Adapting to oxygen: 3-Hydroxyanthrinilate 3,4-dioxygenase employs loop dynamics to accommodate two substrates with disparate polarities

3-Hydroxyanthranilate 3,4-dioxygenase (HAO) is an iron-dependent protein that activates O2 and inserts both oxygen atoms into 3-hydroxyanthranilate (3-HAA). An intriguing question is how HAO can rapidly bind O2, even though local O2 concentrations and diffusion rates are relatively low. Here, a close inspection of the HAO structures revealed that substrate- and inhibitor-bound structures exhibit a closed conformation with three hydrophobic loop regions moving toward the catalytic iron center, whereas the ligand-free structure is open. We hypothesized that these loop movements enhance O2 binding to the binary complex of HAO and 3-HAA. We found that the carboxyl end of 3-HAA triggers changes in two loop regions and that the third loop movement appears to be driven by an H-bond interaction between Asn27 and Ile142. Mutational analyses revealed that N27A, I142A, and I142P variants cannot form a closed conformation, and steady-state kinetic assays indicated that these variants have a substantially higher Km for O2 than WT HAO. This observation suggested enhanced hydrophobicity at the iron center resulting from the concerted loop movements after the binding of the primary substrate, which is hydrophilic. Given that O2 is nonpolar, the increased hydrophobicity at the iron center of the binary complex appears to be essential for rapid O2 binding and activation, explaining the reason for the 3-HAA–induced loop movements. Because substrate binding-induced open-to-closed conformational changes are common, the results reported here may help further our understanding of how oxygen is enriched in nonheme iron-dependent dioxygenases.

It is estimated that ϳ30% of the mass of woody plant material on earth is comprised of aromatic compounds that are known for their unusual stability when compared with similar aliphatic hydrocarbons (1). One strategy devised by nature to overcome the stability of aromatic compounds is to utilize iron-bound oxygen directly to cleave the ring, breaking aromaticity. The ring-cleaving dioxygenase enzymes are abundant and versatile. These enzymes utilize a mononuclear, nonheme iron ion to orchestrate electron transfer between an organic substrate and oxygen molecule with catalytic reaction rates rapid enough for their catalytic intermediates to escape spectroscopic characterization (2). A fundamental question is how these enzymes accomplish rapid O 2 binding, a prerequisite for the observed catalytic rates, whereas local O 2 concentrations in aqueous solution are relatively low (200 -300 M at 1 atm pressure and room temperature), and the rate of oxygen diffusion from air to solution is slow relative to the rate of catalysis.
Here, we chose 3-hydroxyanthranilate 3,4-dioxygenase (HAO), 3 a ring-cleaving dioxygenase, as a model system to study this problem. HAO is a nonheme, iron-dependent extradiol dioxygenase belonging to the cupin superfamily (3)(4)(5). This enzyme cleaves the aromatic ring of its substrate adjacent to the substitution groups. It activates and inserts dioxygen between C3 and C4 of its substrate 3-hydroxyanthranilate (3-HAA), ultimately producing quinolinic acid via the intermediary product ␣-amino-␤-carboxymuconate-⑀-semialdehyde (Fig. 1A). HAO is present in the kynurenine pathway, where it is essential for tryptophan degradation and de novo NAD ϩ biosynthesis in eukaryotic organisms and a few bacteria (6 -10). This enzyme is also found in the 2-nitrobenzoic acid catabolic pathway for energy production in some bacteria (11). HAO from Cupriavidus metallidurans has been shown to operate at a catalytic turnover rate of 25 s Ϫ1 (3,12) until near depletion of O 2 . A second iron-binding site at the protein surface has been proposed to be an iron storage site for the protein to ensure activesite iron occupancy when the enzyme is dealing with a substrate that is also a metal chelator (13).
Previous structural studies of HAO have allowed atomic level understanding of the overall protein structure and the active site architecture (3)(4)(5). A substrate analog, 4-Cl-3-HAA (ClHAA) was tested and defined as an irreversible inhibitor to prevent the active site from binding with the native substrate (12). The crystal structures of HAO from C. metallidurans were also reported previously (3,13). In this study, a closer inspection of the substrate-free (PDB entry 4L2N) and substratebound (PDB entry 1YFY) structures of WT HAO (wtHAO) showed that the enzyme exhibits two distinct conformations (Fig. 1B), which are hereafter termed open and closed states. The substrate-bound structure is in the closed conformation, showing significant structural differences relative to the ligandfree, open state of three specific loop regions surrounding the catalytic iron center, which is composed of a ferrous ion coordinated by a His 2 -Glu 1 motif. The crystal structure of HAO in complex with O 2 and ClHAA (PDB entry 1YFW) (3,12) also shows the closed state with three loops moving toward the active site (Fig. 1C). Therefore, the questions raised here are the following: 1) What is the driving force for the loop movement? 2) How are the loop movements on the three loop regions coordinated? 3) What is the potential biological significance of the loop movement in the context of oxygen binding? To investi-gate these questions, we identified and mutated the essential residues involved in the loop movement, determined kinetic parameters for both oxygen and 3-HAA, and performed X-ray crystallographic studies. The results revealed how the nonheme iron enzyme handles its substrates with disparate polarities through a primary substrate-induced change of the active site hydrophobicity.

Insights inferred from sequence and structural alignments
We noted that three largely hydrophobic loop regions in HAO move toward the catalytic iron center, forming a closed conformation upon HAO binding with the primary substrate (PDB entry 1YFY) compared with the ligand-free counterpart (PDB entry 4L2N) (3,13). Similar structural features were obtained in hundreds of repeated data sets of the ligand-free and substrate-bound structures, indicating that the organic substrate-binding induced closed conformation is an intrinsic feature of the enzyme. The three loop regions consist of residues 21-28 for loop region A (Leu 20 -Leu 21 -Lys 22 -Pro 23 - Most of these residues are highly conserved across the protein sequences of HAO from various origins (Fig. 1D).
A salient feature is that all three loops are composed of mostly hydrophobic residues (bolded); 17 of the total 26 residues (65%) are aliphatic (Fig. S1). Each loop also contains one or two proline residues. Proline is a cyclic hydrophobic residue and is commonly utilized as a helix breaker and an aid in formation of turns. Two consecutive proline residues are present in both loop A and loop C (i.e. Pro 23 -Pro 24 and Pro 147 -Pro 148 , respectively). The proline fragment endows the two loops with increased hydrophobic structure. It is interesting to note that the two negatives make a positive, i.e. the Pro 147 -Pro 148 fragment, a double helix breaker, is part of an ␣-helical structure. Loop B is a highly hydrophobic fragment with only one proline residue (Pro 45 ). Upon 3-HAA binding-induced loop movement, loop A undergoes the largest movement among the three loops with a shift toward the active site of ϳ8.6 Å in average when compared with the corresponding loop regions in the substrate-free form.
Another notable feature of the loop regions in the closed form of HAO is that the side chains of hydrophobic residues of Val 25 , Val 41 , Ile 142 , Val 142 , Leu 139 , and Leu 146 are sequestered toward to the active site, where the catalytic iron ion is located (Fig. S1, B and D). Meanwhile, most of the side chains of the hydrophilic residues point away from the active site: Lys 22 , Arg 28 , His 46 , Lys 140 , Thr 144 , and Asp 145 (Fig. S1, B and C). When the three loops are gathered toward the catalytic iron ion, the ligand-bound iron center becomes significantly more compact and hydrophobic, which is distinct with respect to the more open and spacious conformation observed in the ligandfree structure. The movement of loop A is triggered by the interaction of the substrate carboxylate group with the side chains of Asn 27 and Asn 45 and the backbone of Val 25 , which form the potential H-bond or salt-bridge network in both substrate-bound and ClHAA-bound structures (Fig. 2). Loops A and B appear to move synergistically because of the nearly pairwise backbone residue interactions and like a "double-door" to the active site cavity. As shown in Fig. S1C, an H-bonding network forms among the backbones of residues Pro 23 , Pro 24 , and Gly 26 in loop A and Gly 42 , Pro 44 , and His 46 in loop B, gathering loops A and B toward the active site.

Single-point mutation of Asn 27 and Ile 142 diminished the catalytic efficiency by decreasing the oxygen capturing capacity
One question arising from the structural comparison is how loop C is triggered to move toward the catalytic iron center. This loop region is more distant from the active site in the open conformation. However, the entire ␣-helix to which loop C is connected rotates ϳ10° (Fig. S1B). After binding 3-HAA or ClHAA, the side chain of Asn 27 and the backbone of Ile 142 moves closer to the iron center by forming hydrogen-bonding interactions with each other (Fig. 2) (3). Specifically, the distance between the backbone amide of Ile 142 from loop C and the side chain of Asn 27 from loop A decreases substantially, from ϳ8.0 to 2.6 Å, which results in loops A and C directly moving together and toward the active site. Thus, Asn 27 and Ile 142 appear to be the critical residues for a coordinated conformational change during 3-HAA binding to the iron ion.
To determine the connection and especially the effect of the loop movement on the reaction catalyzed by HAO, we mutated Asn 27 and Ile 142 to proline and alanine. The kinetic parameters of N27A, I142P, and I142A were determined for both substrates, 3-HAA and O 2 in separate experiments. With respect to the organic substrate, 3-HAA, two different buffer environments were involved. One was the oxygen-saturated buffer by bubbling oxygen (Ͼ1.3 mM) during measurement, and the other used regular buffer without any treatment and contained 286 Ϯ 8 M molecular oxygen. Under the regular buffer environment, the mutant N27A showed a 6-fold decrease in the k cat and a 7-fold increase in the K m (3-HAA) relative to the WT enzyme (Table 1), exhibiting a substantially reduced catalytic efficiency, 2.5% of wtHAO. However, using O 2 saturated buffer, the k cat of N27A reached the same level of wtHAO, indicating that the primary reason for a substantial negative impact of the N27A mutation in catalysis with the regular buffer is a decreased oxygen-binding affinity.
The I142A and I142P variants showed 53-and 9.5-fold decreased catalytic efficiency, k cat /K m , toward 3-HAA with the regular buffer compared with wtHAO, respectively. The K m (3-HAA) (239.7 M) of I142A increased 10-fold, whereas the k cat decreased by ϳ5-fold compared with these of wtHAO.

