Evaluation of Anti-biofilm and Antibiotic Synergistic Activities of Silver Nanoparticles Against Some Common Bacterial Pathogens

Introduction: Nowadays silver nanoparticles (AgNPs) are used as antimicrobials due to their well-known biochemical properties. The current study was planned to search the antimicrobial activities of AgNPs alone and in combination with common antibiotics against some field isolates of common bacterial pathogens. Methods: Standard strains of Escherichia coli, Salmonella enteritidis, Staphylococcus aureus and Streptococcus agalactiae, in addition to 9 field isolates for each strain (totally 40 strains, 10 for each) were used. Macrodilution method for determination of minimum inhibitory concentration (MIC) of AgNPs and antibiotics against isolates was used. Biofilm formation was evaluated by microtiter plates. Disc diffusion method was used for assaying bactericidal activities of antibiotics and their combinations with AgNPs against the isolates. Results: Mean MICs of AgNPs for Salmonella serotypes, E. coli, S. aureus and S. agalactiae were 3.125, 6.25, 6.25 and, 12.5 ug/mL, respectively. S. aureus and S. agalactiae showed more sensitivity (increase in fold) to examined antibiotics plus AgNPs compared to Salmonella serotypes and E. coli. The results showed that AgNPs had strong antibacterial and anti-biofilm activities against the examined pathogens. Synergistic and antagonistic effects of AgNPs in combination with tetracyclin, gentamicin, streptomycin, kanamycine, cephalosporin and penicillin were observed in different cases. Conclusion: AgNPs alone or in combination with antibiotics could potentially be used as effective antibacterial and anti-biofilm agents. However, our results showed that synergistic effects of antibiotics combined with AgNPs cannot be considered as a rule.

antimicrobials and are known as main causes of chronic infections in medicine. 4 Biofilms are able to colonize plastic walls of tubes, such as intravenous catheters or tissues like urogenital tract and wounds. 4,5 Most of the studies on AgNPs antimicrobial activities performed on standard bacterial strains and reports for such studies on field isolates of pathogenic bacteria are rare. Moreover, we could find rare reports regarding studies on AgNPs effects on Streptococcus agalactiae. 5 In the present work, we investigated whether the biofilm formation of some field isolates of common bacterial pathogens including S. agalactiae can be influenced by AgNPs at minimum inhibitory concentrations (MIC) and sub-MICs. We also examined the susceptibility of isolates to some antibiotics in combination with AgNPs.

