Antibiofilm Effect of Biogenic Silver Nanoparticles Combined with Oregano Derivatives against Carbapenem-Resistant Klebsiella pneumoniae

Resistant bacteria may kill more people than COVID-19, so the development of new antibacterials is essential, especially against microbial biofilms that are reservoirs of resistant cells. Silver nanoparticles (bioAgNP), biogenically synthesized using Fusarium oxysporum, combined with oregano derivatives, present a strategic antibacterial mechanism and prevent the emergence of resistance against planktonic microorganisms. Antibiofilm activity of four binary combinations was tested against enteroaggregative Escherichia coli (EAEC) and Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC): oregano essential oil (OEO) plus bioAgNP, carvacrol (Car) plus bioAgNP, thymol (Thy) plus bioAgNP, and Car plus Thy. The antibiofilm effect was accessed using crystal violet, MTT, scanning electron microscopy, and Chromobacterium violaceum anti-quorum-sensing assays. All binary combinations acted against preformed biofilm and prevented its formation; they showed improved antibiofilm activity compared to antimicrobials individually by reducing sessile minimal inhibitory concentration up to 87.5% or further decreasing biofilm metabolic activity and total biomass. Thy plus bioAgNP extensively inhibited the growth of biofilm in polystyrene and glass surfaces, disrupted three-dimensional biofilm structure, and quorum-sensing inhibition may be involved in its antibiofilm activity. For the first time, it is shown that bioAgNP combined with oregano has antibiofilm effect against bacteria for which antimicrobials are urgently needed, such as KPC.


Introduction
This manuscript reports, for the first time, the antibiofilm effect of eco-friendly binary combinations composed of oregano compounds (OEO, Car, and Thy), which is considered GRAS (Generally Recognized as Safe) [1], and biogenic silver nanoparticles (bioAgNP)

Biogenic Silver Nanoparticles (bioAgNP) Characterization Biogenic Silver Nanoparticle
Fungal-free solution without AgNO 3 was pale yellow. After adding silver salt, the solution color changed to brownish, and its color intensity increased over the course of time while incubated at 28 • C, suggesting bioAgNP formation. The nanoparticle plasmonic band also indicated that bioAgNP were synthetized; the bioAgNP exhibited a surface plasmonresonance peak centered at 410 nm, while pale yellow fungal-free solution (negative control) showed no absorption peak at this wavelength; the UV-visible absorbance spectra are shown in Figure 1A.
Photon correlation spectroscopy and scanning microscopy analysis confirmed nanoparticle formation. The scanning electron microscopy (SEM) micrograph of bioAgNP shows the spherical shape of nanoparticles ( Figure 1B). The average bioAgNP diameter and zeta potential were 84.10 nm and −15.9 mV, respectively. Nanoparticle size and zetapotential distributions are shown in Supplementary Material in Figures S1 and S2, respectively. Energy-dispersive X-ray fluorescence spectrometer analysis confirmed the presence of Ag in nanoparticle samples (silver concentration obtained by linear regression is shown in Figure S3 in Supplementary Material).  Photon correlation spectroscopy and scanning microscopy analysis confirmed nanoparticle formation. The scanning electron microscopy (SEM) micrograph of bioAgNP shows the spherical shape of nanoparticles ( Figure 1B). The average bioAgNP diameter and zeta potential were 84.10 nm and −15.9 mV, respectively. Nanoparticle size and zeta-potential distributions are shown in Supplementary Material in Figures S1 and S2, respectively. Energy-dispersive X-ray fluorescence spectrometer analysis confirmed the presence of Ag in nanoparticle samples (silver concentration obtained by linear regression is shown in Figure S3 in Supplementary Material).

Sessile Minimal Inhibitory Concentration (SMIC) of Compounds Alone against Both Initial Stage of Biofilm Formation and Preformed Biofilm
As shown in Supplementary Material, OEO, Car, Thy, and bioAgNP alone significantly prevented biofilm formation (Table S1) and decreased metabolic activity of preformed biofilm (Table S2) of both EAEC 042 and KPC-producing K. pneumoniae, compared to untreated control (p < 0.05, Kruskal-Wallis test). SMIC values were determined at 95% or greater inhibition (SMIC ≥95 ) of metabolic activity compared to untreated positive control. Table 1 indicates SMIC ≥95 of each antimicrobial alone for EAEC O42 and KPC-producing K. pneumoniae at both the initial stages of biofilm formation and preformed biofilm; SMIC ≥95 values were determined with the MTT method. OEO, Car, Thy, and bioAgNP alone significantly prevented biofilm formation and decreased metabolic activity of preformed biofilm of both EAEC 042 and KPC-producing K. pneumoniae, compared to the untreated control (p < 0.05, Kruskal-Wallis test). As shown in Supplementary Tables S1 and S2 (data for biofilm at the initial stage of formation and preformed biofilm, respectively), all four antibacterials reduced at least 95% of biofilm viability and caused at least 90% reduction in total biofilm biomass.

Sessile Minimal Inhibitory Concentration (SMIC) of Compounds in Combinations against Both the Initial Stage of Biofilm Formation and Preformed Biofilm
For preformed biofilm or its initial stage of formation, the four combinations showed improved antibiofilm activity against EAEC and KPC strain by reducing SMIC ≥95 of each compound in association ( Table 2). Table 2. Sessile minimal inhibitory concentration (SMIC ≥95 ) of oregano derivatives (mg/mL) and biological silver nanoparticles (bioAgNP, µg/mL) in binary combinations against enteroaggregative Escherichia coli (EAEC 042) and KPC-producing Klebsiella pneumoniae (KPC), at both early stage of biofilm formation and preformed biofilm.  Table 1). NT (not tested):

Antibacterial
The maximum tested concentration of Thy or bioAgNP in combination was 25% of their individual SMIC values because both compounds alone at 0.5 × SMIC already reduce more than 80% of biofilm metabolic activity. However, both Thy and bioAgNP in combinations show greater antibiofilm activity than both compounds individually, as shown in Tables 3 and 4.  Table 3. Antibiofilm effect, shown in terms of biomass and metabolic activity reduction, of oregano derivatives combined with biological silver nanoparticles (bioAgNP) compared to both antimicrobials individually against enteroaggregative Escherichia coli (EAEC 042) biofilms growth in microtiter plates, which were evaluated at an early stage of biofilm formation. Oregano derivatives: OEO (oregano essential oil), Car (carvacrol), and Thy (thymol). * It indicates that the binary combination caused statistically (p < 0.05, Kruskal-Wallis test) a greater reduction in biofilm formation and showed an improved antibiofilm effect compared to both antimicrobials alone at the same concentrations. When the combination has an antibacterial effect similar to the antimicrobials alone, the difference in biofilm reduction is not significant. ±(standard deviation). Table 4. Antibiofilm effect, shown in terms of biomass and metabolic activity reduction, of oregano derivatives combined with biological silver nanoparticles (bioAgNP) compared to both antimicrobials individually against KPC-producing Klebsiella pneumoniae (KPC) biofilms growth in microtiter plates, which were evaluated at an early stage of biofilm formation. We highlight the combination of Thy plus bioAgNP that resulted in SMIC ≥95 reductions of 50% for Thy and 75% for bioAgNP compared to compounds alone against EAEC biofilm formation. For its preformed biofilm, this combination reduced SMIC ≥95 by 75% for Thy and by 87.5% for bioAgNP.