A mechanism for O 2 enrichment in an extradiol dioxygenase
The K m (3-HAA) of I142P increased 4.3-fold. With oxygen-saturated buffer, the apparent constants k cat of I142A and I142P for 3-HAA are 13.9 Ϯ 0.9 and 24.8 Ϯ 2.6 s Ϫ1 , respectively, showing a partial or a full rescue of the catalytic efficiency. Interestingly, both mutants exhibited a near comparable catalytic activity with that of wtHAO only with oxygen supplementation.
Because of the apparent yet significant increasing of hydrophobic environment in the active site, we hypothesize that the major benefit of the loop movement is to attract the other nonpolar substrate, molecular oxygen. We also determined the K m value of O 2 . With a saturating amount of 3-HAA, the K m value increased ϳ9-fold, from 124.8 M for wtHAO to 1144 M for N27A and 1120 M for I142P, whereas the k cat remained similar (Table 1). An attempt to determine the K m for O 2 of I142A mutant failed because of an inability to saturate the enzyme (K m Ͼ 2,227 M).

Mutation of Asn 27 evades substrate-binding-induced loop movement
To assess whether or not the closed conformation would still be formed upon substrate binding after the alteration of the primary residue involved in orchestrating the loop movement, the mutant N27A was crystallized under similar conditions reported for wtHAO (3,13). The holo crystal structures of N27A in the ligand-free form was refined to 1.90 Å ( Table 2). Comparison of the structure of N27A ( Fig. 3A) with wtHAO (PDB entry 4L2N) revealed no apparent deviations, including the metal ligands and second sphere residues, as indicated by a small RMSD value of 0.090 Å between the two structures of all C␣ carbons. We then attempted to obtain a ligand-bound structure of this mutant. N27A crystals were soaked with 3-HAA or ClHAA anaerobically. The crystal structure of N27A in complex with ClHAA was solved at 2.30 Å (Fig. 3B). The omit map in the active site clearly illustrates the location of carboxylate and amine groups of ClHAA because of its strong saltbridge interactions with residue Arg 99 (Fig. S2). However, the phenyl ring and chloride electron densities were incomplete. Compared with ClHAA-bound wtHAO structures (PDB entries 1YFW and 1YFX), there is also negative density map around the chloride of ClHAA (3). Unfortunately, the ES complex structure remained elusive, even after extensive attempts. Compared with the single-crystal electronic absorption spectrum of ligand-free N27A, a N27A crystal soaked with ClHAA for 5 min exhibits an apparent absorbance peak at 328 nm (Fig.  3C), which is characteristic of ClHAA, indicating that ClHAA is indeed bound to N27A. The solution state UV-visible spectra of ClHAA-bound wtHAO and N27A showed similar binding affinity for the inhibitor (Fig. S3). The absorbance maximum of the inhibitor is also centered around 328 nm, which agrees well with the single-crystal UV-visible data.
The electron density map of ClHAA-bound N27A reveals that the inhibitor binds to the active-site iron with an ironhydroxyl distance of 2.7 Å, similar to what was observed in the ClHAA-bound structure of wtHAO. However, the superimposition of ClHAA-bound N27A and wtHAO structures reveals several differences. As compared with wtHAO, the phenyl ring of the inhibitor moved up with an angle of 23°in the mutant structure, whereas the carboxylate group was fixed in both structures by a salt bridge with Arg 99 , and the hydroxyl group of

Effect of Ile 142 on loop regions conformation change
Because the mutation of Ile 142 to proline and alanine also significantly decreased the oxygen binding and activation capacity (Table 1), a question raised was whether I142P and I142A would also be incompetent to present a closed conformation after binding with the substrate or ClHAA. Considering the H-bond interaction is observed between the backbone of Ile 142 and the side chain of Asn 27 , the I142A mutant was A mechanism for O 2 enrichment in an extradiol dioxygenase

in which the sum is over all the i measured reflections with equivalent Miller indices hkl, I(hkl)
is the averaged intensity of these i reflections, and the grand sum is over all measured reflections in the data set. c According to Karplus and Diederichs (28). d All positive reflections were used in the refinement. e According to Engh and Huber (29). f Calculated by using MolProbity (30). g NA, not applicable.

A mechanism for O 2 enrichment in an extradiol dioxygenase
expected to have a more subtle effect than N27A or I142P mutants on the oxygen binding and activation efficiency. The potential effects of deletion of the side chain of Ile 142 on loop regions movement were investigated by solving the ligand-free, enzyme-substrate, and enzyme-inhibitor complex structures.
The superimposed ligand-free structures of I142A (resolution 1.90 Å) and I142P (resolution 2.22 Å) and wtHAO showed high similarity with an RMSD of 0.141 and 0.161 Å from all the C␣ atoms aligned in these structures. After soaking with ClHAA for 10 min, an I142A-ClHAA complex was obtained and refined at 2.31 Å (Fig. 4A and Fig. S4). The single-crystal electronic absorption spectrum of the I142A crystal soaked with ClHAA shows absorbance at 328 nm caused by binding of the inhibitor to the HAO variant (Fig. S5). Like the N27A mutant, the overall structure of the I142A-ClHAA complex also exhibited high similarity with wtHAO with an RMSD of 0.144 Å. The binding mode of ClHAA to the active-site iron ion of I142A was identical to the N27A-ClHAA complex except that a water molecule is still bound to iron ion. However, the distance between the side chain of Asn 27 and Ala 142 backbone was 7.7 Å, making it too far to form a hydrogen-bond interaction. Three loop regions of the I142A-ClHAA complex align well with the open form of ligand-free wtHAO structure (PDB entry 4L2N in Fig. S6). Moreover, the crystal structure of I142A in complex with 3-HAA was obtained and refined to 2.60 Å (Fig. 4B). The electron density map reveals that 3-HAA binds to the active site of I142A in a bidentate chelation pattern. The superimposition of these two structures reveals a 45°rotation of the phenol rings of the substrates in the active site. The loop regions can be well aligned with the substrate-free structure (Fig. S6). The distance between Ala 142 and Asn 27 is 7.6 Å, not eligible for formation of an H-bond.
We also crystallized I142P and soaked its crystals with 3-HAA and ClHAA. As a result, the complex structures of I142P bound with 3-HAA or ClHAA were determined and refined to high resolution ( Table 2). Fig. 4 (C and D) clearly show a completed omit density map for both of ClHAA and 3-HAA in a similar binding mode to each other in the active site but distinct from those observed for the wtHAO. The ligands are anchored in the active site by interacting with the side chains of Arg 99 and Glu 110 . However, they are not directly ligated to the catalytic iron ion. Additionally, the two water ligands remain coordinated to the metal ion. Because residue 142 is part of an ␣-helix structure, and it sits at the start point of the loop C region, the helical structure of I142P in loop C is shorter than that of the wtHAO, resulting in a relatively larger change of the loop C than that in I142A. However, the overall structures of ClHAA-and 3-HAA-bound I142P show high similarity to wtHAO with an RMSD of 0.180 and 0.166 Å, respectively. Moreover, the loop regions well align with the substrate-free, open-form of wtHAO despite a shorter helix in loop C (Fig. S7). The distances for the two complexes between Pro 142 and Asn 27 are 8.5 and 8.4, respectively, and hence these residues are not able to interact with each other. Of note, the I142P alteration leads to a new binding mode for 3-HAA and ClHAA, which explains the 330-to-350 nm shift of the absorbance maximum in the ClHAA-bound I142P solution optical spectrum (Fig. S3).
A perusal of the differences between the open and closed forms of HAO revealed that not only does the backbone of Ile 142 form a hydrogen bond with Asn27 in the closed form, but its bulky, hydrophobic side chain also points toward the active site. Compared with the ligand-free structure, deletion of the side chain of Ile 142 led to a larger cavity with seven ordered water molecules between Ala 142 and the iron center in the ligand-free structure (Fig. S8). The I142P structure also has extra solvent between the Pro 142 and the iron center. The ligand-free structures of wtHAO reported were refined at higher resolutions; therefore the water cluster in the mutant structure is less likely because of resolution differences. It appears that mutation of I142 disrupts a water-free, putative oxygen binding channel connecting protein surface to the catalytic iron ion. Therefore, the K m values for O 2 of I142A and I142P increased dramatically.

Protein dynamics during HAO reaction
Molecular oxygen is known to have a central role in complex living systems. Understanding how nature is able to selectively utilize O 2 , a nonpolar reagent, to perform specific chemistry in aqueous solution at a high rate of catalysis is a question of great fundamental importance and deserving of study (1). It is apparent that the substrate of HAO, 3-HAA, with three polar functional groups, has considerable hydrophilic character. After binding with hydrophilic 3-HAA, how the HAO binary complex attracts a second, hydrophobic substrate is a question of fundamental importance. Here, we demonstrated that binding of 3-HAA induces loop dynamics of HAO to move to make for a more hydrophobic environment to efficiently capture molecular oxygen. The protein dynamic conformational change has loop regions moving by 8.6 Å and totally involves more than 15% of the protein residues in HAO. It is historically believed that O 2 would randomly diffuse into the protein scaffold, and enzymes that utilize O 2 have evolved to develop interior hydrophobic regions for O 2 binding. HAO presents a more intelligent way to facilitate O 2 binding to its active site. The O 2 -binding channel is not permanently composed; it is dynamically formed in response to substrate binding and changes along with the reaction process. Presumably, the open conformation is for exchange of substrate and product, and the closed conformation is for rapidly incorporating oxygen.

