiogram profiling because of their widespread usage in hospitals as medications to combat pseudomonas infections. The isolates shown substantial resistance to multiple tested antimicrobial drugs (p < 0.001) (Table 2).

Table 2 The antimicrobial resistance profile of Pseudomonas aeruginosa isolates. The results are repeated triplicate p < 0.001
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The study revealed that the greatest level of resistance was observed against β-lactam antibiotics, specifically within the penicillin class [piperacillin 63 (80.7%), piperacillin/tazobactam 61 (78.2%)], cephalosporin class [cefepime 57 (73%), ceftazidime 53 (67.9%), and cefoxitin 54 (69.2%)], as well as the tetracycline class [Tigecycline 50 (64.1%)]. Table 2. These findings are consistent with prior research [1, 3, 20]. The increasing prevalence of drug resistance poses a significant challenge for treatment options, particularly with the emergence of high carbapenem resistance among Gram-negative bacilli isolates, which has been extensively reported in various countries. Consequently, carbapenem resistance has become a critical global public health crisis [33]. In the present study, the rate of imipenem resistance against MDR-P. aeruginosa was moderately high at 51.2% (Table 2). This emergence of such resistant isolates is a cause for concern in public health. A study in Isfahan found that out of 106 P. aeruginosa isolates, 62 (58.5%) were resistant to imipenem [34]. This is lower than previous reports, which reported 17.7% resistance [1]. In Iran, imipenem resistance in burn and non-burn patients was 83.2% and 57.5%, respectively [35]. Additionally, 33.3% of isolates showed resistance to meropenem, a highly effective antipseudomonal antibiotic. This finding aligns with studies in Bangladesh and Nepal [20, 36]. Resistance to other classes of antibiotics was linked to resistance against carbapenem antibiotics. Rates of resistance were found to be tigecycline (64.1%), trimethoprim/sulphamethoxazole (62.8%), levofloxacin (38.4%), ciprofloxacin (35.8%), gentamicin (34.6%), and amikacin (33.3%). Interestingly, meropenem and amikacin were effective against approximately 70% of the MDR-P. aeruginosa isolates. Amikacin was found to be the most effective antipseudomonal antibiotic for MDR-P. aeruginosa infections and a suitable treatment option for carbapenem-resistant P. aeruginosa isolates. Amikacin's resistance rate (33.3%) aligns with de Sousa et al.'s findings [2], but a different study reported a high resistance rate (92.4%) in burn infections caused by carbapenem-resistant P. aeruginosa isolates [37]. In brief, 85.8% of strains exhibited resistance to at least three classes of antimicrobials, making them MDR-P. aeruginosa. This result is consistent with the high rates of MDR-P. aeruginosa isolates in Iran, where 89.4% of the isolates were found to be MDR [38]. There is a growing occurrence of MDR-P. aeruginosa isolates worldwide, and this study has found higher rates than prior findings. Research has also indicated that patients infected with P. aeruginosa and Acinetobacter spp. have a significant risk of death, with mortality rates ranging from 40 to 65% [39]. The high resistance to carbapenem antibiotics is concerning from a public health perspective, as these antibiotics are essential for treating infections when lower-class antibiotics are no longer effective [2]. The higher incidence of multidrug-resistant (MDR) patterns among strains in this study may be attributed to factors such as self-medication, empirical usage, and excessive use of carbapenems and third-generation cephalosporins. Additional surveillance initiatives are required in this region to effectively tackle the escalating issue of carbapenem resistance [40].

Biofilm production assay

The study revealed that 78.2% of 78 isolates were biofilm producers, indicating their higher resistance to antibiotics compared to free-floating cells [9].

Figure 1 shows that most strains (65.3%) had strong biofilm formation capacity, while 12.8% and 21.7% exhibited moderate or weak biofilm-forming potential, respectively, using the tissue culture plate method.