Bacteria
For KPC-preformed biofilm, bioAgNP at 3.94 µg/mL and Thy at 0.12 mg/mL (both values represent 0.5 × SMIC ≥95 individually) have not been tested in combinations since both concentrations alone already show a great antibiofilm effect, reducing metabolic activity by around 80%; Thy plus bioAgNP reduced SMIC ≥95 of both antimicrobials by 50% against biofilm on the initial stage of formation.
In a few cases, as shown in Table 2, combinations did not cause SMIC ≥95 reductions, but they still showed a better effect than antimicrobial alone by causing a greater decrease in biofilm metabolic activity and/or total biomass compared to individual treatments (Section 2.4). For example, Car associated with bioAgNP did not reduce SMIC ≥95 of compounds against the KPC strain; however, this combination showed an improved effect compared to separated compounds.

Antibiofilm Effect of Binary Combinations Compared to Antimicrobials Individually against Preformed Biofilm in Microtiter Plates and Its Initial Stage of Formation (Reduction in Biomass and Metabolic Activity)
All tested combinations (OEO plus bioAgNP, Car plus bioAgNP, Thy plus bioAgNP, and Car plus Thy) prevented biofilm formation and inhibited the growth of preformed biofilm by both EAEC 042 and carbapenem-resistant K. pneumoniae. For each bacterium, comparative analysis among each combination and untreated control showed that double antimicrobial treatments significantly reduced total biofilm biomass and biofilm metabolic activity (p < 0.05, Kruskal-Wallis test), as shown in Supplementary Figure S4 for the initial stage of biofilm formation and in Supplementary Figure S5 for preformed biofilm.
All four combinations showed improved antibiofilm activity compared to antimicrobials individually against preformed biofilm and its initial stage of formation. Each combination and its antimicrobials alone at the same concentrations showed that double antimicrobial treatments significantly caused a greater reduction in total biofilm biomass and biofilm metabolic activity in percentage (p < 0.05, Kruskal-Wallis test), as shown in Tables 3-6. Both Tables 3 and 4 present the combinatory effect of antimicrobials on biofilm  at the initial stage, and Tables 5 and 6 present the combination antibiofilm effect against preformed biofilm. Table 5. Antibiofilm effect, which is shown in terms of metabolic activity reduction of oregano derivatives and biological silver nanoparticles (bioAgNP) compared to both antimicrobials individually against enteroaggregative Escherichia coli (EAEC 042) biofilms growth in microtiter plates, which were evaluated at preformed biofilm condition. , and Thy (thymol). * It indicates that the binary combination caused statistically (p < 0.05, Kruskal-Wallis test) a greater reduction in biofilm formation and showed an improved antibiofilm effect compared to both antimicrobials alone at the same concentrations. When the combination has an antibacterial effect similar to the antimicrobials alone, the difference in biofilm reduction is not significant. ±(standard deviation). Table 6. Antibiofilm effect, which is shown in terms of metabolic activity reduction of oregano derivatives and biological silver nanoparticles (bioAgNP) compared to both antimicrobials individually against and KPC-producing Klebsiella pneumoniae (KPC) biofilms growth in microtiter plates, which were evaluated at preformed biofilm condition. Oregano derivatives: OEO (oregano essential oil), Car (carvacrol), and Thy (thymol). * It indicates that the binary combination caused statistically (p < 0.05, Kruskal-Wallis test) a greater reduction in biofilm formation and showed an improved antibiofilm effect compared to both antimicrobials alone at the same concentrations. When the combination has an antibacterial effect similar to the antimicrobials alone, the difference in biofilm reduction is not significant. ±(standard deviation).

Concentrations in
To allow comparison between combined and individual treatments, only concentrations that alone did not have or had a small antibiofilm effect were chosen. We highlight that Thy and bioAgNP, both alone at a subinhibitory concentration (lower than SMIC ≥95 ), did not inhibit EAEC and KPC biofilm formation, respectively, but both antimicrobials in binary combinations with other compounds prevented biofilm formation.       Figure 2A) shows a slightly high-density biofilm, with a great number of cells, bacterial aggregation, formation of microcolonies, and architecture at the initial stage of organization. Figure 2A represents a healthy biofilm at an early stage of maturation since it does not have high biomass density, and its three-dimensional architecture is still under development ( Figure 2A). Four treated samples show damaged biofilm, with less dense biomass, no early architecture organization, extensively decreases in cell density, and smaller and scattered sparsely cellular aggregates, compared to the untreated control; Thy-treated ( Figure 2D), bioAgNP-treated ( Figure 2E), Car plus bioAgNP-treated ( Figure 2G), and Thy plus bioAgNP-treated ( Figure 2H). The OEO-treated sample ( Figure 2B) and OEO plus bioAgNP-treated sample ( Figure 2F) show less dense biofilm, with slightly less cellular density and bacterial aggregation compared to the untreated sample. Car-treated sample ( Figure 2C) and Car plus Thy-treated sample ( Figure 2I) show biofilm with a high cellular density similar to untreated control.   Figure 5 consists of scanning electron micrographs (1000×) of EAEC 042 and KPCproducing K. pneumoniae biofilms grown for 24 h in glass slides. EAEC ( Figure 5A) exhibits biofilm in an immature phase but is more developed than K. pneumoniae ( Figure 5B). After 24 h of growth, E. coli biofilm already shows very initial development of three-dimensional architecture, with cellular aggregates and a remarkable amount of EPS. With the same growth time, K. pneumoniae shows non-aggregated cells and an extremely minimal amount of EPS, still without the three-dimensional structure characteristic of biofilm.   Figure 3A) shows the initial organized three-dimensional architecture, in which extracellular fibril is distributed in an orderly manner, presenting intercellular networks that is typical of healthy biofilm. All antimicrobials affected the development of biofilm architecture, which seems unstructured, resulting in a less dense matrix and loss of cell connections compared to untreated control. Such damages are more intense in the following treated samples: Thy ( Figure 3D), bioAgNP ( Figure 3E), Car plus bioAgNP ( Figure 3G), and Thy plus bioAgNP ( Figure 3H). OEOtreated ( Figure 3B), Car-treated ( Figure 3C), and Car plus Thy ( Figure 3I) show more cellular density compared to control and other treatments; however, intercellular networks are extremely poor. Figure Figure 5A) exhibits biofilm in an immature phase but is more developed than K. pneumoniae ( Figure 5B). After 24 h of growth, E. coli biofilm already shows very initial development of three-dimensional architecture, with cellular aggregates and a remarkable amount of EPS. With the same growth time, K. pneumoniae shows non-aggregated cells and an extremely minimal amount of EPS, still without the three-dimensional structure characteristic of biofilm.