The putative O 2 binding cavity in the active site of HAO
The loop regions covering the active site of HAO form an apparently dynamic hydrophobic cavity. In the ternary com-plex of HAO-ClHAA-O 2 structure, this is where the O 2 binds with one of the oxygen atoms anchored at the catalytic iron ion. The cavity in the ligand-free, open-form HAO structure (Fig.  S9A) appears to be connected with the solvent from two different directions, potentially serving as a tunnel to facilitate ligand binding to the active site. In contrast, the cavity in the closed form wtHAO structure (Fig. S9B) exhibited significant differences as compared with the open form. The radius of the cavity decreases by approximately half, and the cavity no longer connects to the bulk solvent, presumably to minimize the impact of bulk solvent to the reactive intermediates during the catalytic reaction.
As was mentioned previously, Ile 142 and Asn 27 are located ϳ9 and 11 Å away from the catalytic iron ion, respectively. Elimination of the hydrogen-bonding interaction between loops A and C (mutant N27A) creates a variant that does not show the closed conformation upon substrate binding. The catalytic efficiency (k cat /K m ) of N27A for both molecular oxygen and 3-HAA is dramatically decreased compared with WT ( Table 1). The hydrogen bond formed between Asn 27 and Ile 142 is hypothesized to be the driving force to gather the loop regions together, which is triggered by the chelation of 3-HAA or ClHAA. Therefore, the dynamic action of the loop regions are critical for efficiently performing the dioxygenation. Meanwhile, the decreased hydrophobic character of loop region C (mutant I142A) could also lead to the elimination of loop movement, and determination of kinetic parameters with respect to oxygen is not possible. Without the proper positioning of Asn 27 and Ile 142 , the movement of loop regions A, B, and C would not occur, so the hydrophobic environment made by the "closed" form of the loop regions is broken, which leads to the reduction of oxygen capturing capacity of the mutants. Therefore, the major contribution of the loop regions is providing a sealed hydrophobic environment for binding and activation.

The loop regions in HAO from other biological origins
A comparison of available structures of 3-HAA dioxygenases can be found in Fig. S10. With the exception of an extra domain in HAO from mammalian sources, the overall structures show high similarity from a broad range of species. Compared with HAO from C. metallidurans, the RMSD values of HAO from Homo sapiens, Bos taurus, and Saccharomyces cerevisiae are 0.594, 0.813, and 1.072 Å, respectively. Moreover, the loop regions are among the highly conserved regions. Therefore, the loop regions required for the dioxygenation of HAO are one of the conserved structural characteristics during the evolution of HAO. Interestingly, holo-HAO from bovine crystallized in the closed form with a hydrogen bond between Asn 24 and Leu 137 (5). The isoleucine from loop C in prokaryotes is replaced by another similar residue of leucine in HAO from several eukaryotes (Fig. 1D). Based the alignment results, it is reasonable to believe that the dynamic action of loop regions for attracting hydrophilic or hydrophobic substrates has been conserved in HAOs during evolution.

A mechanism for O 2 enrichment in an extradiol dioxygenase How do the nonheme iron enzymes handle two or more substrates with distinct polar natures?
It is common in dioxygenases that flexible loop region movement is induced by interactions with substrates to be more ordered to cover the active site. Such an enzyme-substrate interaction can be observed and visualized by X-ray crystallographic structure determination. In an extradiol dioxygenase LapB, a loop containing His 248 and Tyr 257 was reported to hydrogen bond with its substrate, causing lower flexibility in the ligand-bound form (14). These two residues have essential roles during catalysis. In quercetin 2,3-dioxygenase, after binding its substrate (flavonol), a flexible loop around the active site became more ordered to stabilize the loaded substrate, and then the activation of C2 atom of flavonol was made to bind oxygen (15).
The nonheme iron and ␣-ketoglutarate (␣KG)-dependent enzyme family catalyzes a variety of chemical reactions coupled to the decarboxylation of ␣KG to succinate. They meet the similar challenge of dealing with both a hydrophilic substrate (␣KG or other substrates) and a hydrophobic substrate (oxygen). The common strategy is that they usually react with oxygen after binding a primary substrate. Taurine and ␣KG-dependent dioxygenase catalyzes taurine to aminoacetaldehyde and sulfite. Structural comparison reveals that the binding of taurine induced an ␣-helix region containing hydrophobic residues to move forward to the active site by forming a hydrogenbonding network (16). The strategy of conformational change around the active site is similar with that of HAO. The ␣-helix region would help to form a more hydrophobic environment in the active site and benefit the capture of oxygen for performing the dioxygenation. The CurA halogenase also belongs to the nonheme Fe(II)/␣KGdependent family (17) and catalyzes a cryptic chlorination on a specific acyl carrier protein. Their crystal structures with various states indicate "open" and "closed" conformations. After binding with ␣KG, the disordered "lid" (open form) containing 27 residues on the top of the active site became a more ordered closed form, which was triggered by the salt bridge between ␣KG and Arg 241 . The open form was proposed to organize the binding sequence in which the cosubstrate ␣KG and Cl Ϫ need to bind to the iron ion prior to the oxygen and the acyl carrier protein. Although the lid region does not contain a high percentage of hydrophobic residues, its dynamic action is critical for the chlorination.
Phenylalanine hydroxylase (PAH) is a nonheme iron-and pterin-dependent enzyme that catalyzed the oxidation of phenylalanine to tyrosine. PAH binds pterin and L-Phe first and oxygen (hydrophobic) last (18). Structural study reveals that binding of pterin in the active site of PAH induces a loop region to move in the direction of the iron center by forming hydrogen-bond networks with the residues on the loop. Therefore, the PAH-pterin complex provides an ideal orientation for binding oxygen in between pterin and the iron ion (19).
Unlike the nonheme iron-dependent enzymes mentioned above, the loop regions in HAO do not have an obvious interaction with 3-HAA in the active site; rather they provide a hydrophobic environment for the dioxygenation caused by the high percentage of nonpolar residues in the loop regions. For other dioxygenases, including catechol dioxygenase, 2,3-dihydroxybiphenyl dioxygenase, biphenyl dioxygenase, and cysteine dioxygenase, substrate binding-induced loop region movement has not been reported or does not exist.

Structural requirement for capturing O 2 in iron-dependent proteins
The most well characterized oxygen binding to an iron protein is in heme systems. Hemoglobin is well known as an oxygen-transport metalloprotein in the blood cells, carrying oxygen from the respiratory organs to the rest of the body. Because of the functional requirement, hemoglobin has a high oxygenbinding capacity (20). The relationship between the oxygenbinding and structural information has been investigated since the hemoglobin crystal structures were determined. The histidine gate hypothesis was proposed by Perutz and Mathews (21) to demonstrate the HisE7 in the distal region controls the ligand migration rate by a conformation change from an open to a closed status. Boechi et al. (22) described that the hydrophobic effect around HisE7 is the major driving force for oxygen uptake other than steric hindrance from HisE7. Moreover, Cohen and Schulten (23) compared the characters of O 2 migration pathway in 12 monomeric globins from a broad range of species. The pathway for O 2 migration shows various configurations from different globins; however, the located residues are conserved as mainly containing hydrophobic residues (Trp, Phe, Leu, Ile, etc.). Therefore, the hydrophobic environment of cavity networks of globins facilitate ligands traveling through the protein matrix.

Concluding remarks
Although nature has more than one strategy to solve the low-oxygen-concentration problem in solution for a rapid oxygen consumption reaction at the enzyme active site, the unique feature of protein dynamics induced by substrate binding reported here may be the first structurally defined example for facilitating O 2 binding to the catalytic iron along with the primary organic substrate in each catalytic cycle. With a rapid turnover rate, the O 2 level could drop significantly after several seconds of oxygenation reaction, and the O 2 diffusion to the solution may not be fast enough and thus may cause a seemingly hypoxic condition. If this occurs, the increased hydrophobicity would become more important to attract O 2 . Given the vast number of metal-dependent oxygen utilization enzymes, the protein dynamic-hydrophobicity strategy may be one of nature's common strategies to direct molecular oxygen toward organic substrate-bound metal centers.

Materials
The substrate 3-HAA and the inhibitor ClHAA were purchased from Sigma-Aldrich and Enamine Ltd. (Monmouth Jct., NJ), respectively.

Site-directed mutagenesis and protein preparation
The HAO gene from C. metallidurans was used as the template (GenBank TM accession no. CP000353) (3) to construct single mutants by the PCR overlap extension mutagenesis method. The forward primers were 5Ј-TTGAAGCCGCC-CGTCGGCGCCCGGCAGGTGTGGCAGGAC for N27A and 5Ј-GAGGTGCAGCTCAAGAGCGCCGTCACCGACCTCC-CGCCT for I142A, and 5Ј-GAGGTGCAGCTCAAGAGCCC-GGTCACCGACCTCCCGCC for I142P. The plasmids of mutants were verified and transformed to the expression host cells, Escherichia coli BL21(DE3). The purification strategy of the two mutants' proteins was the same as wtHAO (3). The wtHAO and mutants were anaerobically reconstituted with 3 eq. Fe(NH 4 ) 2 (SO 4 ) 2 and excess Fe 2ϩ was removed by Superdex 75 column before performing kinetic assay and crystallization.

Kinetic assay of wtHAO and mutants toward both substrates
The kinetic assays for wtHAO and mutants toward the substrate of 3-HAA were measured by monitoring the absorbance increase caused by the product formation (␣-amino-␤-carboxymuconate-⑀-semialdehyde, ⑀ 360 nm ϭ 47,500 M Ϫ1 cm Ϫ1 ) with an Agilent 8453 diode-array spectrophotometer (3,13). The O 2 -saturated buffer was made through bubbling with pure oxygen gas during the analysis. The kinetic assays toward the substrate of oxygen were monitored with a Clark-type oxygen electrode (Oxygraph, Hansatech Instruments) with saturating 3-HAA concentration. All assays were done at room temperature.

