Antibacterial Activity of Polyoxometalates Against Moraxella catarrhalis

The antibacterial activity of 29 different polyoxometalates (POMs) against Moraxella catarrhalis was investigated by determination of the minimum inhibitory concentration (MIC). The Preyssler type polyoxotungstate (POT) [NaP5W30O110]14− demonstrates the highest activity against M. catarrhalis (MIC = 1 μg/ml) among all tested POMs. Moreover, we show that the Dawson type based anions, [P2W18O62]6−, [(P2O7)Mo18O54]4−, [As2Mo18O62]6−, [H3P2W15V3O62]6−, and [AsW18O60]7− are selective on M. catarrhalis (MIC range of 2-8 μg/ml). Among the six tested Keggin type based POTs ([PW12O40]3−, [H2PCoW11O40]5−, [H2CoTiW11O40]6−, [SiW10O36]8−, [SbW9O33]9−, [AsW9O33]9−), only the mono-substituted [H2CoTiW11O40]6− showed MIC value comparable to those of the Dawson type group. Polyoxovanadates (POVs) and Anderson type POMs were inactive against M. catarrhalis within the tested concentration range (1-256 μg/ml). Four Dawson type POMs [P2W18O62]6−, [(P2O7)Mo18O54]4−, [As2Mo18O62]6−, [H3P2W15V3O62]6− and the Preyssler POT [NaP5W30O110]14− showed promising antibacterial activity against M. catarrhalis (MICs < 8 μg/ml) and were therefore tested against three additional bacteria, namely S. aureus, E. faecalis, and E. coli. The most potent antibacterial agent was [NaP5W30O110]14−, exhibiting the lowest MIC values of 16 μg/ml against S. aureus and 8 μg/ml against E. faecalis. The three most active compounds ([NaP5W30O110]14−, [P2W18O62]6−, and [H3P2W15V3O62]6−) show bacteriostatic effects in killing kinetics study against M. catarrhalis. We demonstrate, that POM activity is mainly depending on composition, shape, and size, but in the case of medium-size POTs (charge is more than −12 and number of addenda atoms is not being higher than 22) its activity correlates with the total net charge.


INTRODUCTION
Moraxella catarrhalis is a Gram-negative human mucosal pathogen which causes middle ear infections in infants and children and lower respiratory tract infections in adults with chronic pulmonary disease (Karalus and Campagnari, 2000). M. catarrhalis is one of the three major causes of otitis media along with Streptococcus pneumoniae and Haemophilus influenzae (Del Beccaro et al., 1992). Based on culture isolation and serological studies, M. catarrhalis has been implicated as a cause of sinusitis in both children and adults. In addition, M. catarrhalis occasionally causes severe infections such as septic arthritis, bacteremia, cellulitis, osteomyelitis, endocarditis, and pericarditis (Karalus and Campagnari, 2000). The fact that M. catarrhalis was not considered as an important human pathogen until recently has contributed to the limited research aimed to find vaccines for prevention or selective antibiotics for the treatment of respiratory tract infections (Karalus and Campagnari, 2000).
Excessive or improper use of antibiotics led to the development of antibacterial resistance worldwide during the last few decades, suggesting the incidence of these infections may continue to rise. Thus, new active classes of antibiotics are urgently needed for the most common community-acquired respiratory pathogens with emerging antimicrobial resistance. Along with new organic compounds, metal oxides have attracted significant interest over the past decade as they offer alternative modes of antimicrobial action (Dizaj et al., 2014). A particularly attractive sub-class of metal oxides is metal oxide anions, the socalled polyoxometalates (POMs) (Pope, 1983). POMs comprise an array of corner-and edge-sharing pseudo-octahedrally coordinated MO 6 (M most often V, Nb, Mo, W) units that form an ionic core and are amenable to a variety of chemical transformations (Figure 1). Alongside with applications of POMs in catalysis (Wang and Yang, 2015), nanotechnology (Yamase and Pope, 2006), electrochemistry (Sadakane and Steckhan, 1998), material sciences (Proust et al., 2008), and molecular magnetism (Clemente-Juan et al., 2012), POMs have also been proven to exhibit remarkable biological activity. Due to the highly negative charge, strong acidity, geometry, their use in macromolecular crystallography Rompel, 2015, 2017;Molitor et al., 2017) and as antimicrobial, (Yamase, 2005;Li et al., 2016;Bijelic et al., 2018), antiviral (Judd et al., 2001), antitumor (Fu et al., 2015), antidiabetes (Nomiya et al., 2001), and antiamyloid-fibril agents (related to Alzheimer's disease) (Gao et al., 2014) has been reported so far and more attention should be given to the biological and therapeutic effect of POMs.
Polyoxotungstates (POTs), polyoxomolybdates (POMos) and polyoxovanadates (POVs) of different structural types have been shown to exhibit synergy with some conventional antibiotics (Yamase et al., 1996;Tajima, 2001) or direct antibacterial activity (Inoue et al., 2005;Bae et al., 2008) against both Gramnegative and Gram-positive bacteria. In general POVs, especially decavanadate, and large, highly negatively charged POMs exhibit a high activity, whereas for example the activity of Keggin type POMs is bacterial strain dependent (Bijelic et al., 2018).