Fig. 1
figure 1

Biofilm-production capacities among Pseudomonas aeruginosa isolates

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Most pathogenic P. aeruginosa strains can form biofilms, making them difficult to remove from hospital environments and medical equipment. There was a significant (p < 0.0001) difference in biofilm production abilities between tested MDR-P. aeruginosa isolates. The study found that all P. aeruginosa strains, including clinical specimens, can form biofilms under laboratory conditions, although the extent of biofilm formation varies. This aligns with previous studies, where 19.87% of isolates were weak/non-biofilm producers, 20.86% were moderate producers, and 59.27% were strong biofilm producers [3]. In contrast, de Sousa et al. reported higher biofilm production rates, with 92% of P. aeruginosa isolates producing biofilms [2].

Prevalence of carbapenemase genes

The MDR profile, increased incidence, and rapid dissemination of carbapenemases across species through transmissible genetic factors make them the most urgent resistance problem in Gram-negative bacteria [41, 42]. These genes often carry other antimicrobial resistance genes, leading to MDR or extensively drug-resistant traits, limiting treatment options and posing a triple threat. Figure 2 displays the prevalence of carbapenemase genes in 30 isolates that were tested. A study found that all 30 P. aeruginosa isolates tested had one or more carbapenemase genes. The most common carbapenemase gene was blaOXA-48, present in 83.3% of isolates. Other common carbapenemase genes included blaNDM (66.6%), blaIMP (50%), and blaVIM (33.3%), and none of the isolates tested positive for blaKPC (Fig. 3 A, B, C, and D). 76.6% of positive isolates had multiple carbapenemase genes, ranging from two to four. Statistical analysis showed a significant difference (p < 0.001) in carbapenemase resistance genes prevalence among MDR-P. aeruginosa isolates.

Fig. 2
figure 2

Frequencies and distribution of carbapenemase genes in P. aeruginosa isolates

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Fig. 3
figure 3

Detection of carbapenemase genes IMP, and VIM in carbapenemase-producing P. aeruginosa isolates. Lane M represents a 1-kb DNA ladder and lane C is a negative control. A The gel electrophoresis displayed the presence of carbapenemase gene NDM (621 bp). B Lanes 1–5 represent the positive IMP carbapenemase gene. C Gel electrophoresis revealed the presence of blaVIM (390 bp). D The gel electrophoresis showed the presence of the carbapenemase gene blaOXA-48 (438 bp)

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This study found that blaOXA-48 is the most common carbapenemase gene, followed by blaNDM. Subramaniyan and Meenakshi's found that 13.1% of MBL-producing P. aeruginosa strains from the ICU were 13.1% blaVIM-4, 9.8% blaVIM–5, and 3.2% blaVIM–38, with none testing positive for KPC, NDM, and IMP genes [31]. Sheikh et al. identified 236 carbapenem-resistant P. aeruginosa isolates, with 116 carrying MBL genes and 29 testing positive for blaNDM-1 [35]. Similarly, Ramadan et al. found that genes encoding VIM, GES, NDM, and IMP were detected in 50%, 40.9%, 27.3%, and 18.2% of isolates [8]. None of the tested isolates showed the presence of the blaKPC gene, as indicated by earlier research findings [8, 35]. This could be attributed to the existence of additional class A carbapenemases [43]. OXA-48, a highly prevalent worldwide ailment, has reached epidemic proportions in multiple Mediterranean nations, such as Turkey, Iran, Morocco, and Lebanon [35, 44]. The study found that a significant proportion of isolates (76.6%) carried multiple carbapenemase genes, indicating their ability to produce MDR determinants. This is a major concern for antibacterial treatment strategies, as it has been reported in other studies [1, 35].

Biosynthesis of TiO2NPs

The study examined all isolates for TiO2NP production, but only one P. aeruginosa isolate demonstrated the ability to synthesize TiO2NPs.

Characterization of TiO2NPs:

  • 1-visual observation and UV–visible analysis

    The stability and formation of synthesized TiO2NPs in an aqueous colloidal solution were determined through visual observation and UV–visible analysis [21]. The isolated P. aeruginosa strain successfully biosynthesised TiO2NPs by exposing bacterial cultures to TiO2, which was confirmed by the color changes from yellow to white precipitate, indicating the synthesis of TiO2NPs (Fig. 4 A). The color generation in TiO2NPs is due to the excitation of surface plasmon in metal nanoparticles [45]. UV absorption analysis confirmed the synthesis, with a clear absorption peak indicating the anatase phase of nano-TiO2 (Fig. 4 B). The cut-off wavelength was 316 nm, consistent with previous research on TiO2NP synthesis using various bacterial strains.