Crystallization, data collection, processing, and refinement
The mutants were crystallized by the hanging-drop method in an anaerobic chamber as described previously (3,13). After incubation with 20 mM of the inhibitor ClHAA for ϳ5-20 min, the soaked crystals were flash-cooled in liquid nitrogen. The diffraction data sets were collected, processed, and scaled by HKL-2000 (24). The structures of mutants were solved by molecular replacement by using the wtHAO (PDB entry 4L2N) as the template and refined by employing the Phenix 1.11.1-2575 (25) and Coot 0.8.3 (26). PyMOL (27) was used in drawing structural figures.


s2fieldsofstudy: Biology Medicine

text:
The association between IGF1 Gene 3’-UTR polymorphisms and cancer risk

Supplemental Digital Content is available in the text

Introduction
Insulin-like growth factor 1 (IGF1) plays an important role in regulating cellular proliferation and apoptosis. [1] Most circulat-ing IGF1 is bound to insulin-like growth factor binding protein 3 (IGFBP3), which can extend the half-life of IGF1. [2] IGF1 has been implicated in cancer development due to its key role in cell proliferation, differentiation, and apoptosis. [3] Many prospective studies have suggested that elevated IGF1 levels in the circulation can increase cancer risk. [4,5] While nutrition is a key factor that influences IGF1 levels in the circulation, studies of twins have indicated that 40% to 60% of the variation in IGF1 levels in the circulation depends on hereditary factors. [6][7][8][9] Several IGF1 polymorphisms have been identified as risk factors for cancers in genome-wide association studies (GWAS). [10] Three prime untranslated region (3'-UTR) contains important sequences that regulate mRNA transcription, stability, cellular localization, and microRNA binding. [11] Many studies have shown a relationship between IGF1 3'-UTR single nucleotide polymorphisms (SNPs) and cancer susceptibility, but these results are not consistent. [12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31] For example, Jiang et al report that the rs5742714 can increase the risk of gastric cancer, [25] while Ennishi et al maintain that there is no obvious association between rs5742714 and gastric cancer risk. [20] Dong et al report that IGF1 rs6214 can reduce the risk of pancreatic cancer, [31] while Nakao et al's overall analysis indicates that rs6214 does not affect pancreatic cancer risk, but that the polymorphism could increase the risk of pancreatic cancer among patients with body mass indexes (BMIs) of 25 or greater at the age of 20. [21] The inconclusive nature of these results necessitated the present metaanalysis, which will provide a more accurate evaluation of the association between the IGF1 3'-UTR polymorphisms rs5742714, rs6214, and rs6220 and cancer risk.

Ethics statement
As all analyses were based on previously published studies, no ethical approval or patient consent was required.

Search strategy
We performed a literature search for all available articles concerning the association between IGF1 3'-UTR polymorphisms and cancer risk in PubMed, Embase, and Web of Science databases (before April 30, 2018). The following keywords were used: "IGF1 or IGF-1 or insulin-like growth factor 1," "polymorphism or SNP or mutation or variant," and "cancer or carcinoma or tumor." We also identified relevant studies via checking reference lists.

Inclusion and exclusion criteria
The included studies met the following criteria: (1) addressing the relationship between IGF1 polymorphisms and cancer risk, (2) having a case-control or cohort study design, (3) having been published in English, and (4) containing sufficient genotype data.
The exclusion criteria were as follows: (1) not having a case-controlled or cohort study design, (2) being meta-analyses or reviews, and (3) not having sufficient genotype data.

Data extraction and quality score
The 2 authors worked independently to extract information and evaluate the quality of the studies. The following information was extracted: name of first author, publication year, country, ethnicity, type of cancer, genotyping methods, control source, and alleles or genotypes frequency. The quality of the studies was assessed using a quality score form [32] (Supplementary Table 1, http://links.lww.com/MD/C721). Quality scores ranged from 0 to 15. Any disagreement was resolved via discussion.

Statistical analysis
All statistical analyses were performed with the STATA software (Version 12.0, Stata Corporation, College Station, TX). The strength of each association was estimated using ORs and 95% CIs in 5 genetic models: the allele, homozygote, heterozygote, dominant, and recessive models. P values <.05 were considered statistically significant. A Q test and I 2 statistic were used to assess heterogeneity. [33] If the heterogeneity test was P >.1, this would indicate that the heterogeneity was not significant, a fixed-effect model was used to synthesis the OR and 95% CI. [34] Otherwise, the random-effects model was applied. [35] Hardy-Weinberg equilibrium (HWE) for control was calculated via a Chi-squared test. Stratified analyses were conducted by ethnicity, cancer type, control source, and quality score. Sensitivity analyses were carried out to evaluate the stability of the results by omitting a single study each time. Begg test and Egger test were applied to detect potential publication bias. [36,37] 3. Results

Meta-analysis
The associations between IGF1 rs5742714, rs6214, and rs6220 polymorphisms and cancer risk were evaluated using odds ratios (ORs) and their 95% confidence intervals (CIs) under the following 5 genetic models: the allele homozygote, heterozygote, dominant, and recessive models. We also conducted stratified analyses according to ethnicity, cancer type, and quality score.
Only results synthesized from no fewer than 2 studies are shown. In total, our meta-analysis includes 9 studies regarding the rs5742714 polymorphism, which contains 4741 cases and 7267 controls. In overall analysis, no significant association was identified between rs5742714 and cancer risk in any of the 5 models (n = 9, case = 4741, control = 7267, Table 3). In the stratified analysis of ethnicity, no significant association was identified between rs5742714 and cancer risk among the Asian population (n = 7, case = 3395, control = 5863, Table 3). In the stratified analysis of cancer type, no significant association was identified between rs5742714 and the risk of the gastric (n = 2, case = 1283, control = 2135, Table 3) or pancreatic cancer (n = 2, case = 971, control = 2123, Table 3). The results synthesized from studies that scored no less than 12 (n = 7, case = 4213, control = 6730, Table 3) did not display any difference in terms of the results of the overall analysis.
In total, our meta-analysis includes 16 studies regarding the rs6214 polymorphism, which contain 8700 cases and 13,847 controls. In overall analysis, no significant association was identified between rs6214 and cancer risk in any of the 5 models (n = 16, case = 8700, control = 13,847, Table 3). In the stratified analysis of ethnicity, no significant association was identified between rs6214 and cancer risk among the Caucasian (n = 6, case = 4385, control = 6903, Table 3) or Asian (n = 6, case = 2815, control = 5240, Table 3) population. The results of the stratified analysis of cancer type demonstrate that rs6214 reduces the risk of breast cancer under the allele, the heterozygote, and dominant models (n = 4, case = 3550, control = 4617, Table 3 and  Table 3 and Figure 2, AA vs GA + GG: OR, 0.68, 95% CI,0.53-0.87, P = .003). No significant association was identified between rs6214 and the risk of colorectal cancer in this analysis (n = 3, case = 831, control = 2551, Table 3). The results synthesized from studies that scored no less than 12 (n = 12, case = 7302, control = 11,521, Table 3) did not display any difference in terms of the results of the overall analysis.
In total, our meta-analysis includes 9 studies regarding the rs6220 polymorphism, which contain 8127 cases and 10,085controls. In overall analysis, no significant association Table 1 Characteristics of the studies included in the meta-analysis.

First author
Year was identified between rs6220 and cancer risk in any of the 5 models (n = 9, case = 8127, control = 10,085, Table 3). The results of the stratified analysis of ethnicity demonstrate that rs6220 is associated with a significantly increased cancer risk among the Caucasian population under the allele model (n = 4, case = 5524, control = 7077, Table 3, G vs A: OR, 1.06, 95% CI, 1.00-1.13, P = .043). The results of the stratified analysis of cancer type suggest that rs6220 is associated with a significantly increased risk of breast cancer under the homozygote model (n = 3, case = 2859, control = 3765, Table 3, GG vs AA: OR, 1.23, 95% CI, 1.02-1.48, P = .031). The results synthesized from studies that scored no less than 12 showed that rs6220 increased cancer risk under the allele model (n = 7, case = 7614, control = 9569, Table 3, G vs A: OR, 1.06, 95% CI, 1.00-1.11, P = .033). The instability of the rs6220 results in the analyses stratified by score demonstrates that rs6220 tends to increase cancer risk.
The number of studies used in this assessment was limited. Thus, more studies on the associations between the rs5742714, rs6214, and rs6220 polymorphisms and cancer risk are warranted to confirm these conclusions, and the molecular mechanisms via which these polymorphisms function should also be explored.

Sensitivity analysis
Sensitivity was evaluated by deleting each study once at a time. The corresponding ORs were not altered by any single study for rs5742714, rs6214, or rs6220 ( Fig. 3 and Supplementary Table 2, http://links.lww.com/MD/C721), demonstrating that the results were relatively stable in our meta-analysis.

Publication bias
A Begg test and an Egger test were performed to determine the publication biases of the studies. No statistical evidence of publication bias was observed in any of the 5 models for rs5742714, rs6214, or rs6220 (Table 4).

