In vitro activity of biofilm inhibitors in combination with antibacterial drugs against extensively drug-resistant Acinetobacter baumannii

Acinetobacter baumannii is a common pathogen of nosocomial infection, and its ability to form biofilms further contributes to its virulence and multidrug resistance, posing a great threat to global public health. In this study, we investigated the inhibitory effects of five biofilm inhibitors (BFIs) (zinc lactate, stannous fluoride, furanone, azithromycin, and rifampicin) on biofilm formation of nine extensively drug-resistant A. baumannii (XDRAB), and assessed the synergistic antibacterial effects of these BFIs when combined with one of four conventional anti-A. baumannii antibiotics (imipenem, meropenem, tigecycline, and polymyxin B). Each of the five BFIs tested was found to be able to significantly inhibit biofilm formation of all the clinical isolates tested under sub-minimal inhibitory concentrations. Then, we observed synergistic effects (in 22%, 56% and 11% of the isolates) and additive effects (56%, 44% and 44%) when zinc lactate, stannous fluoride and furanone were combined with tigecycline, respectively. When zinc lactate and stannous fluoride were each used with a carbapenem (imipenem or meropenem), in 33% and 56–67% of the isolates, they showed synergistic and additive effects, respectively. Additivity in > 50% of the isolates was detected when rifampicin was combined with imipenem, meropenem, tigecycline, or polymyxin B; and a 100% additivity was noted with azithromycin-polymyxin B combination. However, antagonism and indifference were noted for polymyxin B in its combination with zinc lactate and stannous fluoride, respectively. In conclusion, five BFIs in combination with four antibacterial drugs showed different degrees of in vitro synergistic and additive antibacterial effects against XDRAB.

. Activity of 15 antibiotic drugs against 9 clinical isolates of A. baumannii. a The standards from the CLSI 18

Effects of sub-inhibitory concentrations of BFIs on XDRAB biofilm formation.
With the subinhibitory concentrations of 5 BFIs described above, we tested the effects of these BFIs on biofilm formation. Compared with the no BFI control group, at the sub-inhibitory concentration of each agent, the five BFIs were found to significantly inhibit the biofilm formation of all 9 XDRAB isolates (P < 0.05 or < 0.01) (Fig. 2). The strongest effect was observed with zinc lactate (mean OD 570 decreased of 0.78, from 1.52 to 0.74), followed by   Combination antimicrobial drug susceptibility. Given the observed effects from sub-inhibitory BFIs on biofilm formation, we further tested how a combination use of a BFI with a clinically-relevant anti-A. baumannii antibiotic could interplay. Based on the fractional inhibitory concentration index (FICI) values generated from the combination drug susceptibility testing (Table 2), we observed that when zinc lactate was used in combination with imipenem, meropenem, and tigecycline respectively, 33%, 33%, and 22% of the isolates showed synergistic effects, and 67%, 67%, and 56% showed additive effects. However, when combined with polymyxin B, we detected an antagonistic effect. When stannous fluoride was used with imipenem, meropenem, and tigecycline, synergism was detected in 33%, 44%, and 56% of the isolates, respectively, while additivity was noted in 67%, 56%, and 44% of the isolates. Its combination with polymyxin B resulted in indifferent effects. Additivity was detected when furanone was combined with imipenem, meropenem or tigecycline (100%, 100% or 44% respectively). The combination of furanone and polymyxin B yielded either indifferent or antagonistic effect. Combination of rifampicin with imipenem, meropenem, tigecycline or polymyxin B led to additive effects (78%, 56%, 67% and 100%, respectively). When azithromycin was combined with polymyxin B, we observed an additive effect. Yet, azithromycin showed indifferent effects when combined with imipenem, meropenem or tigecycline.

