Acinetobacter baumannii is a multi-drug resistant (MDR) Gram-negative, aerobic bacillus that has emerged as a major cause of nosocomial infections with mortality rates ranging from 34 to 61% (Karageorgopoulos and Falagas, 2008; Wieczorek et al., 2008; Esterly et al., 2011). In humans, MDR A. baumannii causes a wide-spectrum of clinical conditions, including pneumonia (Leung et al., 2005), blood-stream infections (Wisplinghoff et al., 2004; Centers for Disease Control and Prevention [CDC], 2004), meningitis (Metan et al., 2007), urinary tract infections (Sunenshine et al., 2007), and wound infections (Scott et al., 2007) In addition, reports of other manifestations such as endocarditis, peritonitis, and osteomyelitis associated with A. baumannii have been reported (Olut and Erkek, 2005; Menon et al., 2006) A. baumannii is ranked as one of the most common bacteria associated with intensive care units (Garnacho-Montero and Amaya-Villar, 2010; Ulu-Kilic et al., 2013), and is difficult to treat due to its resistance to most of the currently available antibiotics (Maragakis and Perl, 2008; Doi et al., 2009; Neonakis et al., 2011; Al Mobarak et al., 2014; Ellis et al., 2015) For example, carbapenems, which were once the antimicrobials of choice for treating A. baumannii, are no longer completely effective due to resistance development by the bacterium (Falagas et al., 2006; Bassetti et al., 2008; Abbott et al., 2013; Fonseca et al., 2013). Similarly, although polymyxins have been successfully used to treat A. baumannii infections, strains resistant to these drugs have appeared (Hernan et al., 2009; Lean et al., 2014; Pogue et al., 2015). In light of these reports, the Infectious Diseases Society of America ranked A. baumannii as one of the top priority, antibiotic-resistant pathogens to target due to its rapid propensity to develop drug resistance, and a limited choice of antibiotics available to treat infections caused by this bacterium (Talbot et al., 2006; Shlaes et al., 2013).

A. baumannii is considered to be multidrug resistant, if it exhibits resistance to more than three classes of antibiotics (Falagas et al., 2006). The resistance of A. baumannii to antibiotics has been attributed to multiple mechanisms, including reduced permeability of its outer membrane to antibiotics, constitutive expression of drug efflux pumps, and its ability to acquire and incorporate genetic elements such as plasmids, transposons, and integrons (Giamarellou et al., 2008; Cai et al., 2012). In addition, A. baumannii has a significant ability to produce biofilms on various surfaces (Espinal et al., 2012; Longo et al., 2014), which not only increases the potential of A. baumannii for nosocomial spread, but also contributes to its resistance to antibiotics and virulence (Lee et al., 2008; Rao et al., 2008). Thus, there is a critical need to explore novel strategies for treating A. baumannii infections.

Traditionally, plants have served as a source of novel drugs for treating a variety of diseases in humans (Cowan, 1999). A variety of plant-derived compounds possessing antimicrobial properties against a wide range of microorganisms have been documented (Kon and Rai, 2012; Upadhyay et al., 2014). The antimicrobial effects of four components of ginger against MDR strains of A. baumannii has been reported (Wang et al., 2010). Similarly, the essential oil from coriander was found to exert either synergistic or additive effects with antibiotics such as tetracycline, chloramphenicol, ciprofloxacin, gentamicin, piperacillin, and cefoperazone against A. baumannii (Duarte et al., 2012). Recently, aqueous extract of kiwi (Actinidia deliciosa) and clove (Syzygium aromaticum) were found to exert anti-biofilm activity against A. baumannii (Tiwari et al., 2017). Similarly, it was shown that plant extract from Aegle marmelos and imipenem had synergistic effect against carbapenem resistant strain of Acinetobacter baumannii (Tiwari et al., 2016). In another study, the essential oil from Origanum vulgare possessed potent antimicrobial activity against MDR A. baumannii (Saghi et al., 2015). In addition, previous research from our laboratory indicated that several plant-derived antimicrobials (PDAs), including trans-cinnamaldehyde (TC), an ingredient in cinnamon, eugenol (present in clove) and carvacrol and thymol obtained from oregano oil and oil of thyme, respectively, decreased antibiotic resistance in MDR S. Typhimurium DT 104 (Johny et al., 2010). These investigators observed that TC reduced DT 104’s resistance to five antibiotics, where thymol and carvacrol decreased resistance to three antibiotics.