Effect of Compounds on Violacein Production
The mean MIC values of antibacterials against Chromobacterium violaceum are as follows: 0.15 mg/mL for OEO, 0.08 m/mL for Car, 0.12 mg/mL for Thy, and 23.13 µM for bioAgNP. MIC values were determined by the Clinical and Laboratory Standards Institute standard methodology (with initial bacterial inoculum at 5 × 10 5 CFU/mL) to determine the subinhibitory concentrations that would be tested in the violacein assay. The chosen subinhibitory concentrations of antimicrobials are indicated in Supplementary Material Table S3. Violacein reduction is expressed in percentage and compared to untreated control. Nontreated control is defined as 100% of violacein production.
Oregano-derivative antibacterials and bioAgNP, individually and in combination, reduced violacein production and did not inhibit C. violaceum growth. All treated C. violaceum samples and untreated samples are similar with regard to the number of viable cells (approximately 10 9 CFU/mL). However, violacein production was reduced by 93% (OEO), 94% (Car), 92% (Thy), 81% (bioAgNP), and 95% (Thy plus bioAgNP) compared to nontreated C. violaceum (Supplementary Table S3). Figure 6A qualitatively shows that OEO, Car, Thy, bioAgNP, and Thy plus bioAgNP have inhibitory effects on violacein production by C. violaceum since all treated bacterial cultures visually lack violet pigment or present dramatically reduced violet color compared to untreated control. Figure 6B quantitatively shows significant differences among OEO, Car, Thy, bioAgNP, Thy plus bioAgNP, and untreated control in terms of violacein amount (p < 0.05, Kruskal-Wallis test). Antibiotics 2023, 12, x FOR PEER REVIEW 15 of 33  non-treated C. violaceum (Supplementary Table S3). Figure 6A qualitatively shows that OEO, Car, Thy, bioAgNP, and Thy plus bioAgNP have inhibitory effects on violacein production by C. violaceum since all treated bacterial cultures visually lack violet pigment or present dramatically reduced violet color compared to untreated control. Figure 6B quantitatively shows significant differences among OEO, Car, Thy, bioAgNP, Thy plus bio-AgNP, and untreated control in terms of violacein amount (p < 0.05, Kruskal-Wallis test). Amounts of violacein (%) are the mean ± standard deviation.* Indicates a statistically significant difference (p < 0.05, Kruskal-Wallis test) between treated and untreated control in terms of violacein production.

