Characterization of promising enterobacterial strains for silver nanoparticle synthesis and enhancement of product yields under optimal conditions

2.1 Determination of silver resistance in 105 enterobacterial strains

A total of 105 enterobacterial strains were previously isolated from 15 species of fresh seafood in Thailand and identified to the genus level based on partial 16S rRNA gene sequences. Twenty-four of these strains were further identified to the species level using the VITEK 2 system. The identified genera include Citrobacter (20 strains), Enterobacter (55 strains), Hafnia (two strain), Klebsiella (six strains), Morganella (four strains), Providencia (12 strains), Salmonella (two strains), Serratia (two strains), and Yersinia (two strains) [30], 31]. The minimum inhibitory concentration (MIC) value was determined based on broth culture method of the European Committee for Antimicrobial Susceptibility Testing (EUCAST) [32]. The inocula grown in Lennox LB broth were inoculated into Mueller-Hinton (MH) broth to obtain cultures having an initial concentration of 1.00 × 10⁵ colony forming units (CFU)/mL. Filter-sterilized AgNO₃ was prepared in a two-fold serial dilution series to obtain final concentrations ranging from 4 to 512 mg/L. The cultures were incubated at 37 °C for 20 h. Bacterial growth was determined by turbidity and an optical density at 600 nm (OD₆₀₀) above 0.1. The MIC value was recorded as the lowest concentration inhibiting bacterial growth. The strains able to grow in the presence of AgNO₃ at a concentration exceeding 512 mg/L were classified as silver-resistant [33]. The strains that exhibited resistance at a threshold concentration of 512 mg/L (3 mM) were further tested for their resistance to higher AgNO₃ concentrations at 1,024 mg/L (6 mM) and 1,536 mg/L (9 mM).

2.2 Detection and nucleotide sequencing of silver resistance genes in 105 enterobacterial strains

Genomic DNA of 105 enterobacterial strains were used as DNA templates in polymerase chain reaction (PCR) to detect the presence of 12 silver resistance genes, including copA, cusA, cusC, silA, silB, silC, silE, silF, silG, silP, silR, and silS. The copA and silP genes encode copper/silver-exporting ATPases. The products of cusA, cusC, silA, silB, and silC genes are copper/silver efflux transporters. The silE and silF genes synthesize periplasmic copper/silver-binding proteins. The silG gene likely encodes a periplasmic chaperone to deliver silver ions. The silR and silS genes encode putative transcriptional response regulators [34], [35], [36], [37]. The examined genes, primer nucleotide sequences, and PCR product sizes are shown in Table 1. If not indicated otherwise, the primer sequences were designed using the Primer-BLAST program of the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). The presence and sizes of PCR products were determined by electrophoresis on 1 % agarose gels. The PCR positive bands were eluted from agarose gels using a QIAquick gel extraction kit (Qiagen, Valencia, CA, USA). The purified PCR products were sequenced by Bio Basic, Inc. (Markham, ON, Canada). The resulting nucleotide sequences were aligned with reference sequences in order to identify genes and calculate percentages of sequence identity by using the BLASTn program available on the NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

2.3 Screening of AgNP-synthesizing strains and measurement of AgNP concentrations

The reaction for AgNP synthesis was adjusted from that previously described [40]. The strains were cultured in Lennox LB broth at 37 °C for 36 h under static conditions. The cultures were centrifuged at 11,000 rpm for 10 min at 4 °C in an Eppendorf 5804R centrifuge (Eppendorf, Selangor Darul Ehsan, Malaysia). 10 mL of supernatants were mixed with 10 mL of 1 mM AgNO₃, and the reaction mixtures were left steady at 37 °C for 24 h and 120 h in the dark. The color change from light yellow to brown was the initial confirmation of AgNP synthesis [41]. The visible ultraviolet (UV–vis) spectra of the synthesized AgNPs were determined using a NanoDrop 2000C spectrophotometer (Thermo Scientific, Waltham, MA, USA). The OD values of the reaction mixtures were measured at λ _max using a Cecil CE1011 spectrophotometer (Cecil Instruments, Cambridge, UK). The concentrations of synthesized AgNPs were calculated from a standard curve relating OD values to concentrations of commercial AgNPs with the same λ _max (Sigma-Aldrich, St. Louis, MO, USA).

2.4 Species identification of the selected strain based on the VITEK 2 system

The selected enterobacterial strain, which synthesized AgNPs at the highest concentration, was identified to the species level using the GN card of the VITEK 2 system version 07.01 (bioMerieux, Inc., Durham, NC, USA).