The β-lactam group of antibiotics are the most commonly prescribed antibiotics for the treatment of bacterial infections worldwide (Pitout et al., 1997; Thakuria and Lahon, 2013). A. baumannii is capable of producing β-lactamases that can hydrolyze the β-lactam ring of penicillins, cephalosporins, and carbapenems, thereby conferring resistance to these antibiotics. Therefore, this study investigated the efficacy of TC and eugenol (EG) in increasing the sensitivity of A. baumannii to seven β-lactam antibiotics. In addition, the effect of these PDAs on genes conferring resistance to β-lactam antibiotics in A. baumannii was determined.

The SICs of TC and EG against A. baumannii were found to be 1.1 mM (0.015%) and 1.8 mM (0.03%), respectively, while the MIC was 4 mM (0.05% TC and 0.065% EG) for both PDAs. Although a previously reported MIC of each antibiotic against A. baumannii was used in the study, both A. baumannii isolates grew ∼ by 2.0 log₁₀ CFU/ml after 24 h (antibiotic control), as observed in Figures 1, 2 and Supplementary Figures 1, 2. In the presence of the MIC of TC, the bacterial count after 24 h did not change significantly from the inoculation of level of 6.0 log₁₀ CFU/ml (Figure 1) as expected. However, when A. baumannii was grown in the presence of each antibiotic and TC, its growth after 24 h was significantly decreased in comparison to that in the positive control, antibiotic control and TC control (P < 0.05). In both A. baumannii isolates, the greatest sensitivity was observed to methicillin and lowest sensitivity was seen against ampicillin and meropenem (Figure 1 and Supplementary Figure 1). Similarly, the sensitivity of both A. baumannii isolates to all seven antibiotics was significantly increased in the presence of EG, as indicated by the reduced growth after 24 h (P < 0.05) (Figure 2 and Supplementary Figure 2). In A. baumannii 17978, the greatest sensitivity was observed against ampicillin (Figure 2), whereas A. baumannii 251847 was maximally sensitive to amoxicillin (Supplementary Figure 2).

The results of the efflux pump inhibition assay using EtBr and pyronin Y in A. baumannii ATCC 17978 and A. baumannii 251847 are presented in Figures 3, 4 and Supplementary Figures 3, 4. EtBr and pyronin Y are known substrates for AdeABC and AdeIJK efflux pumps, respectively (Magnet et al., 2001; Xing et al., 2014). The MIC of TC and EG resulted in an increased accumulation of EtBr inside bacterial cells, as indicated by an increase in fluorescence compared to the PBS control (P < 0.05) (Figures 5A–G). CCP, an efflux pump inhibitor used as a positive control (Magnet et al., 2001) also resulted in an increase in fluorescence indicating suppression of efflux pump in A. baumannii. However, a similar increase in the fluorescence was not observed for pyronin Y compared to the PBS control (Figure 4 and Supplementary Figure 4). The results of NPN uptake assay in A. baumannii ATCC 17978 and A. baumannii 251847 are presented in Supplementary Figures 5, 6, respectively. Neither TC nor EG increased NPN uptake, while the EDTA control did show an increase in fluorescence. Further, the samples treated with TC or EG demonstrated a decrease in fluorescence over time (data not shown).