Discussion
The IGF signaling system plays an important role in cell proliferation, differentiation, and apoptosis. [1] IGF1 promotes cell proliferation via the RAS-mitogen-activated protein kinase (MAPK) signaling pathway. [38,39] Moreover, it is also a potent anti-apoptotic molecule that activates the  rs5742714  Total  GG  GC  CC  Total  GG  GC  CC  G  C  G  C  Chia2008  551  449  98  4  683  565  109  9  996  106  1239  127  0.160  15  Suzuki2008  795  629  154  12  721  591  124  6  1412  178  1306  136  0.857  12  Ennishi2011  703  427  238  38  1462  902  479  81  1092  314  2283 Table 3 Meta-analysis of the association between rs5742714, rs6214, and rs6220 polymorphisms and cancer risk. phosphatidylinositol 3-kinase (PI-3K)-AKT pathway. [40,41] Several IGF1 polymorphisms have been found to be associated with elevated IGF1 levels in the circulation, thus increasing the risk of cancer. [12,27] The previous meta-analyses of the relationship between IGF1 polymorphism and cancer focused on studies of IGF1 CA repeat variants. For the first time, we have systematically reviewed and investigated the relationship between the SNPs in the IGF1 gene's 3'-UTR sequences and cancer risk. We have synthesized the results from those groups contained in 2 or more studies. Thus far, for some types of cancer, there is only a single study. We reviewed these studies in the discussion.
In the present meta-analysis, 3 IGF1 polymorphisms were included: rs5742714, rs6214, and rs6220. The criteria for selecting these SNPs were as follows: the SNP should be located in the 3'-UTR region of the IGF1 gene, the SNP should have been reported to have a relationship with cancer risk previously, and the minor allele frequency (MAF) of a selected SNP should be no less than 5% in most of the populations in the 1000 Genomes Project Phase 3 (Supplementary Table 3 The rs5742714 C allele has been reported as a protective mutation in childhood acute lymphoblastic leukemia (ALL) and renal cell carcinoma (RCC). Additionally, the GC and CC genotypes have been reported to reduce the risk of childhood ALL and RCC as compared to the GG genotype. [26,27] Cao et al explored the potential functionality of rs5742714, finding that carriers with the GG genotype had higher levels of IGF1 expression in their renal tissues than carriers with the GC or CC genotype. Further, the rs5742714 C allele was observed to create a microRNA binding site for hsa-mir-580, unlike the G allele. [27] Naoko et al reported that among patients with BMIs 25 or greater at age 20, pancreatic cancer risk was increased with the presence of the C allele for rs5742714. [21] Thus, in the present meta-analysis, we suggest that rs5742714 is not significantly associated with gastric or pancreatic cancer risk. rs6214 is located in the 3'-UTR region of exon 4 in IGF1 and does not cause any amino acid change itself. However, it may have regulatory functions or could be linked with functional alleles at exon 4, leading to a change in the amino acid sequence in the IGF1. [42] Vella et al (2008) tested IGF1 protein levels at birth and at age 7 or 8 years in children who had a different genotype of rs6214. They found that rs6214 polymorphism could increase IGF1 concentrations, but no association was shown between this polymorphism and growth or glucose metabolism. [43] Lu et al reported that rs6214 polymorphism could increase expression of IGF1 mRNA, thus, the difference was not statically significant. [26] Al-Zahrani et al reported that www.md-journal.com that rs6214 was not associated with circulating IGF1 levels among Caucasian women. [12] In fact, the rs6214 polymorphism A allele has been reported to increase childhood ALL risk, [26] as well as increasing esophageal adenocarcinoma (EAC) and head and neck cancer (HNC) risk in women. [23] For the present metaanalysis, it was observed that rs6214 reduced the risk of breast cancer under the allele, the heterozygote, and the dominant models, as well as reducing the risk of pancreatic cancer under the recessive model. It has been reported that the rs6220 G alleles are significantly associated with increasing levels of IGF1, [14] thus increasing prostate cancer risk. Furthermore, rs6220 has been found to reduce the risk of the low-grade gliomas. [16] Interestingly, Al-Zahraniet et al found that there was a statistically significant association between rs6220 and circulating IGF1 levels in females, though, not in males. [12] Moreover, women who have the rs6220 GG genotype had higher IGF1 plasma levels and increased breast density. [29] In the present meta-analysis, it was observed that rs6220 was significantly associated with increasing the risk of breast cancer under the homozygote model. Even if the 3'-UTR sequences cannot translate into proteins, they may contain sequences that are critical for transcriptional regulation, mRNA stability or cellular localization. [17] The biological functions of these polymorphisms in 3'UTR should be explored more in future studies.
The present meta-analysis has several limitations. First, we only included studies published in English. Thus, important studies published in other languages may have been overlooked. Second, the number of studies is relatively small, especially in the stratified analysis. For instance, there is only one study available for rs5742714 regarding ALL, and there is only 1 study available for rs6220 regarding the Asian population, so a pooled study could not be performed for this type of cancer or this ethnicity. Finally, due to the limited information contained in the included articles, we could not analyze adjusted ORs regarding other factors such as gender, age, alcohol intake, and smoking history, which may have influenced the association.
In conclusion, in this study, we systematically reviewed and meta-analyzed the relationship between IGF1 gene 3'-UTR polymorphisms and cancer risk for the first time. We found that rs5742714, rs6214, and rs6220 were not associated with overall cancer risk. In fact, rs6214 reduced the risk of breast and pancreatic cancer, while rs6220 increased the risk of breast cancer. The study also indicated that rs6220 increased overall cancer risk among Caucasian populations. We need welldesigned studies with larger sample sizes to explore the relationship between IGF1 3'-UTR polymorphisms and cancer risk in the future.


extfieldsofstudy: Medicine Chemistry

text:
Induction of Biofilm Formation in Klebsiella pneumoniae ATCC 13884 by Several Drugs: The Possible Role of Quorum Sensing Modulation

Bacterial resistance is caused by several biochemical factors, the formation of biofilm being one of the main causes. This process is triggered by Quorum Sensing (QS), through the production of endogenous molecules, although other substances such as natural products can also do this. In this work, we aimed to determine whether some drugs are involved in the induction of biofilm formation in Klebsiella pneumoniae ATCC 13884, and thus, increase bacterial resistance. For this, the effect of 22 drugs on K. pneumoniae ATCC 13884 growth was determined at sub-plasmatic concentrations; the production of autoinducer lactones was established by HPLC and with a biosensor. The induction of biofilm formation was determined through crystal violet assay at 585 nm in a microplate reader and using urethral catheters. According to the in vitro assays, some drugs were found to induce biofilm formation in K. pneumoniae ATCC 13884. The effect of acetaminophen, hydrochlorothiazide, and progesterone stood out. The first drug caused several changes in the biochemistry of K. pneumoniae ATCC 13884 related to QS: high synthesis of N-hexanoyl-homoserine lactone, increasing bacterial populations by 27% and biofilm formation by 49%, and a more gentamicin resistant biofilm. Furthermore, it increased the colonization area of urethral catheters. Hydrochlorothiazide showed the biggest increase in the induction of biofilm formation of 51%, and progesterone displayed the greatest ability to provoke bacterial mass adherence but had no effects on K. pneumoniae ATCC 13884 bacterial population growth.

Introduction
Antibiotic resistance has become a threat to humanity causing hundreds of thousands of deaths annually and dramatically increasing treatment time and its costs. Klebsiella pneumoniae is the causal agent of several diseases including pneumonia, meningitis, bloodstream infections, and surgical site infections. Furthermore, some strains are resistant to carbapenem antibiotics, including gentamicin and ciprofloxacin [1], and are the most commonly isolated bacterium from medical devices [2,3]. The World Health Organization classified K. pneumoniae as a critical priority and established the need to promote the search for new antimicrobial agents [4].
K. pneumoniae and other bacteria exhibit Quorum Sensing (QS), a mechanism of bacterial communication, which regulates some biochemical processes in planktonic cells, and biofilms [5]. QS is involved in pathogenicity and virulence through modifications in motility, the synthesis of hydrolytic enzymes, toxins, and polysaccharides, as well as biofilm formation [6], among other things. Biofilm formation represents a major resistance mechanism, since it renders bacterial colonies impermeable to antibiotics [7], promotes the transmission of resistance genes, increases the expression of efflux pumps, increases antibiotic metabolism, and favors persister cells [8].
Although increases in recurrent infections have been attributed to processes inherent to pathogen biochemical resistance, QS offers a better understanding of the factors that induce biofilm formation. Therefore, it is essential to determine whether exogenous molecules can induce QS in K. pneumoniae. For this reason, the present study aimed to determine whether some drugs used in the treatment of several diseases could be involved in the formation of K. pneumoniae ATCC 13884 biofilm, and, thereby, increase bacterial resistance. The understanding of this mechanism is likely to result in improved clinical procedures, such as an optimal pharmacologic treatment [9] that avoids exposing patients to these drugs and thus prevents recurrent infections.
This work was conducted through the following steps: (1) The effects of drugs on bacterial growth and biofilm formation were determined. Due to its importance as an OTC drug, the next assays were made only with acetaminophen. (2) The presence of C6AHL autoinducer was established and quantified. (3) The effect of acetaminophen on mature biofilm was analyzed. (4) The architecture of biofilm was observed by microscopy. (5) The effect on the adherence to urethral catheters was also determined.

Drug Selection
Twenty-two drugs were selected from a list of essential medications and were kindly donated by Laboratorios LAPROFF (Sabaneta, Colombia). The drugs were taken from a list of essential drugs and based on their frequency of use. These drugs are mainly used for chronic diseases, or as antibiotics. Later, progesterone and cholic acid were added.

Bacteria
Chromobacterium violaceum CV026 mutant was donated from Dr. Walter Giordano from the University of Rio Cuarto (Argentina). K. pneumoniae subsp. rhinoscleromatis ATCC 13884 was purchased from American Type Cell Collection (Manassas, VI, USA). These were cultured in Luria-Bertani Agar (LB) (Lab M, Lancashire, UK) and incubated at 37 • C (K. pneumoniae) or 28 • C (CV026). For all experiments, the inoculums were prepared in saline solution 0.9%; assays were performed in triplicate unless otherwise stated. The absorbance of violacein was determined at 585 nm in a microplate reader (Thermo Scientific, Waltham, MA, USA).

Equipment
HPLC (Agilent 1200 series system, Santa Clara, CA, USA) coupled with a Quadrupole Mass Spectrometer (VL model) was used and operated in the Electrospray Ionization (ESI) mode, with the Agilent ChemStation 04.03.087 software, equipped with a 4.6 mm × 150 mm, 5 µm ZORBAX Eclipse XDB-C18 analytical column. Analysis was carried out at a temperature of 35 • C with a flow rate of 0.8 mL/min, and an injection volume of 5 µL. Water with 0.1% formic acid and acetonitrile with 0.1% formic acid were used as mobile Phases A and B, respectively. Solvent gradient B from 60% to 90% was used for a run time of 8 min.

Effect of Drugs on K. pneumoniae ATCC 13884 Growth
The effects of drugs on K. pneumoniae growth were determined using the microdilution method, as described by Wiegand [10], but using 100 µL of inoculum, equivalent to 10 6 CFU/mL. The maximum incubation time to obtain a greater bacterial mass was determined using 96-well microplates according to O'Toole [11], but using LB medium and incubating for 30 h, using crystal violet (0.05% in water).

Biofilm Formation
The inoculum was prepared from a colony grown on LB agar for 24 h, in a 0.9% saline solution and adjusted to OD 600nm 0.05; then, 100 µL was added to each well with 100 µL of LB medium containing drugs at non-biocide plasmatic concentrations. The microplates were incubated at 37 • C for 30 h. The attached bacterial mass measurement was carried out as previously stated. The amount of biofilm was calculated by comparison with the growth of untreated bacteria. A violacein absorbance calibration curve was made to quantify the C6-AHL concentration. C6-AHL, at concentrations between 0.06 and 100 µg/mL, was added to 100 µL of sterile K. pneumoniae ATCC 13884 supernatant, 50 µL of LB medium, and 50 µL of C. violaceum CV026 inoculum with DO 600nm of 0.1. The final concentrations of C6-AHL were 0.03, 0.05, 0.10, 0.20, 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25, and 50 µg/mL. This mixture was incubated at 28 • C for 48 h and dried at 50 • C for 12 h. The violacein was extracted with 250 µL of methanol and the absorbance was determined at 585 nm. To establish the relationship between violacein absorbance and concentration of C6-AHL, a simple linear regression model was used, determining the correlation coefficient and the slope.