Discussion
This study shows the antibiofilm effect of oregano-derived compounds and bioAgNP (biologically synthesized using F. oxysporum components) against Enterobacteriaceae strains, such as EAEC 042 and carbapenem-resistant K. pneumoniae. Oregano derivatives combined with bioAgNP present action against the KPC strain, which is identified in the WHO priority pathogen list for which effective antimicrobials are urgently needed [24]. Additionally, such combinations may prevent the emergence of resistance and minimize undesirable organoleptic effects of oregano terpenoids since association may require a lower concentration of each compound compared to their use alone [31]. The four binary combinations presented in this study are eco-friendly since oregano compounds are considered GRAS (Generally Recognized as Safe) [1], and bioAgNP root synthesis is less toxic than chemically synthesized nanoparticles because chemical reagents are not used as reducing or stabilizing agents [49]. Furthermore, F. oxysporum-bioAgNP is stable for several months due to protein capping, which occurs in the biogenic process, as seen by electron microscopy [50].
We report the antibiofilm action of these compounds against both the initial stage of formation and preformed biofilm. Biofilms are heterogeneous in their structure, organization, and metabolic characteristics; their life cycle complexity must be considered for evaluating the results of antibiofilm assays since antimicrobials can exhibit effects against biofilm at different stages [51][52][53].
In this study, three methodologies were used (crystal violet staining, MTT assay, and SEM) for accessing biofilm total biomass, its viability, and structure, respectively; in addition, an initial study about the anti-quorum-sensing effect of compounds was carried out using C. violaceum. The present data highlight the importance of using combined methodologies to access the antibiofilm activity of compounds; each method has its advantages and limitations and evaluates a specific aspect of biofilm, and in combination, they allow more reliable conclusions [51,54].
The violet crystal technique was crucial in our analysis of biofilm at an early stage of formation since the MTT assay alone would not show the efficiency of some antimicrobials to prevent biofilm formation. Some treatments prevent biofilm formation (as they reduce total biomass production measured by the violet crystal) but possibly cause stress in bacterial populations because the MTT test shows treated samples with high metabolic activity similar to the non-treated control.
However, MTT assay was essential for our preformed biofilm study since this technique was more sensitive than violet crystal to show antimicrobial activity against biofilm at the advanced stage of development. The violet crystal test was not feasible for detecting preformed biofilm biomass, but the microscopic technique allowed this analysis to be successful (Figures 2-4).
This research also showed the effect of compounds on the growth of biofilm in a polystyrene microtiter plate (Tables 3-6) and glass slide (Figures 2-4). Different surfaces and environments influence biofilm growth and also may impact biofilm susceptibility to antibiotics [53,55,56].
OEO, Car, Thy, and bioAgNP individually prevented EAEC 042 biofilm formation. All four compounds alone at SMIC ≥95 also inhibited planktonic cell growth of E. coli. Only OEO, Car, and bioAgNP inhibited EAEC biofilm formation at subinhibitory concentrations for planktonic cells (bellow SMIC ≥95 ) ( Table 3), and it suggests that these three compounds show the effect on biofilm formation by interfering both in planktonic cells growth and also in specific pathways of sessile cells. In contrast, Thy-antibiofilm activity may rely on action against planktonic cells since it did not cause a reduction in biofilm biomass and its viability at a concentration below SMIC ≥95 . Planktonic cells and biofilm life styles of single species express different genes, consequently accomplishing different phenotypic profiles [57,58]. Biofilm inhibition at subinhibitory concentrations (for planktonic cells) might be due to the inhibitory effect on the expression of genes related to motility and biofilm formation or the effect on specific biofilm structures and metabolic paths [16,59,60]. In agreement with our data, other studies also reported that OEO, Car [28,61,62], and bioAgNP [40,63,64] prevent E. coli biofilm formation.
For KPC-producing K. pneumoniae, all four tested antibacterials individually prevented biofilm formation; SMIC ≥95 values also inhibited planktonic cell growth. At subinhibitory concentrations for planktonic cells, only OEO, Car, and Thy inhibited the biofilm formation of carbapenem-resistant K. pneumoniae (Table 4). It suggests that oregano compounds show an effect on biofilm formation by interfering both in planktonic cell growth and also in specific pathways of biofilm lifestyle since they reduced total biofilm biomass and its metabolic activity compared to the untreated bacterium. In comparison, bioAgNPantibiofilm activity may rely on action against planktonic cells since these nanoparticles did not cause a reduction in biofilm biomass and its viability at a concentration below SMIC ≥95 . Some researchers also showed that OEO, Car, and Thy inhibited K. pneumoniae biofilm formation [26,30] in agreement with our data.
In this study, EAEC 042 was more sensitive to bioAgNP than KPC-producing K. pneumoniae since these metal nanoparticles prevented biofilm formation at SMIC ≥95 of 0.49 µg/mL for E. coli and 1.97 µg/mL for K. pneumoniae. The literature data show that other biogenic silver nanoparticles prevent biofilm formation by several bacterial species, including E. coli, K. pneumoniae, and Pseudomonas aeruginosa, with a wide range of SMIC [40,63,65,66]. Unlike our data, some studies show that silver nanoparticles, even at subinhibitory concentrations, inhibited biofilm formation by K. pneumoniae [41,63,67]. Thus, comparison of results is difficult since the effective concentration of bioAgNP varies in each study because of nanoparticle diversity in terms of size, morphology, composition, stabilizing agents, and surface charge; furthermore, the use of different techniques for nanoparticle characterization and microbiological analysis may influence conclusion regarding the antimicrobial activity [68][69][70][71]. To reduce factors that limit the comparison of results between different studies, we highlight the importance of standardization of bioAgNP characterization and their microbiological assays, specifically with regard to antibiofilm assays [52,69]. Moreover, different bacterial strains used in several studies may have structural and metabolic differences that make them more or less sensitive to such compounds [28,40,58,72].
At concentrations lower than SMIC ≥95 , OEO and Car prevented biofilm formation of EAEC and K. pneumoniae, but both compounds seem to act in different ways against both strains. For KPC-producing K. pneumoniae, the two oregano compounds reduced both biofilm biomass and biofilm viability. In the case of EAEC, both OEO and Car reduced total biomass, but it seems they caused bacterial stress response since the treated biofilm showed high metabolic activity similar to the non-treated control. It is known that during acid stress, E. coli upregulates some components of the electron transport chain (e.g., several dehydrogenases); under normal growth, such enzymes are involved in generating proton motive force by redox reactions with exportation of protons from the cells [73]. The literature indicates that essential oil can acidify the bacterial cytoplasm, which would justify the increase in metabolic activity detected in this study [74,75]. Furthermore, the mechanism of biofilm formation varies between E. coli and K. pneumoniae [58,72,76]; these differences may contribute to both bacteria responding differently to different treatments. K. pneumoniae initial colonization is a more passive process compared to E. coli; probably, it happens due to lack of motility in K. pneumoniae whose cells are less metabolically active at stages of attaching to surfaces and become progressively active in mature biofilm [77]. Such metabolic differences at the initial stages of biofilm formation may explain why OEO or Car treatments increased EAEC cell viability and decreased it in KPC-producing K. pneumoniae. García-Heredia et al. [28] reported that OEO and Car inhibited biofilm formation by EAEC 042, but both compounds did not prevent EAEC O104:H4 biofilm formation. It indicates that EAEC strains show a difference in their genomic regulation, suggesting that responses to oregano derivatives are not only compound-dependent but may also depend on strain-to-strain variations, in agreement with our results.
The present results suggest that OEO, Car, Thy, and bioAgNP may show specific inhibiting effects on different bacterial species. Several mechanisms may drive their antibiofilm properties, such as reducing fimbriae production, decreasing swarming motility, reducing flagellar biosynthesis, quorum-sensing interruption, inhibition of efflux pumps, and others. This study showed that all oregano compounds and bioAgNP reduced violacein production by C. violaceum ( Figure 6) in agreement with the literature [32,33,[78][79][80][81][82][83], suggesting that disruption of quorum sensing is one of the ways by which they prevent biofilm formation since the production of purple pigment violacein is directly linked to quorum sensing [32]. However, the antibiofilm mechanisms of these compounds must be investigated in detail to evaluate how such compounds modulate the expression of genes that are involved in biofilm formation, for example.
Mature biofilms can protect bacteria living inside against several adverse environmental influences and conditions. Antibiotics or disinfectants frequently fail to remove biofilms from biological or non-biological surfaces, which can represent a source of recurrent infections [84]. Biofilm bacteria often tolerate antibiotics at concentrations 10-10,000-fold greater than planktonic cells [32,85]. For eradicating successfully mature biofilms, it is necessary that antimicrobials penetrate into the aqueous channels of biofilms [84]; OEO and their main components (Car and Thy), despite being lipophilic-volatile substances, caused at least a 95% reduction in metabolic activity of preformed biofilm by both EAEC and KPC-producing K. pneumoniae in a microtiter plate (Table 1).
Preformed biofilms and biofilm under formation condition of both bacterial strains showed similar susceptibility to oregano-derived compounds; for EAEC, SMIC ≥95 val-ues were two-fold greater against pre-established biofilm than biofilm at an early stage of formation. For carbapenem-resistant K. pneumoniae, SMIC ≥95 values were the same for biofilm at both stages. Our previous study showed that OEO, Car, and Thy present similar minimum inhibitory concentration (MIC) against bacterial planktonic cells, including multidrug-resistant strains [31]. In agreement with present data, Reichling [84] and Yadav et al. [86] highlight that several essential oils and individual oil compounds show similar MIC values for planktonic cells and their biofilm. Unlike our data, some studies reported that planktonic bacterial cells are more sensitive to OEO, Car, and Thy than their biofilms [84,87,88]; some researchers have found that Thy inhibited biofilm formation by E. coli [29,61,62]. Result variances among different studies may occur because oregano compounds derive from plants and undergo variations in their chemical composition, which are dependent on climatic and geographical factors, and also extraction methods [32,89].