2.5 Shape and size characterization of the AgNPs synthesized by the selected strains

The reaction mixtures containing the synthesized AgNPs were filtered through membranes with a pore size of 0.45 μm (Sartorius Stedim Biotech, Gottingen, Germany). The filtrates were centrifuged at 11,000 rpm for 20 min at 4 °C in an Eppendorf 5804R centrifuge (Eppendorf, Selangor Darul Ehsan, Malaysia) to precipitate the AgNP pellets. The AgNP pellets were washed three times and dissolved in sterile ultrapure water (Invitrogen, Waltham, MA, USA). Shape and size characterization of the synthesized AgNPs was performed using Tescan Mira3 scanning electron microscopy (SEM) (Tescan Orsay Holding, Brno-Kohoutovice, Czech Republic). Analysis of the shape and size was conducted using ImageJ software [42].

2.6 Condition optimization for the AgNP synthesis by the selected strains

The selected enterobacterial strains were cultured in Lennox LB broth at 37 °C for 36 h with shaking at 150 rpm. The cultures were inoculated to obtain an initial concentration of 1.00 × 10⁵ CFU/mL under the tested conditions. The factors that were varied in the tested conditions were as follows: (1) culture media [Lennox LB, brain heart infusion (BHI), and minimal medium M63. The formulas for these media were described in Ezraty et al. [43], Fisher Scientific [44], and Rewak-Soroczynska et al. [45], respectively, (2) inducer presence (without and with 100 μM AgNO₃), (3) aeration (without and with shaking at 150 rpm), (4) cultivation times (6, 12, 24, 36, and 48 h), and (5) reaction incubation temperatures and times (37 °C and 55 °C for 6, 24, 48, 72, and 120 h). The AgNP concentration measurement was performed as mentioned above.

2.7 Antibacterial activity of the AgNPs synthesized by the selected strains

The AgNPs synthesized by the selected enterobacterial strains were evaluated for antibacterial activity against 17 strains (belonging to 11 species in six genera) of human pathogenic bacteria, including Aeromonas enteropelogenes strain SFSS3, Aeromonas salmonicida strain SFSS7, Aeromonas sobria strain SFSS1, Enterococcus faecalis strains ATCC 29212, S1C1, S1C2, and S3C3, Enterococcus flavescens strains S4C1 and S4C4, Enterococcus hirae strain S3C4, E. coli strains ATCC 25922, S1C1, and S2C1, Pseudomonas aeruginosa strain TISTR 1287, Pseudomonas fluorescens strain TISTR 358, Staphylococcus aureus strain TISTR 2329, and Vibrio parahaemolyticus strain SFTCBS4. The Aeromonas, Enterococcus, Escherichia, and Vibrio strains were obtained from our previous studies [30], 46]. The Pseudomonas and Staphylococcus strains were obtained from the Thailand Institute of Scientific and Technological Research (TISTR). Broth dilution method was performed as previously described [47] with some modifications. One hundred μL of serially diluted inocula of the tested pathogenic bacteria was inoculated into 4 mL of MH broth to obtain an initial concentration of 1.00 × 10⁵ CFU/mL. 2 mL of two-fold serial dilutions of filter-sterilized AgNPs was added to the cultures to obtain final concentrations ranging from 0.84 to 3.35 μg/mL. The cultures were further incubated at 37 °C for 20 h with shaking at 150 rpm. Bacterial growth was determined by turbidity and an OD₆₀₀ above 0.1. The positive and negative controls were prepared without the addition of AgNPs and tested bacteria, respectively.

2.8 Statistical analysis

The data analysis was processed with the SPSS statistical software version 19.0 (IBM Corp., Chicago, IL, USA). All data analyses were performed with triplicate samples. Significant differences among means were evaluated using the analysis of variance (ANOVA) with Tukey’s test in which the p-value <0.05 was indicative of a significant difference.

3.1 Silver resistance in 105 enterobacterial strains

The strains able to grow in the presence of AgNO₃ at a concentration exceeding 512 mg/L were classified as silver-resistant [33]. Based on this definition, 63 out of 105 enterobacterial strains (60.0 %) were silver-resistant. The silver-resistant strains belonged to Enterobacter (34 strains), Citrobacter (12 strains), Providencia (eight strains), Klebsiella (three strains), Morganella (two strains), Yersinia (two strains), Hafnia (one strain), and Serratia (one strain). The 36, 9, and 18 strains were resistant to AgNO₃ up to 512 mg/L (3 mM), 1,024 mg/L (6 mM), and 1,536 mg/L (9 mM), respectively.

3.2 Silver resistance gene patterns in 105 enterobacterial strains

Out of 105 enterobacterial strains, 48 strains (45.7 %) harbored at least one of 12 silver resistance genes, including copA, cusA, cusC, silA, silB, silC, silE, silF, silG, silP, silR, and silS. Ten silver resistance gene patterns are shown in Table 2. Sequencing of PCR-amplified fragments from the representative strains reinforced the presence of these genes based on 96.58–100 % identities to the reference sequences. The sequences of silver resistance genes have been deposited in the NCBI database under GenBank accession numbers PV296195-PV296224.