    Fig. 4
    figure 4

    The positive result of color change and UV–visible spectroscopy of biosynthesized TiO2NPs (A and B), XRD results (C), FT-IR spectra analysis (D), and EDX analysis (E)

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  • Microbial systems, including bacteria, are efficient in producing reducing components for nanomaterial synthesis due to richness in several biomolecules can transform the Ti salts to TiO2NPs [46]. P. aeruginosa was chosen for TiO2 synthesis due to its environmental compatibility, low energy consumption, and cost-effectiveness [47].

  • 2- X-ray diffraction analysis

    The synthesized nanoparticles' chemical composition and crystalline properties were verified using X-ray diffraction (XRD) analysis [21]. The crystal structure of TiO2NPs was examined using an X-ray diffractometer, which confirmed the formation of TiO2NPs from P. aeruginosa culture supernatant, with XRD patterns matching standard diffraction data (JCPDS card number: 98–017-2916). The XRD analysis of synthesized TiO2NPs revealed distinct diffraction peaks at various angles, including 25.30°, 36.96°, 37.81°, 38.56°, 48.04°, 53.88°, 55.07°, 62.68°, 70.31°, 75.09°, and 76.09°, corresponding to the crystal planes 101, 103, 004, 112, 200, 105, 211, 204, 220, 215, and 224, respectively (Fig. 4 C). The synthesized sample has a small size, crystalline structure, and high purity, as indicated by sharp diffraction patterns [26]. The XRD results from Fig. 4 C indicate that the nanoparticle structure primarily consists of the anatase crystalline phase, known for its high photocatalytic activity, and the main peaks' positions align with previous literature findings [26, 48].

  • 3-Fourier Transform Infrared Spectrometry (FTIR)

    Fourier transform infrared (FTIR) spectroscopic analysis was conducted to investigate the interaction between the NPs and capping agents. The FTIR spectra were recorded in the wavenumber range of 400–4000 cm-1. The FTIR spectra of TiO2NPs synthesized by P. aeruginosa exhibited prominent peaks at 3258.14, 2988.16, 2901.38, 1636.3, 1540.85, 1394.28, 1250.61, 1055.84, and 473.43 cm − 1 (Fig. 4 D). The biosynthesized TiO2NPs showed a broad band at 3258 cm − 1, indicating O–H stretching due to the alcoholic group. The peaks at 2988 cm − 1 and 2901 cm − 1 corresponded to the C–H of CH2 and CH3 groups in aliphatic chains, while signals at 1636 cm − 1 and 1540 cm − 1 indicated the presence of amide and amine groups.

  • The FTIR analysis was used to investigate the reduction of TiO2NPs by biomolecules in microbial cells. The peaks at 1394 cm − 1 and 1250 cm − 1 were attributed to C-O stretch vibrations, possibly indicating an alcohol or carboxylic acid group. The bands at 1055 cm − 1 and 473 cm − 1 represented C-O stretching vibrations of aliphatic amines and Ti–O stretching vibrations respectively. These biomolecules may also play a role in nucleation and biosynthesis processes, acting as stabilizing and capping agents [49, 50].

  • 4- Energy-dispersive spectroscopic analysis (EDX)

  • The energy-dispersive spectroscopic analysis confirmed the elemental distribution of Ti and O elements, confirming the formation of TiO2 (Fig. 4 E), in line with previous studies.

  • 5- Morphology and shape of TiO2NPs

    • 1-Transmission electron microscopy (TEM)

      The study analyzed P. aeruginosa nanoparticles using TEM to determine their size, morphology, and distribution at the nanoscale. The TiO2NPs were found to be predominantly spherical and ellipsoidal, with uneven distribution Fig. 5 A. The average diameters and size distributions were determined using ImageJ software, with the majority falling within the 30–40 nm range, accounting for 18% of the distribution (Fig. 5 C). The study's findings, compared to previous studies by Khan, Fulekar, and Eisa et al. [22, 51], were found to align with the literature, with a size distribution histogram indicating sample polydispersity, a common characteristic of nanoparticles synthesized through biosynthetic methods [52]. The study found that most TiO2NPs were spherical or round to ellipsoidal in shape, with both individual NPs and agglomerates observed (Fig. 5 A). The nanoparticles appeared separated from each other, suggesting spiraling peptides around them.