The bioAgNP also eliminated at least 95% of preformed biofilm by both tested strains. However, preformed biofilms were less susceptible to bioAgNP than biofilm at an early stage of development (SMIC ≥95 values are shown in Table 1), in agreement with other studies which suggested that biofilm greater resistance might be partially attributed to nanoparticle aggregation and retarded silver ion and particle diffusion [90][91][92]. For EAEC 042, bioAgNP SMIC ≥95 was 32-fold higher against preformed biofilm than biofilm under formation conditions. For carbapenemase-producing K. pneumoniae, bioAgNP SMIC ≥95 was four-fold greater against pre-established biofilm than biofilm at the initial stage formation.
This present study also showed that the preformed biofilm of E. coli is more tolerant to bioAgNP than K. pneumoniae pre-established biofilm since bioAgNP SMIC ≥95 is higher for EAEC (Table 1); the structural difference of biofilm between the two species may explain this difference in susceptibility to bioAgNP. SEM micrographs of untreated biofilms (24 h of formation) of both bacterial species ( Figure 5) showed that K. pneumoniae presented a more youthful biofilm with lower cell density, little secreted EPS, and less cell aggregation compared to EAEC. Glycocalyx and the EPS matrix of biofilms act as biding sites and limit antimicrobial diffusion through the matrix, reducing drug access to sessile cells [3,93]. In addition, bioAgNP treatment may decrease EPS production in K. pneumoniae [41], contributing to the greater sensitivity of this strain to bioAgNP.
However, oregano terpenoids and silver nanometal exhibit features that may limit their applications as antimicrobials. OEO, Car, and Thy present high volatility and strong organoleptic features [33,94], and bacteria easily develop resistance to silver nanoparticles [44][45][46]95]. We showed previously that E. coli develops resistance to bioAgNP after 12 days of daily treatment [31]. Thus, in order to solve these problems and expand the possibilities for these compound applications, our research group proposes the association between oregano derivatives and bioAgNP. Combinatory antimicrobial therapy is a potent strategy to control antimicrobial resistance, extend antimicrobial agents' life, and also to reduce unwanted characteristics of compounds such as organoleptic features, toxicity, or costs [42,96,97].
All four combinations (OEO plus bioAgNP, Car plus bioAgNP, Thy plus bioAgNP, and Car plus Thy) inhibited the growth of biofilm, both at an early stage of formation and at the maturation phase, by EAEC and carbapenemase-producing K. pneumoniae. Our results are in agreement with the literature data, which show the antimicrobial potential of oregano-derived terpenoids and bioAgNP, both individually combined with conventional antimicrobials or natural compounds, to combat microbial biofilm [32,100,101]. In this present study, none of the four combinations showed antagonistic interaction with regard to antibiofilm activity since they were more efficient than antimicrobials individually by reducing SMIC ≥95 of each compound (Table 2) or decreasing by greater intensity the biofilm biomass production and its viability in cases that SMIC ≥95 reduction did not happen (Tables 3-6).
For EAEC 042, Thy plus bioAgNP reduced SMIC ≥95 against both biofilm formation and preformed biofilm compared to the same antimicrobials individually. Two combinations (OEO plus bioAgNP and Car plus bioAgNP) reduced SMIC ≥95 against preformed biofilm; although both combinations did not reduce SMIC ≥95 for biofilm formation, the combined compound still showed a better effect than isolated antimicrobials to prevent biofilm formation, since they caused significantly greater reduction in biofilm biomass and metabolic activity compared to antibacterials alone at same concentrations. Car plus Thy reduced SMIC ≥95 against E. coli biofilm formation; both compounds in combination also showed an improved effect against preformed biofilm compared to antibacterials alone by reducing its viability in greater intensity.
For KPC-producing K. pneumoniae at an early stage of biofilm formation, three combinations (OEO plus bioAgNP, Car plus bioAgNP, and Thy plus bioAgNP) reduced SMIC ≥95 . Although the association containing Car and Thy did not cause a reduction in SMIC ≥95 to prevent biofilm formation, both compounds, in combination, presented a better effect than both compounds alone, causing a greater reduction in biofilm biomass production and its viability.
For preformed biofilm of KPC strain, the SMIC ≥95 of compounds in combination were not found; the maximum tested concentration of Thy or bioAgNP in combination was 25% of its individual SMIC value because both compounds alone at 0.5× SMIC already reduce around 80% of biofilm metabolic activity. However, two combinations, OEO plus bioAgNP and Car plus Thy, show greater antibiofilm activity than both compounds individually, causing a significantly greater reduction in sessile cell viability. Car plus bioAgNP and Thy plus bioAgNP showed similar antibiofilm activity compared to bioAgNP alone.
In general, this study shows that SMIC ≥95 values reduce up to 50% for OEO and Car, 75% for Thy, and 87.5% for bioAgNP. These percentages of reduction are in agreement with the previous study of our research group in which MIC reduction against planktonic bacterial cells ranged by 50-87.5% for all compounds, showing the additive antibacterial effect of OEO plus bioAgNP, Car plus bioAgNP, Thy plus bioAgNP, and Car plus Thy [31,47].
Other authors also reported that oregano compounds (OEO or Car) combined with eugenol or conventional antibacterial (e.g., ciprofloxacin) present a synergistic effect to prevent and eradicate bacterial biofilms, including resistant strains [89,113]. Otaguiri et al. [100] showed that the same bioAgNP (produced extracellularly with F. oxysporum components) in combination with copaiba essential oil present a synergistic effect against Streptococcus agalactiae biofilm formation, reducing SMIC values of both compounds at least by 75%. This bioAgNP also showed an antibiofilm effect when combined with conventional antimicrobials. Longhi et al. [101] reported that their combination with fluconazole caused a significant decrease in the viability of both the initial and mature biofilm of Candida albicans.
At subinhibitory concentrations (lower than SMIC ≥95 ), the four tested combinations showed antibiofilm activity against both EAEC and KPC-producing K. pneumoniae at the initial stage of biofilm formation (Tables 3 and 4). At concentrations that do not inhibit planktonic cells (bellow SMIC ≥95 ), OEO plus bioAgNP, Car plus bioAgNP, Thy plus bioAgNP, and Car plus Thy reduced total biofilm biomass and its metabolic activity compared to the untreated bacterium, suggesting that such treatments have an effect on specific pathways of biofilm lifestyle. Here we present microscopy and violacein assays as an initial study of the antibiofilm mechanism of these compounds alone and in combination.
SEM assay showed that compounds individually ( Figures 2B-E, 3B-E and 4B-E), mainly Thy and bioAgNP, act against preformed EAEC biofilm on glass slides by affecting biofilm structure which presented reduced total biomass (extensively decreased cell density, less dense matrix, and less intercellular networks, with smaller and more scattered cellular aggregates) and its cells surface exhibited alterations. Both Thy and bioAgNP-treated biofilms presented cells with an irregular wrinkled surface; Thy also caused the sinking of the bacterial cell, and bioAgNP-treated cells also showed smaller sizes than typica E. coli. SEM analysis showed that OEO and Car alone affected EAEC biofilm to a lesser extent compared to other individual treatments. The OEO-treated sample presented slightly reduced biomass density and bacterial aggregation, whose cells exhibited a sinked and irregular wrinkled surface. The Car-treated sample showed biofilm with extremely poor intercellular networks, high cellular density, and cells without remarkable morphological alterations. Kerekes et al. [29] reported that Thy exhibited the best effect against E. coli biofilm among several essential oils, resulting in biofilm with anamorph structure, sparse micro-colonies, and individual cells (no aggregates), in agreement with our data. Guo et al. [92] reported the action of nanosilver against P. aeruginosa biofilm, which exhibited cellular density reduction and distinct EPS matrix formation surrounding bacterial cells with disruption of the cellular membrane.
The scanning microscopy test showed that two combinations (Car plus bioAgNP and Thy plus bioAgNP) stand out by inhibiting preformed EAEC biofilm growth on glass slides (Figures 2-4; images G and H). Car plus bioAgNP and Thy plus bioAgNP samples presented damaged biofilm, with less dense biomass, matrix architecture with disrupted organization, a huge decrease in cell density, and smaller cellular aggregates, which shows loss of cell connections and morphological cell alterations such as reduced size and irregular wrinkled surface compared to untreated control. The OEO plus bioAgNP-treated sample showed less dense biofilm, with slightly less cellular density, fewer cell connections and bacterial aggregation, and altered cells with an irregular wrinkled surface. The Car plus Thy-treated sample showed biofilm with high cellular density without remarkable morphological alterations, but this treatment reduced intercellular networks. The cellular morphological alterations observed by us, such as irregular wrinkled surface and sinking of cellular surface, suggest that oregano derivatives and bioAgNP also affect sessile cells by disrupting the cytoplasmic membrane and cell wall, resulting in leakage of cellular cytoplasmic material in agreement with previous studies that involve planktonic cells [31,47,112,114,115].
In summary, we report in this paper the antibiofilm effect of new antimicrobial compositions against KPC-producing bacteria, which is even highlighted on the WHO Global Priority Pathogens List.Finally, this study also stands out the importance of using combined methodologies to access antibiofilm activity. Different methods trace the heterogeneity in biofilms and allow more reliable conclusions about the antimicrobial effect against sessile bacteria [51,54].
The Both strains, EAEC 042 and KPC-producing K. pneumoniae strain, were chosen because they are great bacterial models for biofilm formation. EAEC 042 was used as a strong biofilm former (positive control; OD 570 > 0.2 in crystal violet assay) and KPC-producing K. pneumoniae as a clinical isolate which is multidrug-resistant and also a strong biofilm former. The E. coli HB101 strain was used as a negative control that does not produce biofilm since it shows OD 570 < 0.1.