Discussion
Our antimicrobial susceptibility testing results show that all nine isolates tested were XDRAB (i.e., non-susceptible to ≥ 1 agent in all tested drug categories but ≤ 2 categories) 19 , with resistance to commonly used antibacterial drugs, including β-lactam plus β-lactamase inhibitor, carbapenems, third/fourth-generation cephalosporins, aminoglycosides, and fluoroquinolones 18 . Some of the isolates were even non-susceptible to tigecycline and/or polymyxin B, which rendered these isolates close to be pandrug-resistant (i.e., non-susceptible to all tested drug categories) 19 . These findings are consistent with the fact that XDRAB has been increasingly isolated in clinical settings globally, which calls for combination therapy 11 . Indeed, the clinical choice of antibacterial drugs for XDRAB infections is limited, and the new therapeutic regime with combination drug use has been pursued 16,17,20,21 . Given that biofilms contribute to bacterial virulence and resistance 22 , we targeted agents with anti-biofilm property for their potential in combination drug use against A. baumannii. At their sub-MIC levels, our results revealed that the five BFIs tested in this investigation showed different degrees of inhibitory effects on the biofilm formation of XDRAB strains, especially the zinc lactate had the strongest effect, followed by stannous fluoride, furanone, rifampicin and azithromycin at our assay conditions. The inhibition of biofilm formation by these BFIs likely occurs through different mechanisms. Studies have shown that zinc compounds can inhibit www.nature.com/scientificreports/ the synthesis of extracellular polysaccharides or the formation of matrix networks, and stannous fluoride can destroy the biofilm structure by loosening the structure of the biofilm matrix 13,23 . Furanone, a quorum-sensing system inhibitor, inhibits the biofilms formation of bacterial by replacing the binding sites of quorum sensing signal molecules 24 . Azithromycin can inhibit the synthesis of alginate in the biofilm of Pseudomonas aeruginosa, thereby destroying the biofilm structure, leading to the formation of channels on the biofilm that may allow the synergistic drug to pass through the biofilm and thus to reach and kill the bacteria inside 25 . Our findings of the effects of sub-inhibitory zinc lactate, stannous fluoride and furanone further expand the understanding of subinhibitory conventional antibiotics including azithromycin and rifampicin in the reduction of A. baumannii biofilm formation 26 . Although BFIs of zinc lactate, stannous fluoride, furanone, and azithromycin have effects on XDRAB, their MIC values are high. Only rifampicin has a low MIC value. However, for the latter, the rapid development of RNA polymerase subunit-encoding rpoB gene mutation-mediated resistance to rifamycins limits the use of rifampicin alone against bacterial infections including A. baumannii 27 . According to the drug treatment principles for XDRAB, carbapenems (imipenem or meropenem), polymyxins (colistin or polymyxin B) and tigecycline are used as basic drugs in combination with other types of antibacterial drugs. Therefore, there is a clinical value in exploring how these BFIs interplay when an anti-A. baumannii antibacterial drug is used in combination with a BFI. The combination drug susceptibility testing results, as shown in Table 2, mainly reveals different levels of synergistic and additive antibacterial effects on XDRAB isolates, which suggests that BFIs may exert their actions via the reduction of biofilm formation and/or direct effect on bacterial growth to interplay with anti-A. baumannii antibiotics against XDRAB. With respect to rifampicin or azithromycin combination use with one of 4 anti-A. baumannii antibiotics, our results are largely in agreement with published in vitro studies [28][29][30] . The synergistic or additive effect from the combination use of zinc lactate, stannous fluoride, furanone or rifampicin with imipenem or meropenem against carbapenem-resistant XDRAB is not totally unexpected because the BFIs exerts different modes of action from that of carbapenems. An in vivo study has demonstrated efficacy of imipenemrifampicin against carbapenem-resistant A. baumannii 31 . However, as our studies have limitations that focus on the measurement of in vitro activity, the clinical significance of these observations remains to be determined.
Certain combinations also showed partly indifferent or antagonistic effects. For example, there were antagonistic and indifferent effects occurring in 100% of the isolates when zinc lactate and stannous fluoride were each combined with polymyxin B, respectively. This could partly be explained by that the tested XDRAB isolates were not resistant to polymyxins and anti-A. baumannii activities of polymyxin B may mask the role of zinc and stannous compounds or these cationic metal compounds may potentially affect polymyxin's mode of action in disrupting bacterial membrane integrity 32 . In other word, the positively charged group of polymyxin B can bind to the negatively charged phosphate in the phospholipids of the bacterial cell membrane, leading to the death of the bacteria 32 , while zinc lactate and stannous fluoride are metal cation biofilms inhibitors therefore they may compete with polymyxin B for drug targets, showing antagonistic or indifferent effect 33 . In contrast, the observation of indifferent effects from azithromycin combination with imipenem, meropenem, or tigecycline could be attributable to the weak anti-A. baumannii activity of azithromycin. In this regard, one study shows that another macrolide, clarithromycin, exerts antagonistic the effect with a carbapenem on P. aeruginosa 34 . However, the additivity between azithromycin and polymyxin is likely due to the membrane disruption of XDRAB by polymyxin B that resulted in improved accumulation of azithromycin into the cells and thus antibacterial activity 32,35 .
Furthermore, it is of importance to consider our in vitro drug combination synergistic results from the pharmacokinetic point of view for their potential clinical implications. Azithromycin and rifampicin are systematically administered antibiotics. While an azithromycin concentration comparable to its high MICs for A. baumannii is unlikely achievable in an in vivo situation, a pharmacokinetic-pharmacodynamic analysis of rifampicin has indicated to readily obtain pharmacokinetic parameters (such as the maximum serum concentration [C max ] value) corresponding to relatively low rifampicin MICs for A. baumannii 36 . Thus, our data are in support for the use of rifampicin as one of the combination agents for treatment of XDRAB infections 37,38 . On other hand, the three chemical BFIs, zinc lactate, stannous fluoride and furanone, are not expected to be administered systematically, partly due to toxicity concerns. However, their inhibitory concentrations can be readily reached in topical or local use such that as zinc lactate and stannous fluoride have been used in oral care formulations (e.g., mouthwash with 1.4 mg/ml for zinc lactate or up to 16 mg/ml for stannous fluoride) 39,40 , which warrants further studies in their potential topical use to combat wound infections associated with A. baumannii 1,41 .
In conclusion, we have presented data demonstrating the inhibitory effects of five BFIs on the biofilm formation of XDRAB at the sub-inhibitory concentrations and interplay between BFIs and anti-A. baumannii antibiotics against XDRAB. Further studies are warranted for their potential clinical implications in combating biofilm-associated bacterial infections.