3.3 AgNP concentrations synthesized by the AgNP-synthesizing strains

After incubation of the reaction mixtures composed of supernatants and 1 mM AgNO₃ for 24 h and 120 h, 22 strains (21.0 %) showed the color change from light yellow to brown, which was the initial confirmation of AgNP synthesis [41]. The synthesized AgNPs showed the λ _max peaks at 420 nm. Therefore, the commercial AgNPs with λ _max of 420 nm (Sigma-Aldrich, St. Louis, MO, USA) were employed to construct a standard curve between OD₄₂₀ values from 0.0 to 1.0 and AgNP concentrations from 0.0 to 10.0 μg/mL. The 22 AgNP-synthesizing strains synthesized extracellular AgNPs in concentrations ranging between 2.78 ± 0.38 and 20.26 ± 0.16 μg/mL. These strains belonged to four genera, including Enterobacter (11 strains), Citrobacter (five strains), Morganella (three strains), and Providencia (three strains). As compared between reaction incubation times of 24 h and 120 h, the AgNP formation of 18 strains was only observed at 120 h, whereas that of four strains could occur at 24 h. The maximum AgNP concentrations were obtained from Citrobacter sp. ENTSF 29-3 (20.01 ± 0.64 μg/mL at a reaction incubation time of 120 h) and Providencia rustigianii SFTCBS3 (20.26 ± 0.16 μg/mL at a reaction incubation time of 120 h). Both strains were therefore selected for further studies.

3.4 Species identification of Citrobacter sp. ENTSF 29-3 based on the VITEK 2 system

Citrobacter sp. ENTSF 29-3, which was previously identified into the genus level based on a partial 16S rRNA gene sequence [31], was subjected to species identification based on the VITEK 2 system. It was identified as Citrobacter freundii with a 97 % probability. P. rustigianii SFTCBS3 was isolated and identified in our previous study [30].

3.5 Shape and size of the AgNPs synthesized by C. freundii ENTSF 29-3 and P. rustigianii SFTCBS3

The SEM photographs (Figure 1) show that both C. freundii- and P. rustigianii-derived AgNPs were spherical with an average size around 17 nm. The particle size distributions of C. freundii- and P. rustigianii-derived AgNPs were in ranges of 11–38 nm and 5–44 nm, respectively.

Figure 1:

SEM photographs displaying the AgNPs synthesized by Citrobacter freundii ENTSF 29-3 (A) and Providencia rustigianii SFTCBS3 (B).

3.6 Optimal conditions for the AgNP synthesis by C. freundii ENTSF 29-3 and P. rustigianii SFTCBS3

The factors, including culture media, inducer presence, aeration, cultivation times, and reaction incubation temperatures and times, were evaluated for their effect on the AgNP synthesis by C. freundii and P. rustigianii. The AgNP concentrations synthesized by both strains grown in three culture media are presented in Table 3. Lennox LB was most suitable for AgNP synthesis by both strains, and a reaction incubation time of 120 h resulted in significantly higher AgNP concentrations than a reaction incubation time of 24 h.

The AgNP concentrations synthesized by both strains grown in the absence and presence of 100 μM AgNO₃ as an inducer are presented in Table 4. The addition of 100 μM AgNO₃ as an inducer in Lennox LB resulted in significantly higher AgNP concentrations from C. freundii and P. rustigianii at reaction incubation times of 120 h and 24 h, respectively.

The AgNP concentrations synthesized by both strains grown under static and aerated conditions are presented in Table 5. The static condition improved AgNP synthesis for both strains at both reaction incubation times (24 h and 120 h).

The AgNP concentrations synthesized by both strains at five different cultivation times are presented in Table 6. A cultivation time of 36 h was most suitable for AgNP synthesis by C. freundii at both reaction incubation times (24 h and 120 h) and by P. rustigianii at 120 h reaction incubation time.

The AgNP concentrations synthesized by both strains at ten combinations of incubation temperature and time are presented in Table 7. A reaction temperature of 55 °C enhanced AgNP formation in both strains at shorter incubation times of 24 h and 48 h.

The most suitable reaction conditions for C. freundii-derived AgNP formation were 37 °C for 120 h, 55 °C for 72 h, and 55 °C for 120 h. The significantly highest concentration of the P. rustigianii-derived AgNPs was obtained when the reaction mixture was incubated at 37 °C for 120 h.

3.7 Bacterial growth inhibition of the AgNPs synthesized by C. freundii ENTSF 29-3 and P. rustigianii SFTCBS3

Both C. freundii- and P. rustigianii-derived AgNPs at 3.35 mg/mL were tested against 17 strains of human pathogenic bacteria. The AgNPs inhibited growth of seven strains: A. enteropelogenes SFSS3, A. sobria SFSS1, E. faecalis S1C2 and S3C3, E. flavescens S4C4, E. hirae S3C4, and S. aureus TISTR 2329.