Oregano-Derived Compounds
OEO (batch 227) was obtained from Ferquima Industry and Commerce of Essential Oil (São Paulo, Brazil). It was extracted by steam distillation, and its main components (72% carvacrol, 2% thymol, 4.5% gamma-terpinene, 4% para-cymene, and 4% linalool) were described in a technical report provided by the company. Carvacrol-W224502 (Car) and thymol-T0501 (Thy) were purchased from Sigma-Aldrich (St. Louis, MO, USA); their densities are 0.95 g/mL and 0.976 g/mL, respectively. Individual solutions of OEO, Car, and Thy were prepared in dimethyl sulfoxide (DMSO, Sigma-Aldrich) for microbiological tests. DMSO maximum concentration in assays was 5% (v/v), and it did not show antibacterial action.

Biogenically Synthetized Silver Nanoparticles (bioAgNP)
The bioAgNP synthesis was performed according to the previously established method [2]. The biosynthesis methodology involved F. oxysporum (strain 551 provided by ESALQ-USP Genetic and Molecular Biology Laboratory-Piracicaba, São Paulo, Brazil). Fungus was grown at 28 • C for 7 days in a medium composed of 0.5% (w/v) yeast extract (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), 2% (w/v) agar malt extract (Acumedia), and distilled water. F. oxysporum biomass was added to distilled water at 0.1 g/mL and incubated at 28 • C for 72 h in agitation (150 rpm). Thereafter, aqueous solution components were separated from biomass by vacuum filtration (qualitative filter having an average pore size from 4 to 12 µm, Unifil). AgNO 3 (Sigma-Aldrich) at 0.01 M was added to this solution, and it was kept at 28 • C for 15 days in the absence of light (static condition). The bioAgNP was obtained after the reduction of silver nitrate by fungal-free solution components. Aliquots of the system were removed for measuring absorption spectra to verify the surface plasmon resonance peak of bioAgNP, using ultraviolet-visible spectrophotometry (Thermo Scientific™ Multiskan™ GO Microplate Spectrophotometer, Marsiling Industrial Estate, Singapore). Washing of bioAgNP was carried out by three steps of centrifugation (27,000× g, 4 • C, 30 min) followed by incubation in an ultrasonic bath (30 min). Ag quantification was performed by Energy-Dispersive X-ray Fluorescence Spectrometer EDX-7000. The nanoparticle's diameter was determined by photon-correlation spectroscopy using ZetaSizer NanoZS (Malvern, UK), and zeta-potential measurement was performed using the same instrument. Transmission electron microscopy (TEM, Zeiss EM900) was used for bioAgNP morphology analysis.

Antibiofilm Assays
The antibiofilm effect of antimicrobials alone and in combination was studied by four methodologies described above (Sections 4.3.1-4.3.3), such as colorimetric techniques (crystal violet and MTT) using microplates, scanning electron microscopy, and quorum sensing inhibition test.