Methods
Bacterial strains. Nine isolates of XDRAB were derived from clinical specimen (septum, endotracheal aspirate or respiratory lavage fluid) of patients from the critical care units, geriatrics, internal medicine and emergency department at the First Affiliated Hospital of Chengdu Medical College in 2018 to 2019 (Chengdu, Sichuan, China). These isolates were identified by standard laboratory methods and ATB New (bioMérieux, France) and also were further verified by PCR of two genes, 16S rRNA (with primers 5′-CAT TAT CAC GGT AAT TAG TG-3′ and 5′-AGA GCA CTG TGC ACT TAA G-3′) and bla OXA-51 (with primers 5′-TAA TGC TTT GAT CGG CCT TG-3′ and 5′-TGG ATT GCA CTT CAT CTT GG-3′) 42,43 . Staphylococcus aureus ATCC29213 and Escherichia coli ATCC25922 used as quality control strains in antimicrobial susceptibility testing were obtained from the American Type Culture Collection (USA).

Bacterial biofilm formation assay under sub-inhibitory concentrations of the biofilm inhibitors.
The biofilm formation assay was performed (with six duplicates for each isolate) using 96-well cell culture plate model with crystal violet staining 47 . The assay was repeated independently twice. Briefly, 170 μl of TSB medium and 10 μl of PBS (control group) or BFI solution (treatment group) were added into each well, followed by the inoculation of 20 μl of bacterial suspension (OD 600 of 0.12). After incubating at 37 °C for 24 h, nonadherent bacterial cells were removed by washing three times with 200 μl of PBS and dried in air. The remaining adherent bacterial cells were stained by adding 200 μl of 0.1% crystal violet. Following incubation of the plates at room temperature for 15 min, the plates were washed three times with PBS and then dried in air. Subsequently, 200 μl of 95% ethanol was added to each well for 5 min and the absorbance of the decolorization solution from each well was measured at the wavelength of 570 nm.
Combination antimicrobial susceptibility testing. The synergistic antibacterial effects of each of the five BFIs with one of the four anti-A. baumannii antibiotics on nine XDRAB isolates were evaluated by microdilution checkerboard method for determining FICI values. The highest concentration of each agent was 2 times its MIC with twofold sequential dilutions, and 8 concentration gradients were tested. Briefly, 170 μl of MHB, 10 μl of each of the series diluents of agents A and B, and 10 μl of freshly prepared bacterial suspension solution were added into the 96-well plate. The checkerboard consisted of columns in which each well contained the same amount of the drug being diluted along the x-axis, and rows in which each well contained the same amount of the drug being diluted on the y-axis, which resulted in each well of a checkerboard containing a unique combination of the two agents being tested. The results were visualized by incubation at 37 °C for 16-20 h. The FICI values were determined for two agents (A and B) by the equation: FICI = A (MIC combined /MIC alone ) + B (MIC combined / MIC alone ), with the interpretive criteria as: FICI ≤ 0.5 for synergy, 0.5 < FICI ≤ 1 for additivity, 1 < FICI ≤ 2 for indifference, and FICI > 2 for antagonism 48,49 .