      Fig. 5
      figure 5

      (A) TEM analysis and (B) FE-SEM analysis of biosynthesized TiO2NPs (C) Histogram of the particle size distribution of the biosynthesized TiO2NPs

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    • 2- Field emission scanning electron microscopy (FESEM)

      The surface morphology and shape of synthesized TiO2NPs were analyzed using SEM, with the results showing uniform spherical shapes and dimensions. This aligns with previous studies [26, 28]. Additionally, the results of Mathesh et al. showed that Spirulina-mediated TiO2 nanoparticles were efficient against multidrug-resistant bacteria due to their spherical form with aggregation in SEM imaging and XRD analysis, which showed 61.4% crystallinity with anatase phase [53]. The SEM images are shown in Fig. 5 B.

The antibacterial efficacy of TiO2NPs

Table 3 presents the disc diffusion method results for evaluating the effects of TiO2NPs with and without different β-lactam antibiotic combinations, on MDR strains of E. coli, Klebsiella sp., A. baumannii, Proteus sp., and P. aeruginosa (Table 3). The bacteria were tested against four antibiotics: imipenem, ceftazidime, piperacillin, and cefoxitin. The results showed that all tested antibiotics were ineffective against the bacteria, which exhibited resistance to these antibiotics, according to the Clinical and Laboratory Standards Institute (2020) (Table 3 and Fig. 6 C).

Table 3 Mean inhibition (mm) and fold area increase of different antibiotics, TiO2NPs, and combined against MDR bacteria. The test is repeated 3 times. *p < 0.01
Full size table
Fig. 6
figure 6

The combined effect between antibiotics and biosynthesized TiO2NPs against Proteus sp. (A), Acinetobacter baumannii (B), and Antibiotic resistance in Proteus sp. (C)

Full size image

The biosynthesized TiO2NPs, at a concentration of 5000 μg/mL, demonstrated a significant (P < 0.01) antibacterial effect against MDR A. baumannii and Proteus sp. strains. The study found that A. baumannii and Proteus sp. showed the largest zone of inhibition (22 mm and 21 mm, respectively), while E. coli, Klebsiella sp., and P. aeruginosa had zones of inhibition of 18 mm, 20 mm, and 19 mm, respectively. The study found that TiO2NPs were more effective against MDR Gram-negative bacilli strains than all tested antibiotics due to their resistance to antibiotic discs (Table 3 and Fig. 6 A and B).

TiO2NPs are a type of metal oxide nanoparticles that have been extensively studied for their antimicrobial properties. They have been found to possess the potential to eradicate both Gram-positive and Gram-negative bacteria [16, 45]. When compared to antibiotics alone, TiO2NPs demonstrated the strongest antibacterial efficacy against manty pathogens [54]. Studies have shown that P. aeruginosa synthesizes TiO2NPs with antibacterial activity, and similar findings have been observed against other bacteria [51, 55,56,57]. A study in Iraq [58] found that TiO2NPs at a concentration of 500 μg/mL showed the highest zone of inhibition against MDR test organisms, with a maximum of 24 mm against Streptococcus pyogenes, showing high sensitivity even at 31.25 μg/mL. Proteus vulgaris showed the least sensitivity. Gram-negative bacteria have higher negative charges and stronger adhesion to positive surfaces, making biosynthesized TiO2NPs more effective against them, regardless of resistance level. It is possible that oxidation and cell death are brought on by the electromagnetic attraction between bacteria and metal oxides. [59]. The bactericidal effect of tested nanoparticles may also be due to reactive oxygen species (ROS), primarily hydroxyl radicals (OH), decomposing bacterial outer membranes, leading to phospholipid peroxidation and oxidative cell death [51]. In addition to rupturing cell membranes, the main role of cytoplasmic ROS generation in NPs' antibacterial actions is to seriously damage bacteria's DNA, which eventually results in the bacterial demise. Furthermore, interactions between NPs and phosphate components within the bacterial cytoplasm may result in the formation of persistent complexes that disrupt essential bacterial enzymes [54, 60].