Detection of C6-AHL by HPLC/MS
A culture of 250 mL of K. pneumoniae ATCC 13884 in an LB medium previously adjusted to OD 600nm of 0.05 was incubated for 2, 4, 6, 8, and 23 h. At the end of each period, each sample was sonicated for 5 min and centrifuged at RCF = 1132 for 10 min and the supernatant was acidified at pH 3.5 with 10% acetic acid, and then lyophilized. The dried material (2 g) was extracted with 80 mL of ethyl acetate. Then, the solvent was vacuum-dried, and the material was re-dissolved in 2 mL of methanol. HPLC analysis was performed as described by Yin [12]. N-hexanoyl-homoserine lactone standard was employed to compare the retention times and a fragment at m/z 200.24 was monitored.

Kinetics of C6-AHL Production
An inoculum of K. pneumoniae ATCC 13884 was prepared in saline solution with an OD 600nm of 0.05 ± 0.01. LB medium was added at a 1:1 ratio (v/v) and the mixture incubated at 37 • C for 24 h; supernatant aliquots were collected at 2, 4, 6, 8, and 23 h. Next, samples were filtered through 0.2 µm pores. From the filtered supernatant, 100 µL were added to 50 µL of C. violaceum CV026 inoculum adjusted to OD 600nm 0.05 ± 0.01 and 50 µL of sterile LB medium. The concentration of C6-AHL in the supernatant was lower than the biosensor detection limit, so all samples were doped with 0.2 µg/mL of C6-AHL to establish the minimum concentration of lactone needed to induce violacein production. After incubating the C. violaceum CV026 biosensor at 28 • C for 48 h, the microplate was dried at 50 • C for 24 h. The dried material was then extracted with 250 µL of methanol and absorbance was determined at 585 nm.

Production of C6-AHL in K. pneumoniae ATCC 13884 after Drug Treatment
The levels of AHLs in the K. pneumoniae ATCC 13884 supernatants after treatment with biofilm-inducing drugs were detected by assessing the levels of violacein synthesis by C. violaceum (CV026). However, to do that, it was first necessary to establish the drugs' ability to activate QS in these bacteria. Therefore, 100 µL of inoculum, adjusted to OD 600nm 0.05, was added to 96-well microplates containing 100 µL of LB medium, with each drug at a final concentration of 1 µg/mL. Samples were incubated for 48 h, then violacein was extracted with 250 µL of methanol and finally absorbance was determined at 585 nm.
Drugs with a high biofilm-inducing effect were added to the K. pneumoniae ATCC 13884 cultures in LB media at the same concentrations exhibited for induction. Samples were incubated at 37 • C for 3 h, the supernatant was filtered through 0.2 µm pores and then evaluated with the C. violaceum CV026, as above. A drug-free culture supernatant containing 0.2 µg/mL of C6-AHL (doping) was used as control. To establish the stability of the biofilms, the antibiotics gentamicin and ciprofloxacin were chosen. The first one was selected because K. pneumoniae ATCC 13884 shows resistance to it [13], and the latter because of its ability to cross the biofilm wall [14] and act upon bacteria embedded there.
A culture of K. pneumoniae ATCC 13884 was incubated for the maximum biofilm formation time, as described above in Section 2.3.1. Kinetics of Biofilm Formation. After the formation of a mature biofilm, the supernatant was removed, and the sample was rinsed with sterile water. A mix of LB medium in 50% saline solution with ciprofloxacin and gentamicin was again added to each well at concentrations of 0.0015-1.0 µg/mL. The plates were incubated at 37 • C for a further 18 h and then washed twice with sterile water. Each well was treated with 0.05% crystal violet to quantify the remaining bacteria and to establish the antibiotic concentration enabling to remove 50% of K. pneumoniae biofilm.

Mature Biofilms Formed under Biofilm-Inducing Drugs
K. pneumoniae ATCC 13884 culture was incubated in the presence of acetaminophen, hydrochlorothiazide, and progesterone for the maximum biofilm formation time. The microplates were rinsed twice with distilled water. Then, 200 µL of gentamicin (concentration 0.1 µg/mL in LB medium) were added. After that, they were incubated at 37 • C for 18 h, and then the supernatant was removed, and each well was rinsed with sterile water and dried at 50 • C. Finally, the samples were treated with 0.05% crystal violet solution to establish the remaining amount of mature biofilm.

Architecture of the Mature Biofilm
Liquid LB medium containing biofilm-inducing drugs and K. pneumoniae ATCC 13884 inoculum in saline solution at OD 600nm 0.05 was placed on 1 cm × 1 cm glass coverslips and incubated at 37 • C for 30 h. Afterwards, the coverslips were submerged once in sterile water. For staining, the coverslips were submerged in 0.1% acridine orange in Plasma Bovine Serum (PBS) at pH 7 for 1 min and then the excess dye was rinsed off with sterile water. The stained biofilm was imaged in an Episcopic-Fluorescence Attachment EFD-3, using a B-2A fluorescence filter cube coupled to with camera.

Effect of Acetaminophen on K. pneumoniae ATCC 13884 Adherence to Urethral Catheters
A piece of 5 cm, 20-caliber PVC catheter Medex ® (Passaic, NJ, USA) was submerged in LB culture medium with K. pneumoniae ATCC 13884 adjusted to OD 600nm of 0.05 in saline solution and containing acetaminophen at plasmatic concentrations (Table 1); the same drug-free medium was used as negative control. Cultures were incubated at 37 • C for 30 h and catheters were submerged in sterile water to remove sessile bacteria. Samples were then dried at 50 • C for 2 h. Pictures were taken with an optical microscope and scanning electron microscope.
For the optic microscope technique, the catheters were submerged in 0.05% crystal violet solution for 5 min and then rinsed with sterile water and dried at 50 • C for 2 h. Afterwards, the catheters were photographed with a binocular microscope using 4× and 10× objectives. Biofilm sizes and their appearances were analyzed by ImageJ program. All experiments were performed in duplicate.
For SEM, after drying, the catheters were covered with nebulized gold and photographed at 200X magnification.

Statistical Analysis
The assays underwent a statistical analysis to compare means using Multiple Range Tests, with a confidence level of 95%, using Statgraphic Centurion software (Version 16.2.04, The Plains, VA, USA).

Results
To determine the effects on quorum sensing mechanisms without interference from the biocide effect, all drugs were analyzed at sub-lethal concentrations; these concentrations were established by finding the concentration range at which bacteria exhibited a growth equal to or higher than that of untreated bacteria. Before measuring the levels of N-Acyl-homoserine lactone, the effect of the drugs was established at concentrations of 1.0 µg/mL directly on the CV026 biosensor, without C6-AHL doping. None of them were found to induce violacein synthesis above the minimal detection limit of absorbance (OD 0.05 UA at 585 nm).
The concentrations of active ingredients were adjusted with LB medium to be within the range of plasmatic concentrations, which were obtained from the Drugbank online database, Version 5.0 (https://www.drugbank.ca/); the specific range for each drug is shown in Table 1. Table 1. Plasma concentration of each drug and its pharmacological application.

Effect of Drugs on K. pneumoniae ATCC 13884 Growth
Gentamicin and tetracycline caused a large increase in the bacterial population, 31% higher than that of the control (Table 2). Amikacin, nitrofurantoin, norfloxacin, levofloxacin, and ciprofloxacin did not affect bacterial growth. Both gentamicin and tetracycline were selected as positive controls for the following experiments. Table 2. Effect of some antibiotics on bacterial growth and biofilm formation.

Antibiotic
Concentration When drugs were assayed, three groups were noted according to their effects. The first group included cetirizine, loratadine, propranolol, metformin, and lovastatin; these drugs did not modify K. pneumoniae ATCC 13884 growth ( Table 3). The second group comprised enalapril, captopril, diclofenac, dexamethasone, and progesterone; these drugs caused a slight increase in bacterial populations (up to 15%). The third group of drugs contained acetaminophen, carbamazepine, verapamil, cholic acid, and hydrochlorothiazide; these drugs increased bacterial growth by 16-31%. Table 3. Effect of some drugs on bacterial growth and biofilm formation.

Kinetics of Biofilm Formation
After incubating K. pneumoniae ATCC 13884 for 54 h, the absorbance of crystal violet at 585 nm was plotted against time ( Figure 1). From these kinetics analyses, the highest bacterial adherence time was 30 h. Afterwards, a reduced adherence was detected, indicating nutrient depletion and biofilm detachment.

Biofilm Formation
The previously mentioned drugs were tested for their ability to induce biofilm formation. Of all 22 drugs assayed, 11 increased biofilm formations (Tables 2 and 3).
The antibiotics amikacin and norfloxacin were the most powerful inducers of biofilm formation, increasing it by 56% and 50% compared to the control, respectively; however, they did not change the bacterial growth. Tetracycline increased biofilm formation by 46% and increased the bacterial population by 31%. Gentamicin at 0.01 µg/mL also increased biofilm formation by 25% and bacterial population by 31%; these results are comparable to those previously reported for K. pneumoniae resistance through the activation of Extended Spectrum Beta Lactamase (ESBL) synthesis and increase in biofilm formation (7). It has been reported that 42.9% of nosocomial isolates of the genus Klebsiella are resistant to this antibiotic [15].
Three other drugs, enalapril, diclofenac, and carbamazepine, had no effect on biofilm formation, despite increasing bacterial growth. Loratadine had no effect on either bacterial growth or biofilm formation.
The steroids dexamethasone and cholic acid did not provoke positive effects on biofilm formation since induction was lower than 14%. However, there is a report that states that an addition of 1% of bile salts to the culture induced biofilm formation in K. pneumoniae [16]. Surprisingly, progesterone exhibited a strong effect on biofilm formation, increasing it by 65%, surpassing tetracycline (46%) despite showing a lower growth induction of only 11%.
The following drugs also had a great impact on the induction of biofilm formation: hydrochlorothiazide (51%), acetaminophen (49%), and captopril (24%). The first two exceeded the inducing effect of tetracycline and simultaneously showed a positive effect on bacterial growth, 17% and 27%, respectively.
The results demonstrate that some drugs play an active role in biofilm formation in K. pneumoniae ATCC 13884. However, there was no positive relationship regarding bacterial growth.