Bacterial Isolates
Standard strains of Escherichia coli ATCC 25922, Salmonella enteritidis RTCC 2465, Staphylococcus aureus RTCC 1907, and S. agalactiae RTCC 1907, in addition to 9 field isolates of each strain (totally 40 strains, 10 for each) were used. E. coli isolates were removed from the dead poultry headed to the Veterinary Clinic of Shahrekord University by regional poultry farms. S. aureus and S. agalactiae were purified from mastitis cow milk in the author's previous works. 6,7 Salmonella serotypes were collected from the animals and preserved in the reservoir at Microbiology Laboratory of Veterinary College. The methods (collection and detection of bacteria) were conducted according to Quinn et al guidelines. 8 AgNPs and MIC Determination Colloidal AgNPs with particles ranging between 10 and 20 nanometers (Sigma, CAS Number 7440-22-4) were prepared from Kian Eksir Co. which imported AgNPs as powder and prepared its colloidal form. A dilution of 100 ug/mL was prepared from the stock solution (1000 ug/ mL) in nutrient broth (Nb) after sonication. Nb was used for all the antibacterial assays. Tube double serial dilution method was used for determination of MIC of AgNPs against isolates, according to guidelines of the Clinical and Laboratory Standards Institute. 9 The bacterial cultures (in tryptic soy broth, TSB, tubes) were incubated aerobically at 37°C for 18-24 hours. The turbidity of the cultures was adjusted to 0.5 McFarland (1.5×10 8 CFU/mL) and then diluted in saline solution in order to obtain an inoculum of 5×10 6 CFU/tube. Positive and negative controls were also set up. The inoculated tubes were aerobically incubated at 37°C, shaking, for about 18 hours. The lowest concentration that inhibited visible growth after incubation was defined as MIC which was confirmed in next step.
Anti-biofilm Assays Biofilm forming abilities of the isolates were evaluated by microplates as described by Tendolkar et al. 10 In brief, the bacteria were cultivated at 37˚C in TSB (Merck, Darmstadt, Germany). The strains were afterward centrifuged at 6000 × g (10 minutes), and the precipitate was resuspended in 5 mL of fresh medium. The optical densities (ODs) were adjusted to an absorbance of 1.00 at 595 nm by spectrophotometer (Jenway, OSA, UK). The cultures were admixed (in ratio of 1:40) in fresh TSB. Then 200 μL of cells was inserted in a single row of a sterile polystyrene microtiter plate. The planktonic cells were aspirated after incubation at 37˚C for 24 hours, and the wells were rinsed three times with sterile phosphatebuffered saline (PBS). The plates were air-dried for 60 minutes at 25˚C. For biofilm tests, 200 μL of aqueous crystal violet (0.2%) was poured in each well and after 15 minutes the wells were washed with sterile PBS to remove the excess dye. Crystal violet was extracted with 200 μL of an 80:20 (vol/vol) mixture of ethyl alcohol and acetone, and its absorbance was read at 595 nm. To assay AgNPs effects on biofilm formation of the isolates, microtiter plate wells were inoculated with NPs containing examined isolates in such a way that each well contained 10 5 CFU/well and loaded with different concentrations of AgNPs (1.6 -25 μg/mL). The plates were incubated at 37˚C, shaking, for 24 hours. Control wells were also considered. Growth was assayed using a microtiter enzyme-linked immunosorbent assay (ELISA) reader (Stat Fax 4200) by monitoring absorbance at 600 nm. Further process was as above. All biofilm and anti-biofilm assays were performed in triplicate. Biofilm production was judged according to Stepanovic et al. 11 In this manner optical density cut-off value (ODc) was calculated as: mean ODs + 3 × standard deviation (SD) of negative control. Accordingly, biofilm producers were classified as: no production when ≤ODc, weak production as ODc < ~ ≤ 2 × ODc, moderate production as 2 × ODc < ~ ≤4 × ODc, and strong production as >4 × ODc, where "~" demonstrates the mean of sample ODs. All strains were tested for biofilm production and 10 isolates in each species that were strong or medium biofilm producers were chosen to calculate the MIC of AgNPs on biofilm formation. Percentage of biofilm inhibition was calculated as explained by Wei et al, 12 using the equation: (1 -A595 of the test/A595 of non-treated control) × 100.
Assays for AgNPs and Antibiotics Combined Effects Antibiotics were selected from 3 groups having different modes of action, including tetracyclin (tetracyclines), gentamicin, streptomycin, kanamycine (aminoglycoside), cephalosporin, and penicillin (beta lactams). To assay bactericidal activities of different antibiotics against the isolates, disc diffusion was exploited on Muller-Hinton agar (MHA) plates. The standard antibiotic discs were purchased from Techno Biolab Co. To determine their combined effects, each antibiotic disc was impregnated with 20 uL solution (1 MIC for each isolate) of AgNPs. Similar tests were performed with antibiotic discs alone. An isolate colony of each strain was cultured in TSB medium (37°C, overnight). The inocula were then exerted to the plates along with the standard and prepared discs. The inhibition area was then determined. All the tests were done in triplicate. Fold area changes were determined by measuring the mean surface area of the inhibition zone. Changes were then calculated as b 2 -a 2 /a 2 , where a and b are inhibition areas for the antibiotic and combination of antibiotic and AgNPs respectively. 3

Results
Mean MICs of AgNPs for S. serotypes, E. coli, S. aureus and S. agalactiae isolates were 3.125, 6.25, 6.25 and 12.5 ug/mL, respectively. All strains were selected based upon their abilities to form strong to medium biofilms. Percentages of biofilm formation, inhibiting 40 isolates in the presence of different concentrations of AgNPs are presented in Table 1.
In the present work, using the disc diffusion method, the combined activities of AgNPs and antibiotics (gentamicin, tetracycline, streptomycin, cephalosporin, kanamycine and penicillin) were studied against 40 isolates of common pathogenic bacteria (S. serotypes, E. coli, S. aureus and S. agalactiae) in comparison to antibiotics alone. The diameters of inhibition zones have been noted in Table 2. S. aureus and S. agalactiae showed more sensitivity (increase in fold) to examined antibiotics plus AgNPs compared to S. serotypes and E. coli. For the 2 recent bacteria only penicillin and tetracyclin plus AgNPs showed fold increasing. Maximum increase in fold area was related to kanamycine and tetracyclin (8 and 2.2 respectively) against S. agalactiae.