The effect of seven antibiotics and two PDAs and their combination on the expression of various antibiotic resistance genes in A. baumannii 17978 is depicted in Figures 5A–G. It was observed that compared to control, the expression of all tested genes was up-regulated (P < 0.05) following A. baumannii growth in the presence of the antibiotics. However, TC significantly down-regulated the expression of major genes conferring resistance to β-lactam antibiotics compared to control (P < 0.05). The genes encoding efflux pump adeA and adeB were down regulated by ∼6- and 9-fold, respectively. Moreover, the expression of genes encoding β-lactamase (blaP) and the multiple drug resistance protein (mdrp) was decreased by ∼3-fold (P < 0.05). Similar to the results observed with TC, all antibiotic resistance genes were down-regulated on exposure to EG compared to control (P < 0.05). The expression of genes encoding efflux pumps, adeA and adeB were down-expressed by 3- and 14-fold, respectively. Similarly, blaP and mdrp were also down-regulated in both strains (P < 0.05). The combination of TC or EG with the antibiotics also resulted in a down-regulation of majority of the tested genes (P < 0.05). Among the combinations of TC or EG with the seven antibiotics, the combination containing aztreonam resulted in a significant down-regulation of all the tested genes compared to control (Figure 5D). The combination of EG with piperacillin (Figure 5C) or penicillin (Figure 5F) also significantly reduced the expression of all the antibiotic resistance genes (P < 0.05).

Rapid emergence of antibiotic resistance in pathogenic microorganisms, especially to multiple antibiotics has ignited research efforts to discover novel antibiotics and develop effective derivatives of currently available antibiotics. However, no promising antibiotics are under development, and the rapidity and complexity of resistance development in pathogens have further exacerbated the situation. In light of this, a potential viable approach was explored to reduce bacterial antibiotic resistance in the development of inhibitors of resistance mechanisms in bacteria (Renau et al., 1999; Walsh and Fanning, 2008). This strategy involves the co-administration of an antibiotic with an “inhibitor,” which counteracts bacterial resistance mechanism(s), thereby rendering the resistant pathogen sensitive to the drug. The advantage of this approach is that it makes it possible to continue the use of current antibiotics, for which in-depth pharmacological and toxicological data are already available. In this regard, PDAs represent a potential natural group of “inhibitors” of bacterial antibiotic resistance mechanisms.

Abundant literature exists on the antimicrobial properties of a variety of plant compounds against a wide range of microorganisms (Burt, 2004; Upadhyay et al., 2014). However, only a handful of studies have addressed their effect on bacterial antibiotic resistance, especially in Gram negative bacteria (Gallucci et al., 2006; Chusri et al., 2009; Johny et al., 2010; Ilić et al., 2014). In the current study, both TC and EG enhanced the sensitivity of A. baumannii to all seven β-lactam antibiotics tested (Figures 1, 2 and Supplementary Figures 1, 2). This is evident from the significant reductions in bacterial counts observed in the samples containing PDA and antibiotics as compared to that in the treatments containing each PDA or antibiotic alone. A checkerboard assay testing a wide range of concentrations of two antimicrobials is commonly used for determining their combinatorial effects on a bacterium (Jayamani et al., 2017; Tharmalingam et al., 2018). However, since our preliminary studies revealed that concentrations of TC and EG below the MIC were not significantly effective in increasing A. baumannii sensitivity to antibiotics, we did not use a checkerboard assay, but instead tested the combinatorial effect of MIC of plant molecules and antibiotics on the pathogen.

A recent study also reported synergistic effect of Eucalyptus camaldulensis with polymyxin B against MDR Acinetobacter baumannii (Knezevic et al., 2016). In another study, Enrofloxacin and cinnamon were observed to possess synergistic effect against Salmonella enterica (Solarte et al., 2017). Further, extracts of medicinal plant Holarrhena antidysenterica with Conessine increased susceptibility of extensively drug resistant A. baumannii to novobiocin and rifampicin (Siriyong et al., 2016). Additionally, no cytotoxic effects on human epithelial cells at the tested concentrations were reported previously with TC and EG (Amalaradjou et al., 2010; Karumathil et al., 2016). Moreover our previous studies revealed that in-feed supplementation of TC and EG in broiler chicks (Kollanoor-Johny et al., 2012) and TC in mice (Narayanan et al., 2017) for 10 days did not result in any toxicity.