Synergistic effect of TiO2NPs with antibiotics by disc diffusion methods

The study examined the effects of TiO2NPs and four β-lactam antibiotics [imipenem (10 μg), ceftazidime (30 μg), piperacillin (100 μg), and cefoxitin (30 μg)] against MDR-Gram-negative bacteria, including E. coli, Klebsiella sp., P. aeruginosa, A. baumannii, and Proteus sp. The disc diffusion method was used to measure the inhibition zones of antibiotics alone and in combination with TiO2NPs at a concentration of 5000 μg/mL [29]. Table 3 shows that the inhibition zone diameter increased when tested antibiotics with TiO2NPs at 5000 μg/mL concentrations against tested isolates. The study measured the inhibition zone produced by antibiotics before and after treatment with TiO2NPs (Table 3 and Fig. 6 A and B), revealing a significant (P < 0.01) increase in the fold area. especially, the zone of inhibition for piperacillin was 6 mm before nanoparticle treatment, and 23 mm after treatment against Proteus sp. and A. baumannii. The combination of antibiotics and TiO2NPs shows synergistic effects, with imipenem showing the lowest antibacterial activity increase in area (13 mm to 25 mm) against Klebsiella sp. The study evaluated the antibacterial activity of antibiotic discs and TiO2NPs in combination with all MDR bacterial isolates. The most significant increase areas (283%) were observed in piperacillin against Proteus sp. and A. baumannii, as well as cefoxitin (266%) against Klebsiella sp. and Proteus sp. (Table 3 and Fig. 6. A and B). The increase in surface areas of inhibition zones varied from 92% up to the considerable amount of 283% and 266% in the presence of TiO2NPs (Table 3). TiO2NPs showed the most significant antibacterial effect against Proteus sp., A. baumannii, in addition, cefoxitin, and their combination with all antibiotics showed increased antibacterial activity compared to individual antibiotics. The combination therapy of TiO2NPs with antibiotics, results in a significant increase in antibacterial activity, possibly by synergistically targeting different bacterial cellular processes and pathways, overcoming antibiotic resistance mechanisms [60]. The rise in infectious diseases associated with antibiotic-resistant pathogenic strains has necessitated the search for alternative antibiotics with potent bactericidal and bacteriostatic properties. Microorganisms have developed complex resistance mechanisms, such as inactivation of antibiotics, alteration of target sites, and transformation of metabolic pathways, due to repeated exposure to antibiotics over generations [61]. Arora et al.'s study found that ceftazidime, an antibiotic, showed a significant synergistic effect (24 mm zone of inhibition) when combined with TiO2NPs, but its antibacterial activity did not improve when used with cefotaxime against MDR-Pseudomonas sp. [62]. Previous studies have shown that TiO2NPs when combined with 23 commercial antibiotics effectively combat MRSA clinical isolates. The combination showed higher activity than individual antibiotics, with amikacin and penicillin G showing the greatest increase in efficacy among tested antibiotics [63]. Because of the active ions, nanoparticles have an incontrovertible antibacterial action and minimal harm to human cells. Additionally, it is a persistent biocide with low volatility and good thermal stability [64]. Hassan et al.'s study found that TiO2NPs mediated by H. lbiecea showed greater bacterial growth inhibition than commercial TiO2NPs against Staphylococcus aureus, E. coli, and Bacillus subtilis, with zones of inhibition of 10.5 mm, 17 mm, and 15 mm respectively [21]. Functionalization of nanoparticles with antibiotics offers a promising platform to combat bacterial resistance, reduce drug dosage and toxicity, and specifically target infection sites, overcoming resistance and minimizing harm to normal cells [65]. TiO2NPs are gaining attention in health and industry due to their photocatalytic properties, low toxicity, and absence of allergic reactions, influenced by factors like particle size and synthesis method [47].