MIC Determination
Minimum inhibitory concentrations (MICs) were determined by the broth microdilution method according to guidelines of the Clinical Laboratory Standards Institute (Wikler, 2009). Double dilutions of tested compounds in 96-well microtiter plates were prepared in the concentration range of 1-256 µg/mL. E. coli (ECM1556) and S. aureus (ATCC 29213) were grown on Mueller-Hinton agar plates (by Becton Dickinson, USA), whereas E. faecalis (ATCC29212) and M. catarrhalis (ATCC 23246) were grown on Columbia agar with 5% defibrinated sheep blood. Inocula were prepared by direct colony suspension method and plates were inoculated with 5·10 −4 CFU/well. Results were determined by visual inspection after 20-22 h of incubation at 37 • C in ambient air. Testing was performed by the standard broth microdilution method with azithromycin (Lode et al., 1996) as the reference antibiotic to assess test validity.

Time-Killing Assay
M. catarrhalis inoculum was prepared by direct colony suspension in sterile saline and the organism density was matched to 1.0 McFarland turbidity standard. The bacterial suspension was further diluted in cation-adjusted Mueller-Hinton Broth in 1:50 ratio to obtain the starting inoculum of 5·10 5 -5·10 6 colony-forming units (CFU)/mL. Tested POMs were added to tubes containing 6 mL of bacterial suspension, in concentrations corresponding to 1×, 5×, and 10×MIC, while the control antibiotic azithromycin was tested with 1× and 10×MIC. One tube was used as a drug-free control. After addition of the POMs, tubes were incubated at 37 • C for 24 h. Viable colony counts were determined at 0, 2, 4, 6, and 24 h. At each timepoint, a 100 µL aliquot was removed from each tube and 10-fold dilutions were prepared in saline, plated on Columbia agar with 5% defibrinated sheep blood in 20 µL aliquots and incubated on 37 • C for 24 h. The lower limit for quantifying colony counts was 200 CFU/mL. Bactericidal activity was defined as a ≥3 log 10 reduction in CFU/mL (Barry et al., 1999).