Discussion
In the present work, we evaluated the antibacterial and anti-biofilm formation activity of AgNPs against 2 gram-negative (Salmonella serotypes and E. coli) and 2 gram-positive (S. aureus and S. agalactiae) bacteria that are considered important pathogens. The mean MICs of AgNPs recorded for gram-negative (3.125-6.25 ug/ mL) and gram-positive (6.25-12.5 ug/mL) bacteria were well comparable with other reports regarding examined species. 13 Thus we could conclude that the AgNPs inhibited growth of the examined isolates, and E coli was more susceptible to AgNPs concentrations compared to S. agalactiae ( Table 1). Most of the reports on AgNPs antimicrobial activities are performed on standard bacterial strains; here we examined some field isolates of pathogenic bacteria in addition to their standard counterpart. In a study by Helmlinger et al, 14 the impact of AgNPs on the biology of prokaryotic and eukaryotic cells was scrutinized. In the mentioned report, it was shown that cells with a higher specific surface area such as platelets faded faster than particles with a smaller specific surface area such as cubes. It is suggested that the antimicrobial effects of AgNPs may be related to silver ions shedding because bacteria are probably unable to ingest the used AgNPs in contrast  to eukaryotic cells. 14 To confirm the latter idea, Wang et al found that gold nanospheres can attach to the membrane of Salmonella typhimurium, but unable to be digested by the bacteria. 15 AgNPs with a concentration range of 1.6-6.25 ug/mL exhibited more than 90% inhibitory effects on biofilm formation of the examined species (Table 1). Generally, the inhibition of the biofilm formation relates to the inhibition of bacterial growth. Studies have shown that chlorhexidin, triclosan and phenolic compounds, among others, may also inhibit biofilm development. 16 Hence, the effective anti-biofilm concentration of AgNPs could be lower than their antibacterial concentration and at the same concentration, higher anti-biofilm activity is expected. Gurranthan et al also reported AgNPs inhibitory concentrations were somehow low in such a manner that could affect cell viability. Gram-positive bacteria of S. aureus and Streptococcus pneumoniae treated with 0.7 μg/ mL of AgNPs showed 90% reduction in biofilm activity. 17 A recent study on Staphylococcus epidermidis showed that anti-biofilm activity of AgNPs was dose-and timedependent. 18 The anti-biofilm activity of AgNPs against antibiotic resistant bacteria was reported in similar studies. 19 As appeared in Table 2, the results clearly showed that antibacterial activity against gram-positive bacteria (S. aureus and S. agalactiae) was more when antibiotics plus AgNPs were used rather than when antibiotics were used alone, as evidenced by increase in fold area. This combinatory effect does not seem to be fully successful for examined Salmonella serotypes and E. coli isolates. Fayaz et al 20 noted that synergistic effect may be a result of conjugative reaction between antibiotic and AgNPs. They showed inhibition of gram-positive bacteria was generally more difficult to be obtained by AgNPs alone, as our results were also in line with their observation. In the case of isolates of S. aureus and S. agalactiae, our results were in line with other findings such as a report by Birla et al. 3 They reported synergistic activities between antibiotics like vancomycin, gentamicin, streptomycin, ampicillin and kanamycin, and AgNPs against Pseudomonas aeruginosa, S. aureus and E. coli. However for Salmonella serotypes and E. coli isolates the story was to some extent different (Table 2), and this finding challenged Gurranthan et al 17 suggestion regarding higher toxicity of individual AgNPs or antibiotics rather than combined form against bacterial cells. De Souza et al reported an antagonistic activity of AgNPs with amoxicillin or oxacillin combinations against a methicillin resistant S. aureus strain. 21 Kazemi et al 22 assessed the efficacy of AgNPs in combination with 4 antibiotics against 50 isolates of mastitis-causing S. aureus. A table in their report shows when AgNPs were combined with streptomycin and erythromycin, 33 and 16 (out of 46) isolates (respectively) showed antagonistic activities for the combinations, however we could not find any reference to this antagonistic activity in their paper. Hari et al observed that in some cases, the combination of antibiotics with AgNPs brought down the inhibitory effect of antibiotics. In their report, AgNPs were recommended with and without antibiotics for controlling the pathogens. 23 Taking together, our results confirmed that synergistic effects of antibiotics combined with AgNPs cannot be considered as a rule. Since AgNPs may have different shapes and sizes, and be modified with different coatings, this may lead to differences in their effects in combination with different antibiotics. There are also other reports that show antibacterial and anti-biofilm activities of antibiotics combined with AgNPs against P. aeruginosa, Shigella flexneri, S. aureus, and S. pneumonia. An enhanced activity for ampicillin and vancomycin against both gram-negative and grampositive bacteria has also been reported. 17 However, reports regarding AgNPs effects on S. agalactiae are rare 5 and as appeared in Table 1, we can conclude that this species is more resistant to antimicrobial activities of AgNPs compared to our other examined isolates.
In conclusion, our results demonstrated that AgNPs shows promising anti-biofilm activity against field isolates of S. serotypes, E. coli, S. aureus and S. agalactiae. These particles can also compete with commercial antibiotics used for the treatment of bacterial infections and sometimes are even better. Regarding examined S. aureus and S. agalactiae isolates, a possible combination of tetracyclin, gentamicin, streptomycin, kanamycine, cephalosporin and penicillin with AgNPs showed enhanced antimicrobial effects and was concluded as synergism. However, synergistic effects of combination of antibiotics and AgNPs cannot be considered as a rule. Further research on animal models is required.

Conclusion
AgNPs alone or in combination with antibiotics could potentially be used as an effective antibacterial and antibiofilm agent. Synergistic effects of antibiotics combined with AgNPs cannot be considered as a rule.

Ethical Approval
The study protocol was approved by Local Ethics Committee of Veterinary College, Shahrekord University.

Competing Interests
Authors declare that they have no potential conflict of interests.