Biofilm Formation
The previously mentioned drugs were tested for their ability to induce biofilm formation. Of all 22 drugs assayed, 11 increased biofilm formations (Tables 2 and 3).
The antibiotics amikacin and norfloxacin were the most powerful inducers of biofilm formation, increasing it by 56% and 50% compared to the control, respectively; however, they did not change the bacterial growth. Tetracycline increased biofilm formation by 46% and increased the bacterial population by 31%. Gentamicin at 0.01 µg/mL also increased biofilm formation by 25% and bacterial population by 31%; these results are comparable to those previously reported for K. pneumoniae resistance through the activation of Extended Spectrum Beta Lactamase (ESBL) synthesis and increase in biofilm formation (7). It has been reported that 42.9% of nosocomial isolates of the genus Klebsiella are resistant to this antibiotic [15].
Three other drugs, enalapril, diclofenac, and carbamazepine, had no effect on biofilm formation, despite increasing bacterial growth. Loratadine had no effect on either bacterial growth or biofilm formation.
The steroids dexamethasone and cholic acid did not provoke positive effects on biofilm formation since induction was lower than 14%. However, there is a report that states that an addition of 1% of bile salts to the culture induced biofilm formation in K. pneumoniae [16]. Surprisingly, progesterone exhibited a strong effect on biofilm formation, increasing it by 65%, surpassing tetracycline (46%) despite showing a lower growth induction of only 11%.
The following drugs also had a great impact on the induction of biofilm formation: hydrochlorothiazide (51%), acetaminophen (49%), and captopril (24%). The first two exceeded the inducing effect of tetracycline and simultaneously showed a positive effect on bacterial growth, 17% and 27%, respectively.
The results demonstrate that some drugs play an active role in biofilm formation in K. pneumoniae ATCC 13884. However, there was no positive relationship regarding bacterial growth.

Detection of C6-AHL through the Biosensor C. violaceum
The curve of violacein absorption vs. C6-AHL concentration showed that violacein production was induced to its maximum peak at approximately 3.0 µg/mL and maintained a linear relationship between 0.03 and 0.39 µg/mL. This range was used to obtain the following equation and to establish C6-AHL concentrations in the supernatants of cultures after treatment with each drug: The equation has a correlation coefficient of 0.8850 and a standard error of 0.0187. The quantity of C6-AHL necessary to activate violacein synthesis in the biosensor was also determined, since the AHL concentration in K. pneumoniae ATCC 13884 supernatants is close to the lower quantification limit of the biosensor. Therefore, the remaining assays with C. violaceum CV026 were doped with 0.2 µg/mL of C6-AHL.

Detection by HPLC/MS
To assess the presence of C6-AHL, the supernatant cultures were analyzed by HLPC/MS, using a standard compound and monitoring the 200.24 fragment [M + H] + . C6-AHL was detected at a concentration of 0.05 ± 0.009 µg/mL in the control sample.

Kinetics of C6-AHL Production in K. pneumoniae ATCC 13884
Violacein synthesis in the C. violaceum CV026 biosensor increased significantly after adding the supernatant of K. pneumoniae ATCC 13884 incubated for 2 h, revealing that the highest rate of C6-AHL synthesis occurs during the exponential phase ( Figure 2). Therefore, analyses of the amount of C6-AHL synthetized in the presence of biofilm-inducing drugs were performed between 2 and 4 h of incubation.

Detection of C6-AHL through the Biosensor C. violaceum
The curve of violacein absorption vs. C6-AHL concentration showed that violacein production was induced to its maximum peak at approximately 3.0 µg/mL and maintained a linear relationship between 0.03 and 0.39 µg/mL. This range was used to obtain the following equation and to establish C6-AHL concentrations in the supernatants of cultures after treatment with each drug: The equation has a correlation coefficient of 0.8850 and a standard error of 0.0187. The quantity of C6-AHL necessary to activate violacein synthesis in the biosensor was also determined, since the AHL concentration in K. pneumoniae ATCC 13884 supernatants is close to the lower quantification limit of the biosensor. Therefore, the remaining assays with C. violaceum CV026 were doped with 0.2 µg/mL of C6-AHL.

Detection by HPLC/MS
To assess the presence of C6-AHL, the supernatant cultures were analyzed by HLPC/MS, using a standard compound and monitoring the 200.24 fragment [M + H] + . C6-AHL was detected at a concentration of 0.05 ± 0.009 µg/mL in the control sample.

Kinetics of C6-AHL Production in K. pneumoniae ATCC 13884
Violacein synthesis in the C. violaceum CV026 biosensor increased significantly after adding the supernatant of K. pneumoniae ATCC 13884 incubated for 2 h, revealing that the highest rate of C6-AHL synthesis occurs during the exponential phase ( Figure 2). Therefore, analyses of the amount of C6-AHL synthetized in the presence of biofilm-inducing drugs were performed between 2 and 4 h of incubation.

Production of C6-AHL in K. pneumoniae ATCC 13884 after Drug Treatments
Supernatants of K. pneumoniae culture treated with acetaminophen at concentrations between 0.3 and 2.5 µg/mL increased violacein production by 10%, which is equivalent to C6-AHL concentrations of 0.35 ± 0.02 µg/mL. Meanwhile, control-induced violacein production was 0.26 ± 0.02 µg/mL of C6-AHL; therefore, acetaminophen at plasmatic concentrations increased the production of this compound by 22%. Other drugs, such as hydrochlorothiazide and progesterone, showed no significant effect on the production of this autoinducer (Table 4). The mature biofilm was exposed to ciprofloxacin and gentamicin to establish the antibiotic concentration necessary to eliminate 50% of the biofilm. Accordingly, the biofilm was up to four times more resistant to gentamicin, as compared with ciprofloxacin (Figure 3), because gentamicin was only able to eliminate 50% of the biofilm at 0.12 µg /mL, while ciprofloxacin achieved this at 0.03 µg/mL. This difference can be explained by ciprofloxacin's ability to cross biofilms, thus performing its antibiotic effect on the embedded bacteria.

Production of C6-AHL in K. pneumoniae ATCC 13884 after Drug Treatments
Supernatants of K. pneumoniae culture treated with acetaminophen at concentrations between 0.3 and 2.5 µg/mL increased violacein production by 10%, which is equivalent to C6-AHL concentrations of 0.35 ± 0.02 µg/mL. Meanwhile, control-induced violacein production was 0.26 ± 0.02 µg/mL of C6-AHL; therefore, acetaminophen at plasmatic concentrations increased the production of this compound by 22%. Other drugs, such as hydrochlorothiazide and progesterone, showed no significant effect on the production of this autoinducer (Table 4).

Minimum Antibiotic Concentration Required to Eliminate Mature Biofilms
The mature biofilm was exposed to ciprofloxacin and gentamicin to establish the antibiotic concentration necessary to eliminate 50% of the biofilm. Accordingly, the biofilm was up to four times more resistant to gentamicin, as compared with ciprofloxacin (Figure 3), because gentamicin was only able to eliminate 50% of the biofilm at 0.12 µg /mL, while ciprofloxacin achieved this at 0.03 µg/mL. This difference can be explained by ciprofloxacin's ability to cross biofilms, thus performing its antibiotic effect on the embedded bacteria.

Effect of Antibiotics on Mature Biofilm Formed by Drugs
To demonstrate the strength of the induced biofilm, mature biofilms were treated with a dose of gentamicin required to eliminate 50% of the biofilm, that is, 0.12 µg/mL. At higher concentrations of acetaminophen, 2.5 and 1.25 µg/mL, the biofilm was more resistant to gentamicin, nearly 47% of the control (Figure 4). However, the biofilm which formed at low concentrations of acetaminophen was less resistant-even less resistant than those formed at low concentrations of hydrochlorothiazide and progesterone. These drugs produced more stable biofilms which were resistant to gentamicin but at lower concentrations (0.02 and 0.06 µg/mL, respectively).

Effect of Antibiotics on Mature Biofilm Formed by Drugs
To demonstrate the strength of the induced biofilm, mature biofilms were treated with a dose of gentamicin required to eliminate 50% of the biofilm, that is, 0.12 µg/mL. At higher concentrations of acetaminophen, 2.5 and 1.25 µg/mL, the biofilm was more resistant to gentamicin, nearly 47% of the control (Figure 4). However, the biofilm which formed at low concentrations of acetaminophen was less resistant-even less resistant than those formed at low concentrations of hydrochlorothiazide and progesterone. These drugs produced more stable biofilms which were resistant to gentamicin but at lower concentrations (0.02 and 0.06 µg/mL, respectively). Figure 4. Effect of gentamicin on the stability of the biofilm formed under biofilm-inducing drugs at plasmatic concentrations. Mature biofilm formed after 30 h of drug applications was treated with gentamicin 0.1 µg/mL, and then, the mature biofilm remainder was quantified. In acetaminophen, a drug-dependent decrease was noticed at higher concentrations of 2.5 and 1.25 µg/mL. * Statistically significative difference.

Effect on Mature Biofilm Architecture
Three images at 10× magnification were taken of each biofilm sample formed on coverslips after staining with 0.1% acridine orange. The biofilm size is indicated by the bright area and the population density is indicted by brightness color. In the presence of 0.6 µg/mL acetaminophen ( Figure 5A), the K. pneumoniae ATCC 13884 biofilms enlarged their colonization area and reduced the inter-bacterial spaces. As result of the increase in population density, a more compacted structure was formed. As can be seen in the control images ( Figure 5B), the samples which were not treated formed smaller aggregates with fewer bright areas and flatter configurations were formed. Mature biofilm formed after 30 h of drug applications was treated with gentamicin 0.1 µg/mL, and then, the mature biofilm remainder was quantified. In acetaminophen, a drug-dependent decrease was noticed at higher concentrations of 2.5 and 1.25 µg/mL. * Statistically significative difference.

Effect on Mature Biofilm Architecture
Three images at 10× magnification were taken of each biofilm sample formed on coverslips after staining with 0.1% acridine orange. The biofilm size is indicated by the bright area and the population density is indicted by brightness color. In the presence of 0.6 µg/mL acetaminophen ( Figure 5A), the K. pneumoniae ATCC 13884 biofilms enlarged their colonization area and reduced the inter-bacterial spaces. As result of the increase in population density, a more compacted structure was formed. As can be seen in the control images ( Figure 5B), the samples which were not treated formed smaller aggregates with fewer bright areas and flatter configurations were formed.