Antibacterial Activity of Preyssler and Dawson Type POTs and POMos
The antibacterial activity of the 29 POMs against the Gramnegative M. catarrhalis was evaluated by means of MIC ( Table 1). The highest activity with a MIC range of 1-8 µg/ml was observed for POMs with Preyssler type ( Figure 1A) and Dawson ( Figure 1B) structure.
Moreover, the most active POM on M. catarrhalis, namely the Preyssler anion P 5 W 14− 30 ( Figure 1A) (MIC = 1 µg/ml), was additionally tested on the Gram-positive organisms S. aureus and E. faecalis and the Gram-negative E. coli, which are major human pathogens that cause a wide range of clinical infections ( Table 2). P 5 W 14− 30 exhibited good activity against S. aureus with MIC = 16 µg/ml and E. faecalis with MIC = 8 µg/ml, which is the same as for the clinically applied drug azithromycin (Lode et al., 1996), however, it performed inactive against the Gramnegative E. coli. The chitosan-P 5 W 30 nanoassembly has already demonstrated high anticancer activity, which is considered to arise due to high number of phosphorous and tungsten atoms (Shah et al., 2014). Remarkably, the Se-containing lacunary anion Se 2 W 14− 29 , which is of comparable size and equally charged, exhibited significantly lower MICs (64 µg/ml). This indicates the importance and the influence of the structure, shape, and composition for the antibacterial activity, justifying more detailed studies to elucidate the structure-activity relationship.
Except for P 2 Mo 6− 18 , S 2 Mo 4− 18 , and As 2 W 6− 18 , all Dawson type POMs (Figure 1B) tested in this study exhibited potential antibacterial activity exhibiting a MIC within the range of 2-8 µg/ml. Among the Dawson type group, P 2 W 6− 18 and its

POSITIVE CONTROL
Azithromycin (Lode et al., 1996) 0.06 POMs combined in groups according to their structural type and within the group listed from higher to lower activity. *m -number of addenda atoms.
triple-protonated equally charged vanadium-substituted analog P 2 W 15 V 6− 3 ( Figure 1B) have proven to be the most promising with a MIC of 2 µg/ml suggesting that VO 6 sites in Dawson type mixed polyoxovanadatotungstates (POVTs) lattice do not have any significant impact on the antibacterial activity, which was observed earlier for Keggin POVTs as they were remarkably more active against S. pneumoniae than their corresponding POTs (Fukuda and Yamase, 1997). In the Dawson pair P 2 W 6− 18 (MIC = 2 µg/ml) and P 2 Mo 6− 18 (MIC > 256 µg/ml), the POMo is considered as inactive, whereas for As 2 W 6− 18 (MIC > 256 µg/ml) and As 2 Mo 6− 18 (MIC = 4 µg/ml) the opposite effect is observed. Dawson related compounds, namely AsW 7− 18 ( Figure 2C), with  (Figure 2B), which has a pyrophosphate anion enclosed (Kortz and Pope, 1994), demonstrated higher activity against bacteria (MIC values are 8 and 4 µg/ml, respectively) than classical As 2 W 6− 18 and P 2 Mo 6− 18 (Figure 2A). The presence of highly bioactive and toxic arsenic trioxide in the first case should play a significant role, but difference in the coordination of the heteroatoms in both cases leads to a change of the "rugby-ball-shaped" (Figure 2A) Dawson structure to a "hour-glass" shaped anion (Figures 2B,C), which also may be related to discrepancies in antibacterial activity. These anomalies in the activity of isostructural POTs and POMos indicate that both the hetero-and addenda atoms play a significant role in the bioactivity and that the appropriate combination of these atoms must be decisive for the antibacterial activity.
The superiority of the Dawson structure among four different structural groups of polyoxomolybdates in the inhibition of a tartrate-resistant acid phosphatase (ACP) from Leishmania donovani and the tartrate-sensitive ACP from human seminal fluid (prostatic ACP) has been reported previously (Saha et al., 1991). As 2 Mo 6− 18 was the most potent inhibitor and exhibited the highest degree of selectivity against both ACPs. Here, As 2 Mo 6− 18 is proved to be a potent antibacterial agent with the third lowest MIC value of 4 µg/ml against M. catarrhalis.