A. baumannii has been reported to exhibit resistance to β-lactam antibiotics through several mechanisms, including the production of β-lactamases, changes in penicillin-binding proteins (PBPs), altering the structure and number of porin proteins, decreased membrane permeability, and by use of efflux pumps that exit antibiotics out of the bacterial cell (Vila et al., 2007; Manchanda et al., 2010; Bonnin et al., 2013; Tang et al., 2014). Moreover, the presence of efflux pumps and MDR proteins in A. baumannii contribute significantly to both intrinsic and acquired resistance to antibiotics (Lomovskaya and Bostian, 2006). A. baumannii genome encodes a wide array of multidrug efflux systems, including AdeABC, a resistance-nodulation-division (RND) family-type pump (Damier-Piolle et al., 2008; Wieczorek et al., 2008; Yoon et al., 2013; Sun et al., 2014). The substrates for this pump include beta-lactams, aminoglycosides, erythromycin, chloramphenicol, tetracycline, fluoroquinolone, trimethoprim, and EtBr (Magnet et al., 2001; Higgins et al., 2004; Heritier et al., 2005; Peleg et al., 2007). The three component structures of AdeABC include the inner membrane fusion protein (AdeA), transmembrane component (AdeB) and an outer membrane protein (AdeC), with the inactivation of adeB resulting in the loss of pump function and multidrug resistance (Magnet et al., 2001).

In order to determine if TC or EG exerted an inhibitory effect on the aforementioned efflux pumps in A. baumannii, an efflux pump inhibition assay was performed with EtBr and pyronin Y along with CCP, a documented efflux pump inhibitor (Magnet et al., 2001; Chusri et al., 2009). EtBr and pyronin Y are known substrates for AdeABC and AdeIJK efflux pumps, respectively (Magnet et al., 2001; Damier-Piolle et al., 2008; Chusri et al., 2009; Xing et al., 2014). Both TC and EG resulted in the inhibition of AdeABC efflux pump in the two tested A. baumannii strains, as evident from the increase in fluorescence in the treated samples compared to PBS control (Figure 3 and Supplementary Figure 3). However, the PDAs did not exert any inhibitory effect on the action of AdeIJK efflux pump in A. baumannii, as evident from the lack of difference in fluorescence units between PDA-treated and PBS control samples (Figure 4 and Supplementary Figure 4). Similarly, the results from the NPN uptake assay revealed that TC and EG did not increase A. baumannii’s outer membrane permeability, as seen from Supplementary Figures 5, 6, where no increase in fluorescence was observed in PDA-treated A. baumannii as against the samples treated with EDTA, a known outer membrane permeabilizer in Gram-negative bacteria (Helander and Mattila-Sandholm, 2000; Alakomi et al., 2006). These results indicate that TC and EG increased the sensitivity of A. baumannii to the tested antibiotics at least in part by inhibiting the efflux pump, AdeABC.

For ascertaining if TC or EG exerted an inhibitory effect on any of the antibiotic resistance genes conferring resistance to β-lactam antibiotics in A. baumannii, we performed a RT-qPCR on mRNA extracted from A. baumannii following growth in the presence and absence of the PDAs. The results from the RT-qPCR revealed that TC and EG in combination with or without each antibiotic significantly down-regulated the expression of the majority of genes that confer resistance to β-lactam antibiotics (Figures 5A–G). Among the various genes screened, those encoding efflux pumps, adeA and adeB were maximally down-regulated by both PDAs. These results concur with the results from the EtBr efflux pump inhibition assay, and suggest that TC and EG enhanced the efficacy of the seven β-lactam antibiotics against A. baumannii by thwarting the various resistance mechanisms, especially those involving the efflux pumps.

The results of this study suggest the potential use of TC and EG in conjunction with the currently available β-lactam antibiotics for the treatment for MDR A. baumannii infections and could lead to development of new treatment options with reduced antibiotic dosage. However, efficacy studies in suitable animal models are warranted before recommending their clinical usage.

KV is the corresponding author and primary contact during manuscript submission, review, and publication process. The work was done under his supervision as the principal investigator. He significantly contributed to the design, drafting, revisions, and interpretation of data. The manuscript is being submitted with his final approval for publication. MN is the submitting author. DK and AK-J designed the study. DK and JG conducted the experiments. DK and MN prepared the manuscript for submission.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.