Biofilm Quantification by Chemical Methods (Crystal Violet and MTT)
The antibiofilm effect of oregano-derived compounds (OEO, Car, and Thy) and bioAgNP, individually and in combination, were evaluated at two stages of bacterial biofilm formation as follows: early stage (from 0 to 24 h of biofilm growth) and biofilm maturation phase (from 24 to 48 h). Both techniques were performed according to previously described methods, with necessary modifications, as follows: crystal violet test [116,117] and dimethylthiazol diphenyl tetrazolium bromide (MTT) assay [117,118].
For assessing the prevention of biofilm formation, antimicrobials and bacteria were added concomitantly to the microtiter plate. Briefly, bacterial-isolated colonies grown in nutrient-agar (Himedia, Mumbai, India) medium were suspended in phosphate-buffered saline (0.1 M PBS, pH 7.2) to standardize the inoculum density. This suspension was adjusted to achieve turbidity equivalent to 0.5 McFarland standard, which corresponds approximately to 1.5 × 10 8 colony-forming units (CFU)/mL. Bacteria and antimicrobials were added concomitantly to wells of 96-well polystyrene microtiter plate; a volume of 0.02 mL of equivalent 0.5 McFarland suspension was added to 0.18 mL Dulbecco's Modified Eagle's Medium (DMEM, Sigma-Aldrich) containing antimicrobials individually or in combination. Before adding antimicrobials, DMEM was supplemented with 0.45% glucose (Sigma-Aldrich). The plate was incubated at 37 • C for 24 h under static conditions to allow bacterial form biofilm. Two identical microtiter plates were prepared; one for the crystal violet-staining procedure and the other for the MTT assay.
For evaluating the effect of compounds against preformed biofilm, firstly, non-treated bacteria were added to a microtiter plate and incubated for 24 h to allow biofilm attachment and growth, then preformed biofilm was treated with antimicrobials. Briefly, the bacterial inoculum was prepared as previously described; 0.02 mL of equivalent 0.5 McFarland suspension and DMEM supplemented with 0.45% glucose (0.18 mL) were added to wells of a 96-well polystyrene microtiter plate, followed by incubation at 37 • C for 24 h. Thereafter, unattached cells and medium were removed, and biofilm biomass was rinsed three times with PBS. Then 0.2 mL of DMEM alone (untreated control) or DMEM containing antimicrobials (individually and in combination) were added to preformed biofilm, followed by post-incubation at 37 • for 24 h.
After 24 h of treatment (for both biofilm at the initial stage and preformed biofilm), planktonic cells and DMEM were aspired off, and adherent biomass was rinsed three times with PBS. For the crystal violet assay, biomass was stained with 0.2% (w/v) crystal violet solution for five min; then, three washing steps were carried out to remove unbound dye. Finally, after adding 0.2 mL of ethanol 95% (v/v) to each well containing the bound dye, the biofilm was quantified by a microplate reader at 570 nm (Thermo Scientific™ Multiskan™ GO Microplate Spectrophotometer). For viability assay, MTT solution (0.1 mL per well at 0.25 mg/mL) was added to each well, and the microplate was incubated at 37 • C for 2 h. Thereafter, 0.1 mL of solubilization solution was added to each well to dissolve formazan crystals. After 15 min homogenization, the plate was read at 570 nm using the same microplate spectrophotometer.
Untreated bacteria, inoculated on DMEM alone or containing DMSO at 5% (v/v), were used as a positive control (PC, defined as 100% biofilm metabolic activity). DMEM alone was used as sterility control (SC). The percentage of biofilm inhibition (total biomass and metabolic activity reductions) for each antimicrobial treatment was calculated using Equation (1).
The sessile (biofilm) minimum inhibitory concentrations were determined at 95% or greater inhibition (SMIC ≥95 ) of metabolic activity compared to untreated positive control. Experiments were carried out in quintuplicate on at least three different occasions. Details of methodologies are described below.

Quorum Sensing Inhibition
Test Based on C. violaceum C. violaceum CCT 3468 was used as a model for quorum sensing inhibition assay since violacein production involves quorum sensing. Before quantitative analysis of violacein production, the minimum inhibitory concentration (MIC) of compounds (individually and in combination) and their subinhibitory concentrations were determined. In addition, quantification of viable bacterial cells was performed for treated and untreated C. violaceum. Details of the violacein assay are described below.

Determination of Subinhibitory Antibacterial Concentrations
Before the violacein inhibition assay, subinhibitory concentrations of each compound against C. violaceum were determined by the broth microdilution method. Determination of MIC of each antimicrobial individually (OEO, Car, Thy, and bioAgNP) was performed according to the Clinical and Laboratory Standards Institute guidelines [119], with necessary modifications. For antimicrobial combination (Thy plus bioAgNP), MIC values were determined by double-antimicrobial gradient as described by Traub and Kleber [120], with necessary modifications. Briefly, to standardize the inoculum density for the susceptibility test, C. violaceum isolated colonies grown in Luria Bertani (LB, Himedia) agar medium were suspended in PBS 0.1M (pH 7.2) to achieve turbidity equivalent to 0.5 McFarland standard, as previously described for microtiter assays. The equivalent 0.5 McFarland suspension was diluted 1:100 in LB (Himedia) broth to obtain a concentration of approximately 10 6 CFU/mL. A volume of 0.05 mL of bacterial inoculum at 10 6 CFU/mL was added to 0.05 mL of LB containing antimicrobial individually or in combination. Lastly, bacteria at 5 × 10 5 CFU/mL in LB containing antimicrobials were incubated at 28 • C for 24 h with shaking (130 rpm). MIC was defined as the lowest antimicrobial concentration that inhibited visible growth after 24 h of treatment at 28 • C. The assay was carried out in triplicate, at least on three different occasions.
For antimicrobials tested individually, concentrations ranged as follows: (i) 0.07-9.5 mg/mL for OEO, (ii) 0.08-9.76 mg/mL for Car, (iii) 0.008-1 mg/mL for Thy, and (iv) 0.49-63 µg/mL for bioAgNP. For the combination assay, the concentration range was 0.01-0.06 mg/mL for Thy and 0.49-1.97 µg/mL for bioAgNP. LB alone and LB containing each antimicrobial separately were tested as sterility controls. Untreated bacteria inoculated on LB broth alone and containing DMSO at 5% (v/v) were tested as growth control.

Violacein Inhibition Assay
At first, quantification of viable cells in C. violaceum-treated samples was performed. C. violaceum was grown in LB broth at 28 • C for 72 h (130 rpm); every 24 h, the medium was renewed by transference of 0.1 mL of each previous culture into LB broth (4.9 mL). For the violacein inhibition assay, C. violaceum overnight culture was diluted 1:10 in LB broth; then six samples were prepared by adding 2.5 mL of diluted bacterial inoculum to 2.5 mL of LB alone (untreated control) or LB containing antimicrobial (subinhibitory concentrations) individually or in combination, whose concentrations were as follows: (i) OEO at 0.15 mg/mL, (ii) Car at 0.15 mg/mL, (iii) Thy at 0.12 mg/mL, (iv) bioAgNP at 15.75 µg/mL, and (v) combination with Thy at 0.03 mg/mL plus bioAgNP at 0.49 µg/mL. The untreated and treated bacterial samples were incubated at 28 • C for 24 h (130 rpm). After 24 h treatment, each sample was evaluated with regard to the number of C. violaceum viable cells according to the National Committee for Clinical Laboratory Standards [121]; Antibiotics 2023, 12, 756 25 of 32 0.01 mL from serial dilutions (in PBS 0.1M, pH 7.2) of treated and non-treated cultures were subcultured in LB agar for CFU/mL determination.
The amount of violacein produced by each sample (treated and non-treated C. violaceum) was qualitatively analyzed (turbidity and color of bacterial cultures were analyzed by visual inspection) according to Blosser and Gray [122]. Briefly, bacterial cells of each sample were pelleted (5500× g, 10 min, 25 • C) and resuspended with 0.2 mL 0.1 M PBS (pH 7.2). Bacterial cells were lysed by adding 0.2 mL of 10% (w/v) sodium dodecyl sulfate (SDS), mixing for 10 s with a vortex mixer, and cells were maintained at room temperature for 5 min. For violacein extraction, 0.9 mL of water-saturated butanol (1:3) was added to cell lysate, followed by mixing for 5 s. The final solution was centrifuged (13,000× g, 5 min). The upper n-butanol phase containing violacein was collected and transferred to a 96-wells plate. The absorbance of extracted violacein was measured at 595 nm in a microplate reader (Thermo Scientific™ Multiskan™ GO Microplate Spectrophotometer). Untreated bacteria inoculated on LB broth alone or containing DMSO at 5% (v/v) were used as a positive control (defined as 100% of violacein production). The percentage of violacein produced by treated cells was calculated using Equation (2). For each sample, the percentage of inhibition of violacein production was determined based on the positive control, subtracting the percentage of violacein from 100. The assay was carried out in triplicate, at least on three different occasions.