Antibacterial Activity of Keggin-and Anderson Based Type POTs
Keggin type POTs are known to exhibit antibacterial activities, for example, by increasing the susceptibility of certain bacteria strains toward β-lactam antibiotics (Yamase et al., 1996). In this study the strongest activity was shown for the Keggin based CoTiW 6− 11 ( Figure 1D) exhibiting a MIC value of 16 µg/ml. Interestingly, despite consisting of the same isomer of Keggin unit, the classical PW 3− 12 ( Figure 1C) and the two mono-substituted PCoW 5− 11 and CoTiW 6− 11 (Figure 1D) showed completely different activities. The most negatively charged CoTiW 6− 11 is the most active compound; however, the charge dependency is not observed in the case of the other two Keggin anions-PW 3− 12 with a total charge of −3 exhibited a MIC of 128 µg/ml and PCoW 5− 11 with a total charge of −5 exhibited a value >256 µg/ml. Thus, we assume a decisive role for the accessible TiO 6 unit in CoTiW 6− 11 (Figure 1D) in the activity against M. catarrhalis. CoTiW 6− 11 was previously also shown as the most potent NTPDase inhibitor among six different POTs, (Müller et al., 2006).
The inorganic and organically functionalized Anderson type POTs and POMos ( Figure 1I) are inactive against M. catarrhalis ( Table 1). The inactivity of this type of POM was previously observed for Helicobacter pylori, which as well as M. catarrhalis is most sensitive to larger POMs (Yamase et al., 1996). It is tempting to speculate that the combination of compact size and small charge of Anderson type anion (Blazevic and Rompel, 2016) is the reason of its antibacterial inactivity.

Antibacterial Activity of Isopolymetalates
Among the investigated isopolyanions only two POTs (W 10− 12 ( Figure 1H) and W 12− 22 ) and octamolybdate Mo 4− 8 ( Figure 1G) showed a MIC value >256 µg/ml. It should be noted, that decavanadate tested in this study (V 6− 10 ) did not show antibacterial activity (MIC >256 µg/ml), which confirms the selective activity of the most common vanadates V 6− 10 and V 4 O 4− 12 against Streptococcus pneumoniae with MIC values in the range of 4-32 µg/ml (positive control with conventional antibiotics: 2-32 µg/ml; Fukuda and Yamase, 1997). We also included tetranuclear vanadium tartrates (V 4 -L-tart 4− and V 4 -D-tart 4− ) in our study as they, similarly to V 6− 10 , are one of the few vanadate species with proved stability and hydrolytic immunity in aqueous solutions over time (Schwendt et al., 2007). However, both POVs were inactive toward M. catarrhalis.

The Relationship Between the Composition of POMs and Its Activity Against M. catarrhalis
By analyzing the data in and CoTiW 6− 11 ). As already noted above, there are at least three factors affecting the antibacterial activity: size, charge, chemical composition, and their combination. In order to understand the structure-activity-relationship (SAR) we minimized the influence of one of these factors and compared the main characteristics for phosphorus-containing Keggin PW 3− 12 (Figure 1C), Dawson P 2 W 6− 18 (Figure 1B), and Preyssler P 5 W 14− 30 ( Figure 1A) POTs (Table 3). Leastways for these fully saturated (not lacunary) POTs with the same heteroatom PW 3− 12 , P 2 W 6− 18 , and P 5 W 14− 30 there is a clear dependence in the increase in antibacterial activity with an FIGURE 2 | Ball and stick representation of (A) classical Dawson type anion P 2 Mo 6− 18 (Briand et al., 2002); (B) anion in P 2 O 7 Mo 4− 18 (Kortz and Pope, 1994); (C) anion in AsW 7− 18 (Jeannin and Martin-Frere, 1979). Color code: addenda atom Mo (B) or W (C), dark blue spheres; heteroatom P (B) or As (C), yellow spheres; O, red spheres. increase in charge and size and no correlation with respect to the redox potential.
No simple SAR was found for all tested POMs, however, narrowing the data set only to the largest tested group, namely POTs with a charge <-12 and with a number of addenda atoms not being higher than 22 it became possible to correlate the antibacterial activity and the charge of the POT (Figure 3). The presented dependence may indicate for medium-sized POTs (but not for POTs with number of addenda atoms more than 22) a stronger effect against M. catarrhalis of anions exhibiting a charge of −8 to −6.
Cells of M. catarrhalis have on their surface low molecular weight lipopolysaccharides (LPS), also called lipooligosaccharides (LOS), which contribute to the increased hydrophobicity of its outer membrane and to the high susceptibility to hydrophobic antimicrobial agents such as macrolides (Gotoh et al., 1989;Tsujimoto et al., 1999). However, M. catarrhalis shows susceptibility not only to hydrophobic agents, but also to hydrophilic agents such as β-lactam antibiotics (Gotoh et al., 1992). The increased susceptibility of these strains toward β-lactams is probably due to the higher permeability of the outer membrane toward these agents. POMs, as examples of super chaotropic anions, can adsorb onto lipid monolayers via electrostatic and/or hydrophobic interaction depending on the charge of the lipid layer (Kobayashi et al., 2017). The model experiments with three differently charged Keggin anions show that dominant interaction equally depends both on the charge density of POMs and on the lipid density (Kobayashi et al., 2017).  Figure 1B) and AsW 7− 18 ( Figure 2C) demonstrate Dawson structure, CoTiW 6− 11 ( Figure 1D) and SiW 8− 10 ( Figure 1E) are Keggin-based anions, whereas W 10− 12 ( Figure 1H) and W 12− 22 -isopolytungstates (see Table 1).