Antibiofilm activity of TiO2NPs

The National Institutes of Health and Center for Disease Control report that 65–80% of infections are caused by microbes that form biofilms, with the most common being P. aeruginosa, E. coli, and Staphylococcus aureus [66]. The study reveals that biosynthesized TiO2NPs have antibiofilm properties against 25 MDR-P. aeruginosa strains, effectively inhibiting the formation of biofilms. The study revealed that TiO2NPs effectively inhibited the attachment and formation of biofilms by tested P. aeruginosa strains in a dose-dependent manner. The study found that biosynthesized TiO2NPs significantly (p < 0.001) reduced the attachment of strong biofilm-producing P. aeruginosa compared to the control group. Treatments with different concentrations of TiO2NPs (19.531, 39.0625, 78.125, 156.25, 312.5, 625, 1250, and 2500 μg/ml) reduced biofilm activity by 40%, 42%, 51%, 62%, 75%, 86%, 90%, and 94% compared to control cultures without TiO2NPs (Fig. 7).

Fig. 7
figure 7

The antibiofilm inhibition percentage of biosynthesized TiO2NPs on strong biofilm-producing-P. aeruginosa

Full size image
Fig. 8
figure 8

A displays complete 16S rRNA gene nucleotide sequences for P. aeruginosa, from a newly obtained isolate from Erbil-Kurdistan Region-Iraq, assigned NCBI GenBank accession number ON678251. Peak of sequencing of 16S rRNA gene of P. aeruginosa (A). Phylogenetic neighbor- joining trees of 16S rRNA amplified from the tested isolates of P. aeruginosa. The bacteria recovered in the present study are marked with an asterisk (B)

Full size image

TiO2NPs disrupt biofilm formation by potentially interfere with quorum sensing mechanisms in bacteria, which are essential for biofilm formation and communication among bacterial cells. By disrupting these pathways interacting with biofilm matrix components, leading to destabilization and inhibition of biofilm development [67]. The mechanism of action of TiO2NPs involves targeting sulfhydryl groups in the cell membrane, forming S-TiO2 bonds, and suppressing the electron transport chain and enzymes essential for biofilm formation [13]. Abdulazeem et al.'s study on bacterial biofilm eradication using TiO2NPs revealed that treatment with MIC concentrations reduced biofilm growth compared to a control group without NPs [9]. Landage et al. found that TiO2NPs reduced biofilm formation by 40–50% in both Gram-negative and Gram-positive bacterial strains, particularly in P. aeruginosa [26]. Alavi & Karimi's study found that Ag-TiO2 nanocomposites reduced biofilm roughness, a virulence factor linked to quorum sensing mechanisms in P. aeruginosa ATCC 27853 [68].The fusion of nanoparticles with the bacterial cell membrane is what causes the significant antimicrobial effects of nanoparticles, which cease bacteria from growing to the point where they can form a biofilm. This is another reasons for the increased anti-biofilm effects of nanoparticles [69].

TiO2NP-producing P. aeruginosa isolate's 16S rRNA gene sequence and phylogenetic analysis

The BLAST alignment program confirmed the isolate's alignment agreement, revealing that the 16S rRNA sequence of P. aeruginosa shares 100% homology with strains MK156198, KY582738, MT832126, ON014782, and MZ191664 from Iraq, Egypt, and Singapore, respectively. The 16S rRNA gene sequencing of a P. aeruginosa isolate showed high homology with other local and global strains, with a range of 100% to 99.89% (Table 4 and Fig. 8. B).

Table 4 Percentage distribution of P. aeruginosa according to blast of GenBank NCBI of partial 16S rRNA gene
Full size table

This homology was observed with P. aeruginosa strains from China, Japan, and Nigeria, and is consistent with other P. aeruginosa isolates in the GenBank database [30].