Scanning Electron Microscopy (SEM) Study of Antibiofilm Effect of Compounds
The effect of oregano-derived compounds (OEO, Car, and Thy) and bioAgNP, individually and in combination, against the preformed biofilm of EAEC 042 was analyzed by SEM. Firstly, the bacterial inoculum was prepared as described in Section 4.3.1. Bacteria (0.1 mL of equivalent 0.5 McFarland suspension) and DMEM supplemented with 0.45% glucose (0.9 mL) were added to wells (which contained uncoated glass slides at the bottom) of a 24-well polystyrene microtiter plate, followed by incubation at 37 • C for 24 h in agitation (120 rpm) to allow cell attachment and biofilm growth. Thereafter, unattached cells and medium were removed, and biofilm biomass was rinsed three times with PBS 0.1 M (pH 7.2). Then 0.2 mL of DMEM alone (untreated control) or DMEM containing antimicrobials (individually and in combination) were added to the preformed biofilm, followed by incubation at 37 • C for 24 h (120 rpm).
After 24 h treatment, planktonic cells and DMEM were aspired off, adherent biomass was rinsed three times with PBS 0.1M (pH 7.2), and preparation of samples (treated and untreated bacteria) for SEM analyses was performed accordingly [123], with necessary modifications. Previously, four solutions for microbial glycocalyx fixation were tested (data not shown), and the solution containing alcian blue as a cationic dye was chosen as the most suitable one for EAEC 042. Firstly, glass slides with adherent biomass (treated and untreated samples) were immersed for 20 h (at 4 • C) in 1 mL of 0.1M sodium cacodylate buffer (pH 7.2) containing 2.5% (v/v) glutaraldehyde, 2% (v/v) paraformaldehyde, and 0.15% (w/v) alcian blue. After primary fixation in aldehyde with alcian blue, the samples were washed (three washing steps of 10 min each) in 0.1 M cacodylate buffer (pH 7.2), following post-fixation in OsO 4 1% for 2 h at room temperature. All reagents for both chemical fixations were provided by Electron Microscopy Sciences. Post-fixed samples were then rinsed (three times for 10 min each) in 0.1 M cacodylate buffer (pH 7.2) and dehydrated in an ethanol gradient (Sigma-Aldrich) (30,50,70,90, and 100 • GL), critical point-dried using CO 2 (BALTEC CPD 030 Critical Point Dryer), coated with gold (BALTEC SDC 050 Sputter Coater) and observed under scanning electron microscope (FEI Quanta 200).

Conclusions
This study shows for the first time the antibiofilm effect of bioAgNP combined with oregano compounds (OEO, Car, or Thy) against E. coli and K. pneumoniae, including KPCproducing strains for which new antibiotics are urgently needed. Binary-compound combinations improved the antibiofilm effect of antimicrobials alone, disrupting preformed biofilm and preventing its formation. We highlight the great antibacterial activity of Thy associated with bioAgNP, which inhibited the growth of biofilm on both polystyrene and glass surfaces, reduced SMIC ≥95 of each compound, decreased biofilm metabolic activity and biomass, disrupted its three-dimensional structure, and altered its cell morphology; Thy plus bioAgNP also reduced violacein production by C. violaceum, indicating that disruption of quorum sensing may be one of its antibiofilm mechanisms. Next, a more detailed examination of oregano plus bioAgNP must be performed to provide information with regard to the antibiofilm mechanism of action (at a molecular level) and its antibiofilm efficacy in vivo and in non-laboratory situations. However, terpenoids derived from oregano associated with bioAgNP (synthesized with F. oxysporum) successfully combat biofilm-associated bacteria and may overcome existing antibiotic resistance so they could be applied in several sectors of industry, clinical, and hospital settings, such as formulation of surface cleaners, food packaging, cosmetic products, wound care supplies, for treating infection in burns, among others.

Supplementary Materials:
The following supporting information can be downloaded at: https://ww w.mdpi.com/article/10.3390/antibiotics12040756/s1, Figure S1. Size distribution by intensity (%) of bioAgNP provided by photon correlation spectroscopy; the average diameter of nanoparticles was 84.10 nm, and the polydispersity index (PDI) was 0.269. Figure S2. Zeta potential distribution of bioAgNP, for which an average value was −15.9 mV. Figure S3. Calibration curve used to determine the concentration of silver in bioAgNP after washing steps. Figure S4. Effect of oregano-derived compounds and bioAgNP, individually and in combination, on biofilm growth of enteroaggregative Escherichia coli (EAEC 042) and KPC-producing Klebsiella pneumoniae evaluated at an early stage of biofilm formation. Figure S5. Effect of oregano-derived antibacterials and bioAgNP, individually and in combination, on biofilm growth of enteroaggregative Escherichia coli (EAEC 042) and KPC-producing Klebsiella pneumoniae evaluated at a later stage under preformed biofilm condition. Table S1. Antibiofilm effect of all tested concentrations of oregano derivatives and bioAgNP against biofilm growth in microtiter plates, which were evaluated at an early stage of biofilm formation. Table S2. Antibiofilm effect of all tested concentrations of oregano derivatives and bioAgNP against biofilm growth in microtiter plates, which were evaluated at preformed biofilm condition. Table S3. Quantitative reduction of violacein produced by Chromobacterium violaceum treated with oreganoderived antibacterials and bioAgNP, alone and in combination. Viable cell number (log CFU/mL) of untreated (control) and treated-C. violaceum is also shown.
Author Contributions: S.S., conception and drafting of the study, design and planning of experiments, carrying out experiments, data acquisition, analysis and interpretation, and writing of this article; F.M.M.B.T. and M.C.L.N., identification and characterization of Klebsiella pneumoniae carbapenemaseproducing K. pneumoniae strain that was fundamental for the present study; L.A.P., assistance and guidance in bioAgNP biosynthesis, mainly with fungal growth conditions; A.G.d.O., assistance and guidance in electron microscopy assays; N.D., conception of bioAgNP biosynthesis methodology, nanoparticles characterization; G.N., assistance and guidance in bioAgNP biosynthesis, data analysis, and interpretation, and critical review of the article; R.K.T.K., conception and advisor of this study, data analysis and interpretation, critical review of the article, and final approval of the version to be published. All authors have read and agreed to the published version of the manuscript.