Time-Killing Studies
In order to assess whether the tested compounds kill the bacteria (bactericidal effect) or prevent its growth (bacteriostatic effect), time-kill study was performed. Killing kinetics for three the most active compounds: Preyssler P 5 W 14− 30 ( Figure 1A) and two Dawson P 2 W 6− 18 and P 2 W 15 V 6− 3 ( Figure 1B) POTs were FIGURE 4 | Time-kill curves for Dawson P 2 W 6− 18 and P 2 W 15 V 6− 3 , Preyssler P 5 W 14− 30 POTs and azithromycin at minimum inhibitory concentration (MIC) (red), 5-fold (blue) and 10-fold MIC concentration (green) against M. catarrhalis ATCC23246 strain. Control represents uninhibited bacterial growth (black). determined against M. catarrhalis. POTs were tested at three concentrations, corresponding to 1×, 5×, and 10×MIC. The bactericidal activity of the agents was defined for at least a 3 log 10 reduction in viable colony counts. In the control (sample without antibiotic), the numbers of the viable strain were kept within the cultivation of 24 h relative to those at 0 h. Figure 4 represents time-killing curves for compounds P 5 W 14− 30 , P 2 W 6− 18 , and P 2 W 15 V 6− 3 . All tested POMs show bacteriostatic effects, resulting from a little change in viable colony numbers within 24 h despite the concentration being equal to 10-fold MIC (Figure 4). Although it would seem preferable for an antibiotic to kill the offending bacteria rather than to merely inhibit it, the clinical importance of an in vitro bactericidal action being better than a bacteriostatic action has rarely been documented. The superiority of bactericidal over bacteriostatic action in the treatment of gram-positive bacterial infections is intuitive rather than based on rigorous scientific research (Pankey and Sabath, 2004).
2) The Preyssler type POT P 5 W 14− 30 ( Figure 1A) showed the highest antibacterial activity against M. catarrhalis (MIC = 1 µg/ml) and further MIC investigation against S. aureus and E. faecalis proved its antibacterial potential.
3) Based on MIC values, Dawson-type POMs (see Figure 1B) exhibited highest activity and selectivity against M. catarrhalis. 4) Among Keggin-type POMs (see Figures 1C-E), only the mono-substituted CoTiW 6− 11 ( Figure 1D) showed MIC comparable to that of the Dawson-type group. 5) POVs and Anderson type POMs ( Figure 1I) were inactive (MIC > 256 µg/ml) against M. catarrhalis strain. 6) According to time-killing studies three the most active POTs (Preyssler P 5 W 14− 30 and Dawson P 2 W 6− 18 and P 2 W 15 V 6− 3 ) showed bacteriostatic effect against M. catarrhalis. 7) POM activity mainly depends on composition, shape and size, but in the case of medium-size POTs correlates with the total net charge.