Conclusions

Multi-drug resistant P. aeruginosa isolates require complex therapy strategies due to their high resistance to a variety of antimicrobial agents. Precise detection of bacterial infections resistant to carbapenem is essential for managing patient care and preventing contamination. The results of this study demonstrate the effectiveness of successfully biosynthesized TiO2NPs. The combination of antibiotics and biosynthesized TiO2NPs demonstrated potent antibacterial and anti-biofilm effects particularly for controlling MDR- P. aeruginosa. This combination therapy can effectively eliminate the pathogen's antibiotic resistance and biofilm formation, posing a significant global public health threat. The synergistic activity of biosynthesized TiO2NPs with antibiotics presents a valuable warrant for further investigation in clinical applications.

Availability of data and materials

The datasets generated and/or analysed during the current study are available in the [TiO2NP-producing P. aeruginosa isolate's 16S rRNA gene sequence] repository, [https://data.mendeley.com/datasets/8xxfgwt4z6/1]. The nucleotide sequence of the P. aeruginosa strain was submitted to the National Centre for Biotechnology Information (www.ncbi.nlm.nih.gov), assigned GenBank accession number ON678251.

Abbreviations

A. baumannii :

Acinetobacter baumannii

E. coli :

Escherichia coli

FE-SEM:

Field Emission Scanning Electron Microscopy

FTIR:

Fourier-Transform Infrared Spectroscopy

K. pneumonia :

Klebsiella pneumonia

MBL:

Metallo-beta-lactamase

MDR:

Multidrug-Resistant

NP:

Nanoparticles

OD:

Optical density

P. aeruginosa :

Pseudomonas aeruginosa

PCR:

Polymerase Chain Reaction

S-TiO2 bonds:

Sulfhydryl-TiO2 Bonds

TEM:

Transmission Electron Microscopy

TiO2NPs:

Titanium Dioxide Nanoparticles

TSB:

Tryptic Soy Broth

XRD:

X-ray Diffraction

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Acknowledgements

The authors express their gratitude to the Research Centre's management and staff for their valuable support.

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Authors and Affiliations

  1. Department of Clinical Analysis, College of Pharmacy, Hawler Medical University, Erbil, Iraq

    Sayran Hamad Haji & Aryan R. Ganjo

  2. Medical Research Center of, Hawler Medical University, Erbil, Iraq

    Sayran Hamad Haji

  3. Department of Physiology and Microbiology, College of Medicine, Hawler Medical University, Erbil, Iraq

    Tola A. Faraj

  4. Department of Medical Analysis, Faculty of Applied Science, Tishk International University, Erbil, Iraq

    Aryan R. Ganjo & Tola A. Faraj

  5. Department of Medical Laboratory Technology, Kalar Technical College, Garmian Polytechnic University, Kurdistan Region, Kalar, Iraq

    Mohammed H. Fatah

  6. Department of Microbiology, Par Hospital, Erbil, Iraq

    Sakar B. Smail

Authors
  1. Sayran Hamad Haji
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  2. Aryan R. Ganjo
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  3. Tola A. Faraj
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  4. Mohammed H. Fatah
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Contributions

SHH and ARG designed the study and supervised the whole work. SBS participated in sample collection, and other practical parts. SHH performed data analysis, drafted part of the manuscript, ARG proofread and finalized the manuscript. TAF and MHF contributed to drafting the manuscript. All the authors have read and approved the final manuscript.

Corresponding author

Correspondence to Aryan R. Ganjo.

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Ethics approval and consent to participate

The study was approved by Hawler Medical University's College of Pharmacy's ethics committee [No. 13082022-8-12], Written informed consent was obtained from the participants prior to participation in the study. Informed consent was obtained from the parents or legal guardians of all participants under the age of 18 prior to their inclusion in the study. Consent was documented through the signed consent forms, which were collected prior to participations. All procedures were carried out in accordance with relevant guidelines and regulations.

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The authors declare no competing interests.

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Haji, S.H., Ganjo, A.R., Faraj, T.A. et al. The enhanced antibacterial and antibiofilm properties of titanium dioxide nanoparticles biosynthesized by multidrug-resistant Pseudomonas aeruginosa. BMC Microbiol 24, 379 (2024). https://doi.org/10.1186/s12866-024-03530-y

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  • DOI: https://doi.org/10.1186/s12866-024-03530-y

Keywords

BMC Microbiology

ISSN: 1471-2180

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