Effect of the Drugs on K. pneumoniae ATCC 13884 Adherence to Urethral Catheters
Quantification of the colonized area for each treatment was carried out using the ImageJ program. Thus, the colonized area of the bacteria under acetaminophen treatment was 12.17%, while under control conditions it was 2.07%. For both samples, an area of 1,735,237 µm 2 was evaluated. In the presence of acetaminophen, the bacteria colonized a greater area on the PVC catheters ( Figure  6A). In addition, more compact structures were observed in comparison with the biofilm produced without drug treatment ( Figure 6B), with more dispersed aggregates.
Analysis of the scanning electron microscope images (Figure 7) shows that the biofilm which formed under acetaminophen conditions displayed large colonization areas that were made up of compact masses of bacteria in prominent islets in comparison to those cultures without this drug. Figure 6. Optical microscope image of the K. pneumoniae ATCC 13884 biofilm assembled on the urethral catheter: (A) biofilm formation during acetaminophen treatment; and (B) without treatment with acetaminophen; both stained with crystal violet at 0.05%. Quantification of the area for each treatment was carried out using the ImageJ program, processing the sample by the particle content. Thus, the colonized area in the control sample was 2.07%, while in the acetaminophen sample it was 12.17%.

Effect of the Drugs on K. pneumoniae ATCC 13884 Adherence to Urethral Catheters
Quantification of the colonized area for each treatment was carried out using the ImageJ program. Thus, the colonized area of the bacteria under acetaminophen treatment was 12.17%, while under control conditions it was 2.07%. For both samples, an area of 1,735,237 µm 2 was evaluated. In the presence of acetaminophen, the bacteria colonized a greater area on the PVC catheters ( Figure 6A). In addition, more compact structures were observed in comparison with the biofilm produced without drug treatment ( Figure 6B), with more dispersed aggregates.

Effect of the Drugs on K. pneumoniae ATCC 13884 Adherence to Urethral Catheters
Quantification of the colonized area for each treatment was carried out using the ImageJ program. Thus, the colonized area of the bacteria under acetaminophen treatment was 12.17%, while under control conditions it was 2.07%. For both samples, an area of 1,735,237 µm 2 was evaluated. In the presence of acetaminophen, the bacteria colonized a greater area on the PVC catheters ( Figure  6A). In addition, more compact structures were observed in comparison with the biofilm produced without drug treatment ( Figure 6B), with more dispersed aggregates.
Analysis of the scanning electron microscope images (Figure 7) shows that the biofilm which formed under acetaminophen conditions displayed large colonization areas that were made up of compact masses of bacteria in prominent islets in comparison to those cultures without this drug. Figure 6. Optical microscope image of the K. pneumoniae ATCC 13884 biofilm assembled on the urethral catheter: (A) biofilm formation during acetaminophen treatment; and (B) without treatment with acetaminophen; both stained with crystal violet at 0.05%. Quantification of the area for each treatment was carried out using the ImageJ program, processing the sample by the particle content. Thus, the colonized area in the control sample was 2.07%, while in the acetaminophen sample it was 12.17%. Figure 6. Optical microscope image of the K. pneumoniae ATCC 13884 biofilm assembled on the urethral catheter: (A) biofilm formation during acetaminophen treatment; and (B) without treatment with acetaminophen; both stained with crystal violet at 0.05%. Quantification of the area for each treatment was carried out using the ImageJ program, processing the sample by the particle content. Thus, the colonized area in the control sample was 2.07%, while in the acetaminophen sample it was 12.17%.
Analysis of the scanning electron microscope images (Figure 7) shows that the biofilm which formed under acetaminophen conditions displayed large colonization areas that were made up of compact masses of bacteria in prominent islets in comparison to those cultures without this drug.

Discussion
As mentioned before, the WHO has made an urgent call to search for new antibiotics and new treatments to control many pathogenic bacteria, including K. pneumoniae. This bacterium, along with Escherichia coli, are pathogens commonly found in urinary infections as well as forming biofilms on urethral catheters [16]. There is a significant number of strains of these bacteria resistant to first line antibiotics such as gentamicin and ciprofloxacin [17]. However, in addition to the alternatives proposed by WHO, it is also necessary to determine the biochemical mechanisms involved in the pathogenicity, virulence, and resistance of these bacteria, especially quorum sensing. In fact, up to the present time, there have been no published reports on the influence of drugs on the formation of these biofilms. Investigations into these mechanisms could help explain recurrent infections in patients undergoing surgical procedures.
Of the 22 drugs assayed, 11 were found capable of increasing biofilm formation and modifying K. pneumoniae resistance, including gentamicin, tetracycline, amikacin, norfloxacin, loratadine, captopril, verapamil, dexamethasone, and progesterone. The effects of progesterone, in addition to that of acetaminophen and hydrochlorothiazide, stand out among them.
Progesterone displayed the greatest ability in the induction of biofilm formation (65%) but had a smaller effect on K. pneumoniae ATCC 13884 bacterial population growth (11%). Similarly, hydrochlorothiazide, a diuretic widely used in combination with antihypertensive drugs, had a great effect on the induction of biofilm formation (51%) and a smaller effect on bacterial growth (17%). Acetaminophen caused several changes in K. pneumoniae ATCC 13884 cultures related to quorum sensing, such as inducing the synthesis of the N-hexanoyl-homoserine lactone (C6-AHL) autoinducer by up 22%, increasing bacterial populations by 27%, inducing biofilm formation by 49%, and forming a more gentamicin-resistant biofilm. Furthermore, it increased the colonization area of the urethral catheter. Non-modified drugs are mainly excreted in the feces and urine at concentrations of approximately 0.09-0.6 µg/mL [18]. Therefore, intact drugs at low concentrations in urine might be enough to activate the virulence and resistance mechanisms of K. pneumoniae ATCC 13884.
The hypocholesterolemic lovastatin did not cause modifications in bacterial growth or biofilm formation at concentrations of 0.024-3.1 µg/mL; however, previous studies showed that statins are

Discussion
As mentioned before, the WHO has made an urgent call to search for new antibiotics and new treatments to control many pathogenic bacteria, including K. pneumoniae. This bacterium, along with Escherichia coli, are pathogens commonly found in urinary infections as well as forming biofilms on urethral catheters [16]. There is a significant number of strains of these bacteria resistant to first line antibiotics such as gentamicin and ciprofloxacin [17]. However, in addition to the alternatives proposed by WHO, it is also necessary to determine the biochemical mechanisms involved in the pathogenicity, virulence, and resistance of these bacteria, especially quorum sensing. In fact, up to the present time, there have been no published reports on the influence of drugs on the formation of these biofilms. Investigations into these mechanisms could help explain recurrent infections in patients undergoing surgical procedures.
Of the 22 drugs assayed, 11 were found capable of increasing biofilm formation and modifying K. pneumoniae resistance, including gentamicin, tetracycline, amikacin, norfloxacin, loratadine, captopril, verapamil, dexamethasone, and progesterone. The effects of progesterone, in addition to that of acetaminophen and hydrochlorothiazide, stand out among them.
Progesterone displayed the greatest ability in the induction of biofilm formation (65%) but had a smaller effect on K. pneumoniae ATCC 13884 bacterial population growth (11%). Similarly, hydrochlorothiazide, a diuretic widely used in combination with antihypertensive drugs, had a great effect on the induction of biofilm formation (51%) and a smaller effect on bacterial growth (17%). Acetaminophen caused several changes in K. pneumoniae ATCC 13884 cultures related to quorum sensing, such as inducing the synthesis of the N-hexanoyl-homoserine lactone (C6-AHL) autoinducer by up 22%, increasing bacterial populations by 27%, inducing biofilm formation by 49%, and forming a more gentamicin-resistant biofilm. Furthermore, it increased the colonization area of the urethral catheter. Non-modified drugs are mainly excreted in the feces and urine at concentrations of approximately 0.09-0.6 µg/mL [18]. Therefore, intact drugs at low concentrations in urine might be enough to activate the virulence and resistance mechanisms of K. pneumoniae ATCC 13884.
The hypocholesterolemic lovastatin did not cause modifications in bacterial growth or biofilm formation at concentrations of 0.024-3.1 µg/mL; however, previous studies showed that statins are able to inhibit K. pneumoniae growth at concentrations far higher than plasmatic ones [19]. On the other hand, metformin showed no effects on bacterial growth, but decreased biofilm formation by 21%, similar to ciprofloxacin, levofloxacin, and nitrofurantoin.
The other drugs assayed did not change the previously mentioned effects, although it has been reported that ibuprofen and diclofenac at plasmatic concentrations of 1.0 and 20 µg/mL, respectively, inhibit the formation of biofilms on abiotic surfaces [20].
Previously, it was established that the use of a catheter is a risk factor that increases the possibility of acquiring K. pneumoniae infections [21], as bacterial adherence and proliferation on abiotic surfaces causes persistent hospital infections. For instance, nearly 70% of hospitalized patients who have a urethral catheter for several days develop urinary infections due to the formation of biofilms on these devices [16]. In this paper, the effects of acetaminophen in the induction of bacterial colonization in catheters and changes in the architecture of the biofilm have been demonstrated. All of these factors favor microbial resistance to antibiotics, and, therefore, recurrence of infections.
All these results demonstrate the role of some drugs in modulating the biochemical behavior of K. pneumoniae. A complete understanding of the mechanism of action of these drugs in quorum sensing could generate new therapeutic alternatives, such as avoiding their use in high-risk patients. Furthermore, new specific inhibitors targeting this process could be designed; these working alongside current antibiotics could have the ability to restore the original power of the antibiotics through the inhibition of biofilm formation. Moreover, some bacterial development inhibitors by modulation of quorum sensing have been classified as anti-pathogenic [22,23] and anti-virulent [24] instead of antibiotic.
Finally, in light of the above, quorum sensing cannot be evaluated based only on the effect of one parameter of microbial biochemistry or by the modulation of violacein production in the biosensor C. violaceum, as is usually described in the literature, since microbial behavior is highly specific to each type of bacteria and autoinducer and their biochemical processes.

Conclusions
In summary, drugs such as acetaminophen, hydrochlorothiazide, and progesterone are modulators of quorum sensing in K. pneumoniae ATCC 13884 and could be involved in bacterial resistance through the induction of a more resistant biofilm, impermeable to antibiotics. Finally, the same methodology could be used to explore the effects of drugs on other microorganisms such as E. coli. Similarly, it could be used to analyze the role of other molecules involved in other human activities, including foods and cosmetics, which should be considered as potential inducing agents of microbial resistance.